AUTOMATED CELL CLASSIFICATION ORGANIZATION

专利摘要:
automated organization for cell classification. it is a device and methods for automatically performing organization steps for flow cytometry operations. the invention provides the spatial determination of a flow stream and the subsequent automatic alignment of analysis devices and / or collection vessels. the automatic determination of flow current properties provides automatically configured flow cytometer parameters.
公开号:BR112015022073B1
申请号:R112015022073-8
申请日:2014-04-11
公开日:2020-10-06
发明作者:Pierce O. Norton；Vladimir Azersky
申请人:Becton, Dickinson And Company；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED ORDERS
[0001] Pursuant to Title 35, paragraph 119 (e), of the United States Code, this application claims priority from the filing date of Provisional Patent Application No. US 61 / 811,465 filed on April 12, 2013, the disclosure of which has been incorporated into this document as a reference. INTRODUCTION
[0002] Flow cytometers known in the art are used to analyze and classify particles in a fluid sample, such as cells in a blood sample or particles of interest in any other type of biological or chemical sample. A flow cytometer typically includes a sample reservoir for receiving a fluid sample, such as a blood sample and a coating reservoir that contains a coating fluid. The flow cytometer transports the particles (hereinafter referred to as "cells") in the fluid sample as a cell stream to a flow cell, while also directing the coating fluid to the flow cell.
[0003] In the flow cell, a liquid coating is formed around the cell stream to transmit a substantially uniform speed to the cell stream. The flow cell hydrodynamically concentrates cells within the stream to pass through the center of a laser beam in a flow cell. The point at which cells cross the laser beam is commonly known as the question mark. When a cell moves through the question mark, this causes the laser light to disperse. The laser light also excites components in the cell stream that have fluorescent properties, such as fluorescent markers that have been added to the fluid sample and adhered to certain cells of interest or fluorescent microspheres mixed in the stream. The flow cytometer includes an appropriate detection system consisting of photomultiplier tubes, photodiodes or other light detecting devices, which are concentrated at the point of intersection. The flow cytometer analyzes the detected light to measure the cell's physical and fluorescent properties. The flow cytometer can further classify cells based on these measured properties. The flow stream exits the flow cell through a nozzle with a nozzle diameter that is suitable for the fluidic systems and desired rating rate.
[0004] To classify cells by an electrostatic method, the desired cell must be contained in an electrically charged droplet. To produce droplets, the flow cell is rapidly vibrated by an acoustic device, such as a piezoelectric element. The volume of a droplet is conventionally estimated by the hydrodynamic properties of the flow stream and the nozzle dimensions. To charge the droplet, the flow cell includes a charge element whose electrical potential can be quickly changed. Because the cell stream exits the flow cell in a substantially downward vertical direction, the droplets also propagate in that direction after they are formed. Droplets, loaded or not, need to be collected in a sample collection vessel that is properly directed to collect one or more flow currents generated by the baffles. In this way, the droplets and the cells contained in them can be collected in appropriate collection vessels downstream of the plates.
[0005] Known flow cytometers similar to the type described above are described, for example, in Patent Documents Nos. U.S. 3,960,449, 4,347,935, 4,667,830, 5,464,581, 5,483,469, 5,602,039, 5,643,796 and 5,700,692, the entire contents of which have been incorporated by reference to this document. Other types of known flow cytometers are the FACSVantageTM, FACSortTM, FACSCountTM, FACScanTM and FACSCaliburTM systems manufactured by Becton Dickinson and Company, the assignee of the present invention.
[0006] Although this method generally allows the flow cytometer to dispense classified cells in collection vessels and then classify the cells of interest with reasonable precision, the method requires a substantial amount of user input at the time of organization. The flow stream and collection vessels are manually aligned in a conventional manner. Fluid parameters such as flow rate and coating fluid composition must be compatible with an appropriate nozzle diameter. SUMMARY
[0007] Aspects of the present disclosure include systems for adjusting one or more parameters of a flow cytometer. Systems in accordance with certain modalities include an imaging sensor configured to capture one or more images from a field of detection of a flow current from the flow cytometer and a processor configured to generate a data signal from the one or more images captured in a manner the system to adjust one or more flow cytometer parameters in response to the data signal.
[0008] In certain modalities, the systems in question are configured to reduce the need for user input or manual adjustment during sample analysis with a flow cytometer. In some embodiments, systems of interest can be partially or completely automated so that settings for flow cytometer parameters are controlled by the processor. In certain embodiments, the systems in question are configured to adjust one or more parameters of the flow cytometer without any human input.
[0009] In certain embodiments, the present disclosure provides a system for automatically locating a current position in a liquid flow of a flow cytometer comprising a first camera, adapted to detect a current position in a first detection field and to generate a first signal representative of the current position and a first stage, the first stage being connected operationally with the first camera and configured to move in an XY plane in response to the first signal.
[0010] The system can additionally include a second camera adapted to detect a current position in a second detection field and to generate a second signal representative of the current position, the first and second detection fields of the first and second cameras being substantially orthogonally oriented in the XY plane with the first stage being operationally connected to the second camera and configured to move the XY plane in response to the second signal in addition to the first signal. In some embodiments, a laser is mounted or a collection device is mounted on the first pallet.
[0011] In some modalities, the system may include a second platform, a collection device being mounted on the second platform and the second platform is configured to move in the XY plane in response to the first signal and the second signal. The system may further comprise an electrical system configured to adjust an electrical charge in the flow current in response to the second signal from the second camera. The operational connection between the cameras and the platforms can be mediated by a processor connected to the first camera and the first and second cameras and the first platform and the processor is configured to receive signals from the first and second cameras and calculate an ideal position to the first dais. In some modalities, the operational connection is mediated by a processor connected to the first and second cameras and the second stage and configured to receive the signals from the first and second cameras and calculate an ideal position for the second stage. In some embodiments, the chain may include a series of drops.
[0012] A system according to certain modalities is provided to automatically determine a nozzle opening diameter with a first camera, adapted to detect a current dimension in a first detection field and to generate a first signal representative of the current dimension and a processor that has memory with instructions in it, configured to determine a value for the nozzle opening diameter from the current dimension and transmit the value to a flow cytometer. The chain dimension can be the width of the chain. In some embodiments, the flow cytometer can be configured to automatically adjust a series of parameters after receiving the transmitted value. The series of parameters can be selected from the group comprising hydrostatic pressure, drop load, deflection stress, load correction value, drop delay, drop frequency, drop amplitude and loading phase.
[0013] Aspects of the development also include methods for adjusting one or more parameters of a flow cytometer. Methods according to certain modalities include capturing one or more images of a flow current from the flow cytometer in a detection field, determining one or more properties of the flow current in the detection field, generating a data signal that corresponds to a or more properties of the flow current and adjust one or more parameters of the flow cytometer in response to the data signal.
[0014] Aspects of the present disclosure also include computer-controlled systems to practice the methods in question, the systems additionally including one or more computers that have processors configured to automate one or more steps of the methods described in this document. In some embodiments, systems include a computer that has a computer-readable storage medium with a computer program stored on it, and the computer program when loaded on the computer includes instructions for capturing one or more images of a stream stream. flow cytometer in a detection field; algorithm to determine the spatial position of the flow current in the detection field; algorithm to generate a data signal that corresponds to the spatial position of the flow current and instructions to adjust one or more parameters of the flow cytometer in response to the data signal. In certain cases, systems include a computer that has a computer-readable storage medium with a computer program stored on it, and the computer program when loaded on the computer includes instructions for capturing one or more images of a stream stream. flow cytometer in a detection field; algorithm to determine the physical dimensions of the flow current in the detection field; algorithm to generate a data signal that corresponds to the physical dimensions of the flow current and instructions to adjust one or more parameters of the flow cytometer in response to the data signal. BRIEF DESCRIPTION OF THE FIGURES
[0015] The invention can be better understood from the detailed description below read in conjunction with the accompanying drawings. Included in the drawings are the following figures:
[0016] Figure 1 depicts a schematic illustration of a system according to certain modalities.
[0017] Figure 2 depicts a flow chart that illustrates steps to practice methods of the present disclosure according to certain modalities.
[0018] Figure 3 depicts a flowchart that illustrates steps to practice methods of the present disclosure according to certain modalities. DETAILED DESCRIPTION
[0019] Before the present invention is described in more detail, it should be understood that the invention is not limited to the particular described modalities, therefore it can vary. It is also understood that the terminology used in this document is intended to describe particular modalities only and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
[0020] Where a range of values is provided, it is understood that each intervention value, from the tenth of the unit of the lower limit unless the context clearly indicates otherwise, between the upper and lower limit of that range and any other declared value or intervening in that determined range, is included in the invention. The upper and lower limits of these smaller ranges can be included independently in the smaller ranges and are also included in the invention, subject to any limit specifically excluded in the covered range. Where the range covered includes one or both of the limits, ranges that exclude one or both of the included limits are also included in the invention.
[0021] Unless otherwise defined, all technical and scientific terms used in this document have the same meaning as is commonly understood by a person skilled in the art related to that invention. Although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention, representative methods and illustration materials are now described.
[0022] All publications and patents cited in the specification have been incorporated into this document by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated into this document by reference to reveal and describe the methods and / or materials in connection with which publications are cited. The citation of any publication is due to its disclosure prior to the filing date and should not be constructed assuming that the present invention is not entitled to a date prior to such publication by virtue of the foregoing invention. In addition, the publication dates provided may differ from the current publication dates which may need to be independently confirmed.
[0023] It is noted that, as used in this document and the appended claims, the singular forms "one", "one" and "a / o" include referents in the plural unless the context clearly states otherwise. It is further noted that claims can be traced to exclude any optional elements. Accordingly, this statement is intended to serve as an antecedent basis for the use of such exclusive terminology as "only", "only" and the like in connection with the indication of elements of the claim or use of a "negative" limitation.
[0024] As will become evident to those skilled in the art when reading this disclosure, each of the individual modalities described and illustrated in this document has distinct components and resources which can be readily discernible from or combined with the resources of any of the other diverse modalities without departing from the scope or spirit of the present invention. Any method mentioned can be carried out in the order of events mentioned or in any other order that is logically possible.
[0025] As summarized above, the present disclosure provides systems configured to automate adjustments of one or more parameters of a flow cytometer. In additional descriptive embodiments of the disclosure, systems configured to adjust one or more parameters of a flow cytometer are first described in more detail. Then, methods for adjusting one or more parameters of a flow cytometer with the systems in question are described. Computer controlled systems which automate adjustments to one or more parameters of a flow cytometer are also provided. SYSTEMS FOR ADJUSTING PARAMETERS OF A FLOW CYTOMETER
[0026] Aspects of the present disclosure include systems configured to adjust parameters of a flow cytometer. The term “adjust” is used in this document in its conventional sense to refer to one or more functional parameters of the flow cytometer. As described in more detail below, the desirable adjustment can vary in terms of consumption optimizations, where in some cases the desired adjustments are adjustments that result in improved efficiency of some desirable parameter, for example, improved accuracy of cell classification, collection of improved particle, component malfunction identification (e.g., clogged flow cell nozzle), energy consumption, particle loading efficiency, more accurate particle loading, improved particle deflection during cell sorting, among other adjustments . In modalities, the systems in question are configured to reduce the need for user input or manual adjustment during sample analysis with a flow cytometer. In certain embodiments, systems of interest can be fully automated so that the settings for parameters of a flow cytometer are controlled by a processor. “Fully automated” means that adjustments made in response to the data signal corresponding to one or more parameters of the flow current and derived from one or more images captured from the flow current require little or no human intervention or manual entry into systems in question. In certain embodiments, the systems in question are configured to adjust one or more parameters of the flow cytometer based on the data signal that corresponds to one or more parameters of the flow current without any human intervention.
[0027] As summarized above, systems include one or more imaging sensors configured to capture images of a flow cytometer flow stream in one or more detection fields. “Detection field” means the region of the flow current that is imaged by one or more imaging sensors. Detection fields may vary depending on the properties of the flow stream being interrogated. In embodiments, the detection field can measure 0.001 mm or more of the flow current, such as 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 2 mm or more, such as 5 mm or more, and includes 10 mm or more of the flow stream. For example, where the systems in question are configured to determine a physical dimension (for example, width) of the flow current, the detection field can be a planar cross-section of the flow current. In another example, where the systems in question are configured to determine the spatial position of the flow current, the detection field can be a predetermined length of the flow current, such as, for example, to determine the angle made by the flow current with respect to the geometric axis of the flow cell nozzle.
[0028] The detection field interrogated by the systems in question may vary depending on the flow cytometer parameter that is adjusted. In some embodiments, the detection field includes the nozzle orifice of the flow cell. In other embodiments, the detection field includes the location of the flow stream where the droplets containing the particles of interest are charged (ie, the "interruption" point where the flow stream begins to form distinct droplets). In still other embodiments, the detection field includes the region where charged particles are deflected by deflector plates during cell classification.
[0029] Systems include one or more imaging sensors configured to capture images of a flow stream in a detection field. The imaging sensor can be any suitable device capable of capturing and converting an optical image to an electronic data signal, which includes, but is not limited to, coupled devices, semiconductor charge coupled devices (CCD), active pixel sensors (APS), complementary metal-oxide semiconductor image sensors (CMOS) or N-type metal-oxide semiconductor image sensors. In some embodiments, the imaging sensor is a CCD camera. For example, the camera can be an electron multiplier CCD camera (EMCCD) or an enhanced CCD camera (ICCD). In other modalities, the imaging sensor is a CMOS type camera.
[0030] Depending on the number of detection fields being interrogated and parameters of interest of the flow cytometer, the number of imaging sensors in the systems in question may vary as desired. For example, the systems in question may include one imaging sensor or more, such as two imaging sensors or more, such as three imaging sensors or more, such as four imaging sensors or more, such as five imaging sensors or more and includes up to ten imaging sensors or more. In certain embodiments, systems include an imaging sensor. In other modalities, systems include two imaging sensors. Where systems include more than one imaging sensor, each imaging sensor can be oriented with respect to the other (as referenced in an XY plane) at an angle in the range of 10 ° to 90 °, such as from 15 ° to 85 °, such as from 20 ° to 80 °, as from 25 ° to 75 ° and includes from 30 ° to 60 °. In certain modalities, each imaging sensor is oriented orthogonally (according to the reference in an X-Y plane) to each other. For example, where the systems in question include two imaging sensors, the first imaging sensor is oriented orthogonally (according to reference in an X-Y plane) to the second imaging sensor.
[0031] Where the systems in question include more than one imaging sensor, each imaging sensor can be the same or a combination of sensors. For example, where the systems in question include two imaging sensors, in some embodiments the first imaging sensor is a CCD-type device and the second imaging sensor is a CMOS-type device. In other modalities, both the first and the second imaging sensors are CCD type devices. In still other modalities, both the first and the second imaging sensors are CMOS-type devices.
[0032] In some modalities, the imaging sensors are stationary and maintain a single position on the flow cytometer. In other embodiments, the imaging sensors can be configured to move along the flow current path. For example, the imaging sensor can be configured to move upstream and downstream along the flow stream by capturing images in a plurality of detection fields. For example, systems may include an imaging sensor that is adapted to capture images in two or more different detection fields along the flow stream, such as 3 or more detection fields, such as 4 or more detection fields and includes 5 or more detection fields. Where the imaging sensor is configured to move along the flow current, the imaging sensor can be moved along the flow current path either continuously or at different intervals. In some embodiments, the imaging sensor is moved continuously. In other embodiments, the imaging sensor can be moved along the flow current path at different intervals, such as, for example, by 1 mm or larger increments, such as 2 mm or larger increments and includes 5 mm or larger increments .
[0033] Where the imaging sensor is configured to capture images at different positions along the flow current path, the imaging sensor can be configured to capture images continuously or at different intervals. In some cases, imaging sensors of interest are configured to capture images continuously. In other cases, imaging sensors are configured to take measurements at different intervals, such as capturing an image of the flow current every 0.001 millisecond, every 0.01 millisecond, every 0.1 millisecond, every 1 millisecond, at every 10 milliseconds, every 100 milliseconds and includes every 1,000 milliseconds, or some other interval.
[0034] As described in more detail below, the imaging sensor is configured to capture one or more images of the flow current in each detection field. For example, the imaging sensor can be configured to capture 2 or more images of the flow stream in each detection field, such as 3 or more images, such as 4 or more images, such as 5 or more images, such as 10 or more images, such as 15 or more images and includes 25 or more images. Where a plurality of images is captured in a detection field, the processor (as discussed below) may include a digital imaging processing algorithm for joining the plurality of images.
[0035] Depending on the desired current flow rate and image resolution, the imaging sensor can have an exposure time of 100 ms or less when reading the entire sensor, such as 75 ms or less, such as 50 ms or less , such as 25 ms or less, such as 10 ms or less, such as 5 ms or less, such as 1 ms or less, such as 0.1 ms or less, such as 0.01 ms or less, such as 0.001 ms or less, such as 0.0001 ms or less, such as 0.00001 ms or less and includes an exposure time of 0.000001 ms or less. For example, the exposure time of the imaging sensor in a detection field that captures images of the flow current in the nozzle orifice of the flow cell may have an exposure time ranging from 0.0001 ms to 10 ms, such as from 0.001 ms to 5 ms, as well as from 0.01 ms to 2 ms and includes from 0.1 ms to 1 ms. The exposure time of imaging sensors in a detection field that captures images of the flow cytometer flow stream downstream of the nozzle orifice can have an exposure time ranging from 0.0001 ms to 10 ms, such as 0.001 ms to 5 ms, such as 0.01 ms to 2 ms and includes 0.1 ms to 1 ms.
[0036] In certain embodiments, imaging sensors in the systems in question may have 1M of active pixels or more, such as 1.5M of or more, for example, 2M or more, 2.5M or more or 3M or more. In some respects, a pixel corresponds to a current physical dimension of about 0.3 pm. Depending on the detection field, in some cases, imaging sensors have a sensor area of 150 mm2 or more, such as about 150 mm2 to about 175 mm2, about 175 mm2 to about 200 mm2, 200 mm2 to about 225 mm2, about 225 mm2 to about 250 mm2, about 250 mm2 to about 300 mm2, about 300 mm2 to about 400 mm2, about 400 mm2 to about 500 mm2, about 500 mm2 to about 750 mm2, about 750 mm2 to about 1,000 mm2 or about 1,000 mm2 or more.
[0037] The imaging sensor can be positioned at any suitable distance from the flow current of the flow cytometer as long as the detection field has the capacity to capture an image of the flow current. For example, the imaging sensor can be positioned 0.01 mm or more of the flow current, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 2.5 mm or more, such as 5 mm or more, such as 10 mm or more, such as 15 mm or more, such as 25 mm or more and includes 50 mm or more of chain flow cytometer flow.
[0038] In some modalities, the imaging sensor is positioned at an angle to the geometric axis of the flow current. For example, the imaging sensor can be positioned at an angle to the geometric axis of the flow current that ranges from 10 ° to 90 °, such as from 15 ° to 85 °, such as from 20 ° to 80 °, such as such as 25 ° to 75 ° and includes 30 ° to 60 °. In certain embodiments, the imaging sensor is positioned at an angle of 90 ° to the geometric axis of the flow stream.
[0039] In some cases, the imaging sensor additionally includes an optical adjustment protocol. By "optical adjustment" it is understood that capturing images of the detection field by the imaging sensor can be changed as desired, such as to increase or decrease the captured dimensions or to improve the optical resolution of the image. In some cases, optical adjustment is an enlargement protocol configured to increase the size of the detection field captured by the imaging sensor, such as 5% or more, such as 10% or more, such as 25% or more , such as 50% or more and includes increasing the detection field of the imaging sensor to 75% or more. In other cases, optical adjustment is a reduction protocol configured to decrease the size of the detection field captured by the imaging sensor, such as 5% or more, such as 10% or more, such as 25% or more , such as 50% or more and includes decreasing the width of the slit-shaped microsphere to 75% or more. In certain embodiments, optical adjustment is an enhanced resolution protocol configured to enhance the resolution of captured images, such as 5% or more, such as 10% or more, such as 25% or more, such as 50 % or more and includes improving the resolution of captured images to 75% or more. Capturing images of the detection field by the imaging sensor can be adjusted with any convenient optical adjustment protocol, which includes, but is not limited to, the lens, mirrors, filters and combinations thereof. In certain embodiments, the imaging sensor includes a focused lens. The focused lens, for example, can be a reduction lens. In other embodiments, the focusing lens is a magnifying lens.
[0040] Imaging sensors of the present disclosure may additionally include one or more wavelength separators. The term “wavelength separator” is used in this document in its conventional sense to refer to an optical protocol for separating polychromatic light into its component wavelengths for detection. Wavelength separation, according to certain modalities, can include blocking or selectively passing specific wavelengths or wavelength bands of polychromatic light. To separate light wavelengths, transmitted light can be passed through any convenient wavelength separation protocol, which includes, but is not limited to, colored glass, bandpass filters, interference filters, dichroic mirrors, networks diffraction, monochromators and combinations thereof, among other wavelength separation protocols. Depending on the detection field, the light source and the current flow that are displayed, systems may include one or more wavelength separators, such as two or more, such as three or more, such as four or more, such like five or more and includes 10 or more wavelength separators. In one example, imaging sensors include a bandpass filter. In another example, imaging sensors include two or more bandpass filters. In another example, imaging sensors include two or more bandpass filters and a diffraction grating. In yet another example, imaging sensors include a plurality of bandpass filters and a monochromator. In certain embodiments, imaging sensors include a plurality of bandpass filters and diffraction grids configured in a filter wheel arrangement. Where imaging sensors include two or more wavelength separators, wavelength separators can be used individually or in series to separate polychromatic light into component wavelengths. In some embodiments, wavelength separators are arranged in series. In other embodiments, wavelength separators are arranged individually so that one or more measurements are conducted using each of the wavelength separators.
[0041] In some embodiments, systems include one or more optical filters, such as one or more bandpass filters. For example, in some cases the optical filters of interest are bandpass filters that have a minimum bandwidth in the range of 2 nm to 100 nm, such as from 3 nm to 95 nm, such as from 5 nm to 95 nm, such as from 10 nm to 90 nm, such as from 12 nm to 85 nm, such as from 15 nm to 80 nm and includes bandpass filters that have minimum bandwidths in the range of 20 nm to 50 nm. In other cases, optical filters are long-pass filters, such as, for example, long-pass filters which attenuate light wavelengths of 1,600 nm or less, such as 1,550 nm or less, such as 1,500 nm or less, such as 1,450 nm or less, such as 1,400 nm or less, such as 1,350 nm or less, such as 1,300 nm or less, such as 1,000 nm or less, such as 950 nm or less, such as 900 nm or less , such as 850 nm or less, such as 800 nm or less, such as 750 nm or less, such as 700 nm or less, such as 650 nm or less, such as 600 nm or less, such as 550 nm or less, such as 500 nm or less and includes a long-pass filter that attenuates light wavelengths of 450 nm or less. In still other cases, optical filters are short-pass filters, such as, for example, short-pass filters which attenuate light wavelengths of 200 nm or more, such as 250 nm or more, such as 300 nm or more, such as 350 nm or more, such as 400 nm or more, such as 450 nm or more, such as 500 nm or more, such as 550 nm or more and includes short-pass filters which attenuate light wavelengths 600 nm or more.
[0042] In other modalities, the wavelength separator is a diffraction grating. Diffraction networks may include, but are not limited to, transmission, but are not limited to transmission, dispersive or reflective diffraction networks. Adequate spacing of the diffraction grating may vary depending on the light source configuration, detection field and imaging sensor and other optical adjustment protocols present (for example, lens with focus), in the range of 0.01 pm to 10 pm, such as from 0.025 pm to 7.5 pm, such as from 0.5 pm to 5 pm, such as from 0.75 pm to 4 pm, such as from 1 pm to 3.5 pm and includes from 1, 5 pm to 3.5 pm.
[0043] In some embodiments, each imaging sensor is operably coupled to one or more light sources to illuminate the flow current in the detection field. In some embodiments, the light source is a broadband light source that emits light that has a wide range of wavelengths, such as, for example, with a range of 50 nm or more, such as 100 nm or more , such as 150 nm or more, such as 200 nm or more, such as 250 nm or more, such as 300 nm or more, such as 350 nm or more, such as 400 nm or more and includes the range of 500 nm or more. For example, a suitable broadband light source emits light that has wavelengths from 200 nm to 1500 nm. Another example of a suitable broadband light source includes a light source that emits light that has wavelengths from 400 nm to 1,000 nm. Any convenient broadband light source protocol can be employed, such as a halogen lamp, deuterium arc lamp, xenon arc lamp, broadband light source coupled to stabilized fiber, a band LED wide with continuous spectrum, superluminescent emitting diode, semiconductor light emitting diode, broad spectrum white LED light source, integrated white light source with multi-LED, among other broadband light sources or any combination thereof.
[0044] In other modalities, the light source is a narrow band light source that emits a particular wavelength or a narrow range of wavelengths. In some cases, narrowband light sources emit light that has a narrow range of wavelengths, such as, for example, 50 nm or less, such as 40 nm or less, such as 30 nm or less, such as 25 nm or less, such as 20 nm or less, such as 15 nm or less, such as 10 nm or less, such as 5 nm or less, such as 2 nm or less and includes light sources which emit a length of specific light wave (ie, monochrome light). Any convenient narrowband light source protocol can be employed, such as a narrow wavelength LED, laser diode or a broadband light source coupled to one or more optical bandpass filters, diffraction grids, monochromators or any combination thereof.
[0045] The systems in question may include one or more light sources, as desired, such as two or more light sources, such as three or more light sources, such as four or more light sources, such as five or more. more light sources and includes ten or more light sources. The light source may include a combination of types of light sources, for example, where two light sources are employed, a first light source may be a broadband white light source (for example, white LED light from broadband) and a second light source may be a light source near the broadband infrared (for example, LED near the broadband IR). In other cases, where two light sources are employed, a first light source can be a broadband white light source (for example, broadband white LED light) and the second light source can be a narrow-spectrum light (for example, a near-IR LED or narrow-band visible light). In still other cases, the light source is a plurality of narrowband light sources and each emits specific wavelengths, such as an arrangement of two or more LEDs, such as an arrangement of three or more LEDs, such as an arrangement of five or more LEDs, which includes an arrangement of ten or more LEDs.
[0046] In some embodiments, light sources emit light that has wavelengths in the range of 200 nm to 1,500 nm, such as from 250 nm to 1,250 nm, such as from 300 nm to 1,000 nm, such as from 350 nm to 900 nm and includes 400 nm to 800 nm. For example, the light source may include a broadband light source that emits light that has wavelengths from 200 nm to 900 nm. In other cases, the light source includes a plurality of narrowband light sources that emit wavelengths in the range of 200 nm to 900 nm. For example, the light source can be a plurality of narrow band LEDs (1 nm to 25 nm) and each emits light independently having a wavelength range between 200 nm and 900 nm. In some embodiments, the narrowband light source is one or more narrowband lamps that emit light in the 200 nm to 900 nm range, such as a narrowband cadmium lamp, cesium lamp, helium lamp, lamp mercury lamp, cadmium-mercury lamp, potassium lamp, sodium lamp, neon lamp, zinc lamp or any combination thereof.
[0047] In certain modalities, the light source is a strobe light source where the flow current is illuminated with periodic flashes of light. Depending on the light source (eg flash lamp, pulsed laser) the frequency of strobe light may vary and may be 0.01 kHz or more, such as 0.05 kHz or more, such as 0.1 kHz or more , such as 0.5 kHz or more, such as 1 kHz or more, such as 2.5 kHz or more, such as 5 kHz or more, such as 10 kHz or more, such as 25 kHz or more, such as 50 kHz or more and includes 100 kHz or more. In these embodiments, the strobe light can be operably coupled to a processor that has a frequency generator that regulates strobe frequency. In some cases, the frequency generator is coupled to the droplet conduction generator so that strobe light is synchronized with the droplet generation. In other cases, the strobe frequency generator is operably coupled to one or more optical sensors so that the strobe frequency is synchronized with the image capture frequency. In certain cases, suitable strobe light sources and suitable frequency controllers include, but are not limited to, those described in U.S. Patent Document 5,700,692 and 6,372,506, the disclosures of which are incorporated herein by reference. Pulsed and strobe light sources are also described in Sorenson, et al. Cytometry, Volume 14, No. 2, pages 115 to 122 (1993); Wheeless, et al. The Journal of Histochemestry and Cytochemistry, Volume 24, No. 1, pages 265 to 268 (1976), the disclosures of which are hereby incorporated by reference.
[0048] As summarized above, the systems include one or more processors operably coupled to the imaging sensors where the processors are configured to generate a data signal from the captured images and to adjust one or more parameters of the flow cytometer in response to data signal. In modalities, the processor is configured to execute memory instructions to adjust one or more parameters of the flow cytometer based on the data signal derived from the captured images. Parameters of the flow cytometer which are adjusted according to the modalities of the present disclosure include, but are not limited to hydrostatic pressure, drop charge voltage, deflector plate voltage, charge correction value, drop delay, frequency of drop conduction, drop amplitude and loading phase. In certain embodiments, the processor is operably coupled to one or more support platforms and the position of the support platforms can be adjusted in response to the data signal derived from the captured images.
[0049] In modalities, processors include memory that has a plurality of instructions for performing the steps of the methods in question (as described in more detail below), such as lighting a flow cytometer flow stream in a detection field with a light source, capture one or more images of the flow current, generate a data signal that corresponds to one or more properties of the flow current based on the captured images, and adjust parameters of the flow cytometer in response to the data signal. The systems in question may include both hardware components and software components, with the hardware components taking the form of one or more platforms, for example, in the form of servers, so that the functional elements, that is, those elements of the system that perform specific tasks (such as managing information input and output, information processing, etc.) of the system can be performed by running software applications on and by one or more computer platforms represented in the system. The processor includes memory that has instructions stored in it to perform the steps of the methods in question which include lighting a flow cytometer flow stream in a detection field with a light source, capturing one or more images of the flow stream , generate a data signal that corresponds to one or more properties of the flow current based on the captured images and adjust parameters of the flow cytometer in response to the data signal.
[0050] In modalities, the processor is configured to generate a data signal that corresponds to one or more properties of the flow of captured images. In detection fields where the flow current is continuous, the processor can be configured to generate a data signal that corresponds to the spatial position of the flow current, the dimensions of the flow current such as width of the flow current, as well as rate flow and flow turbulence. In detection fields where flow current is made up of different droplets, the processor can be configured to generate a data signal that corresponds to the spatial position of the flow current, drop size including diameter and drop volume, conduction frequency of drop, amplitude of drop as well as uniformity of drop size and frequency. In certain embodiments, the processor can be configured to generate a data signal that corresponds to the ratio of the flow current size compared to the expected flow current size based on empirical characteristics of the flow cytometer and user input data . In other embodiments, the processor can be configured to evaluate captured images to determine whether a current flow is present or absent in a particular detection field. In yet other embodiments, the processor can be configured to evaluate captured images of the flow stream to determine the size of the flow cell nozzle orifice.
[0051] In some embodiments, the processor is operably coupled to an imaging sensor that captures images of the flow current in a detection field and generates a data signal that corresponds to the spatial position of the flow current. For example, the processor can take the captured images of the flow current in the detection field and map the spatial position of the flow current in an X-Y plane. In some cases, the position of the flow stream in the X-Y plane is compared to the spatial position of the vertical geometric axis of the flow cell nozzle to determine the position of the flow current with respect to the vertical geometric axis formed by the flow cell nozzle. Based on the spatial position of the flow current determined in the detection field, the processor generates a data signal that corresponds to the spatial position of the flow current.
[0052] In these modalities, the data signal corresponding to the spatial position of the flow current can be used by the processor to automatically adjust one or more parameters of the flow cytometer. In some cases, the data signal is used to adjust the position of a support platform that has one or more containers for collecting particles, such as for cell classification. In certain embodiments, the processor generates a data signal that corresponds to the position of the flow stream and adjusts the position of a support strata so that the collection containers on the support strata are aligned with the path of the flow stream. For example, the processor can be configured to map the position of the flow stream in each detection field in an XY plane, map the position of the container in the XY plane and match the position of the container in the XY plane with the position of the flow stream in the XY plane to align the collection vessel with the flow stream. In some cases, the systems in question are configured to map the position of the flow current in two detection fields. In these cases, the processor maps the spatial position of the flow current in the first detection field in an X-Y plane and maps the spatial position of the flow current in the second detection field in the X-Y plane. Based on the mapped positions of the flow current in the first and second detection fields, the processor is configured to generate a data signal that corresponds to the spatial position of the flow current in the flow cytometer.
[0053] In these modalities, the processor generates a data signal that corresponds to the spatial position of the flow current and automatically adjusts the position of a support platform on an X-Y plane in order to optimize the flow current collection. For example, optimizing collection may include a reduction in the number of flow stream particles not collected by the containers in the support strata due to a misalignment of the flow stream with the collection containers. For example, the number of particles not collected by containers on the support strata due to misalignment is reduced by 5% or more as compared to a container on an unadjusted support strata in response to the data signal, such as by 10% or more, such as 15% or more, such as 20% or more, such as 25% or more, such as 35% or more, such as 50% or more, such as 75% or more, such as 90% or more , such as 95% or more and including 99% or more. Otherwise, the processor in certain cases automatically aligns the position of the support platform in response to a data signal that corresponds to the spatial position of the flow stream so that the number of particles collected by the container is increased by 5% or more as compared to a container on a support platform not adjusted in response to the data signal, such as 10% or more, such as 15% or more, such as 20% or more, such as 25% or more, such as 35 % or more, such as 50% or more, such as 75% or more, such as 90% or more, such as 95% or more and including 99% or more. In other cases, the adjustment of the position of the support platform that has containers for collecting charged particles during cell classification can be increased as compared to the collection with an unadjusted support platform in response to the data signal 2 times or more, such as 3 times or more, such as 5 times or more and including 10 times or more.
[0054] In some modalities, a support platform is positioned downstream of deflector plates and includes containers to collect classified cells that have been separated based on load (that is, positive, negative and neutral). In some cases, the support structure may include three or more containers. In other cases, the support structure includes a single container partitioned into three or more compartments to collect the classified cells. An imaging sensor is configured to capture images of the flow current in a detection field downstream of the baffles and a processor operationally coupled to the imaging sensor generates a data signal that corresponds to the spatial positions of the flow currents. In these modalities, the processor takes the captured images of each flow stream and maps the spatial position of the flow stream in an X-Y plane. In some cases, the position of the flow current in the X-Y plane is compared to the position of the flow current before entering the baffles to determine the deviation due to the effects of the baffles. In these embodiments, the processor can generate a distinct data signal that corresponds to the position of the neutral particle flow stream, the negative particle flow stream and the positive particle flow stream, or any combination thereof. In one example, the processor generates a data signal that corresponds to the current flow of neutral particles after deflection by the deflector plates. In another example, the processor generates a data signal that corresponds to the current flow of negative particles after deflection by the deflector plates. In yet another example, the processor generates a data signal that corresponds to the position current flow of positive particles that come from the baffles. In yet another example, the processor generates a data signal that corresponds to the current flow positions of the positive particles, the negative particles and the neutral particles.
[0055] Based on the spatial positions determined for each flow current, the processor automatically adjusts one or more parameters of the flow cytometer. For example, the data signal can be used to adjust the position of a support platform that has containers for collecting positive particles, negative particles and neutral particles. In such cases, the processor generates a data signal that corresponds to the spatial positions of each flow stream (ie, neutral particle current, positive particle current and negative particle current) and automatically adjusts the position of the support platform to align collection containers with each flow stream in order to optimize collection. For example, the position of the support platform can be automatically adjusted to align collection containers with each flow stream so that the number of particles collected by the containers is increased by 5% or more as compared to a container in a support structure. not adjusted in response to the data signal, such as 10% or more, such as 15% or more, such as 20% or more, such as 25% or more, such as 35% or more, such as 50% or more, such as 75% or more, such as 90% or more, such as 95% or more and including 99% or more.
[0056] As summarized above, systems according to the modalities of the present disclosure include one or more processors that are automated to adjust parameters of a flow cytometer based on data signals derived from images captured from the flow stream of the flow cytometer. In certain embodiments, flow cytometer parameters that can be adjusted include coating fluid pressure, hydrostatic pressure, droplet charge voltage, deflection plate voltage, charge correction value, drop delay, drop conduction frequency , drop amplitude and loading phase.
[0057] In some embodiments, the processor can be configured to adjust the hydrostatic pressure in response to a data signal that corresponds to one or more properties of the flow current determined based on the captured images. In some cases, the hydrostatic pressure may be increased such as 689.47 Pa (0.1 psi) or more, such as 3,447.37 Pa (0.5 psi) or more, such as 6,894.74 Pa (1 psi) or more, such as 34,473.72 Pa (5 psi) or more, such as 68,947.44 Pa (10 psi) or more, such as 172,368.62 Pa (25 psi) or more, as in 344,737.24 Pa (50 psi) or more, such as 517,105.86 Pa (75 psi) or more and including increasing the hydrostatic pressure by 689,474.48 Pa (100 psi) or more. For example, the hydrostatic pressure can be increased by 1% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 25% or more, such as 50% or more, such as 75% or more and including increasing the hydrostatic pressure by 90% or more. In other cases, the hydrostatic pressure may be reduced such as 689.47 Pa (0.1 psi) or more, such as 3,447.37 Pa (0.5 psi) or more, such as 6,894.74 Pa (1 psi) or more, as in 34,473.72 Pa (5 psi) or more, as in 68,947.44 Pa (10 psi) or more, as in 172,368.62 Pa (25 psi) or more, as in 344,737.24 Pa (50 psi) or more, as in 517,105.86 Pa (75 psi) or more and including increasing the hydrostatic pressure by 689,474.48 Pa (100 psi) or more. For example, hydrostatic pressure can be reduced by 1% or more, such as 5% or more, such as 10% or more, such as 15% or more, such as 25% or more, such as 50% or more, such as 75% or more and including reducing the hydrostatic pressure by 90% or more.
[0058] In still other modalities, the processor can be configured to adjust the drop charge voltage in response to a data signal that corresponds to one or more properties of the flow current determined based on the captured images. In some cases, the drop charge voltage is increased, such as 0.01 V or more, such as 0.05 V or more, such as 0.1 V or more, such as 0.5 V or more , such as 1 V or more, such as 5 V or more, such as 10 V or more, such as 15 V or more, such as 25 V or more, such as 50 V or more and including - increase the drop charge voltage by 75 V or more. For example, the drop charge tension can be increased by 1% or more, such as by 5% or more, such as by 10% or more, such as by 15% or more, such as by 25% or more, such as 50% or more, such as 75% or more and including increasing the drop charge tension by 90% or more. In other cases, the drop charge voltage is reduced, such as 0.01 V or more, such as 0.05 V or more, such as 0.1 V or more, such as 0.5 V or more , such as 1 V or more, such as 5 V or more, such as 10 V or more, such as 15 V or more, such as 25 V or more, such as 50 V or more and including - increase the drop charge voltage by 75 V or more. For example, the drop charge tension can be reduced by 1% or more, such as by 5% or more, such as by 10% or more, such as by 15% or more, such as by 25% or more, such as 50% or more, such as 75% or more and including reducing the drop charge tension by 90% or more.
[0059] In still other modalities, the processor can be configured to adjust the deflection plate voltage in response to a data signal that corresponds to one or more properties of the flow current determined based on the captured images. In some cases, the deflection plate voltage is increased, such as 5 V or more, such as 10 V or more, such as 50 V or more, such as 100 V or more, such as 250 V or more, such as at 500 V or more, such as at 1,000 V or more and including increasing the drop charge voltage by 2,000 V or more. For example, the drop charge tension can be increased by 1% or more, such as by 5% or more, such as by 10% or more, such as by 15% or more, such as by 25% or more, such as 50% or more, such as 75% or more and including increasing the deflection plate tension by 90% or more. In other cases, the drop charge voltage is reduced, such as 0.5 V or more, such as 5 V or more, such as 10 V or more, such as 50 V or more, such as 100 V or more, such as 250 V or more, such as 500 V or more, such as 1,000 V or more and including reducing the deflection plate voltage by 2,000 V or more. For example, the drop charge tension can be increased by 1% or more, such as by 5% or more, such as by 10% or more, such as by 15% or more, such as by 25% or more, such as 50% or more, such as 75% or more and including reducing the deflection plate tension by 90% or more.
[0060] In still other modalities, the processor can be configured to adjust the drop conduction frequency in response to a data signal that corresponds to one or more properties of the flow current determined based on the captured images. In some cases, the drop conduction frequency is increased, such as 0.01 Hz or more, such as 0.05 Hz or more, such as 0.1 Hz or more, such as 0.25 Hz or more, such as 0.5 Hz or more, such as 1 Hz or more, such as 2.5 Hz or more, such as 5 Hz or more, such as 10 Hz or more and including at 25 Hz or more. For example, the frequency of gout conduction can be increased by 1% or more, such as by 5% or more, such as by 10% or more, such as by 15% or more, such as by 25% or more, such as 50% or more, such as 75% or more and including increasing the frequency of gout conduction by 90% or more. In other cases, the drop conduction frequency is reduced, such as 0.01 Hz or more, such as 0.05 Hz or more, such as 0.1 Hz or more, such as 0.25 Hz or more, such as 0.5 Hz or more, such as 1 Hz or more, such as 2.5 Hz or more, such as 5 Hz or more, such as 10 Hz or more and including 25 Hz or more. For example, the frequency of gout conduction can be reduced by 1% or more, such as by 5% or more, such as by 10% or more, such as by 15% or more, such as by 25% or more, such as 50% or more, such as 75% or more and including reducing the frequency of gout conduction by 90% or more. In still other embodiments, the processor can be configured to adjust the drop delay in response to a data signal that corresponds to one or more properties of the flow current determined based on the captured images. In some cases, the drop delay is increased, such as 0.01 microseconds or more, such as 0.05 microseconds or more, such as 0.1 microseconds or more, such as 0.3 microseconds or more , such as 0.5 microseconds or more, such as 1 microsecond or more, such as 2.5 microseconds or more, such as 5 microseconds or more, such as 7.5 microseconds or more and including increase the drop delay by 10 microseconds or more. For example, the gout delay can be increased by 1% or more, such as by 5% or more, such as by 10% or more, such as by 15% or more, such as by 25% or more, such as 50% or more, as well as 75% or more and including increasing the gout delay by 90% or more. In other cases, the drop frequency is reduced, such as 0.01 microseconds or more, such as 0.05 microseconds or more, such as 0.1 microseconds or more, such as 0.3 microseconds or more , such as 0.5 microseconds or more, such as 1 microsecond or more, such as 2.5 microseconds or more, such as 5 microseconds or more, such as 7.5 microseconds or more and including reduce drop delay by 10 microseconds or more. For example, the gout delay can be reduced by 1% or more, such as by 5% or more, such as by 10% or more, such as by 15% or more, such as by 25% or more, such as 50% or more, as well as 75% or more and including reducing gout delay by 90% or more.
[0061] In still other modalities, the processor can be configured to adjust the drop amplitude in response to a data signal that corresponds to one or more properties of the flow current determined based on the captured images. In some instances, the drop amplitude is increased, such as 0.01 volts or more, such as 0.025 volts or more, such as 0.05 volts or more, such as 0.1 volts or more, such such as 0.25 volts or more, such as 0.5 volts or more and including increasing the drop amplitude by 1 volt or more. For example, the amplitude of gout can be increased by 1% or more, such as by 5% or more, such as by 10% or more, such as by 15% or more, such as by 25% or more, such as 50% or more, as well as 75% or more and including increasing the amplitude of gout by 90% or more. In other examples, the drop amplitude is reduced, such as 0.01 volts or more, such as 0.025 volts or more, such as 0.05 volts or more, such as 0.075 volts or more, such as 0.1 volts or more, such as 0.25 volts or more and including reducing the drop amplitude by 1 volt or more. For example, the amplitude of gout can be reduced by 1% or more, such as by 5% or more, such as by 10% or more, such as by 15% or more, such as by 25% or more, such as 50% or more, as well as 75% or more and including reducing the drop range by 90% or more.
[0062] In some embodiments, the processor is operationally coupled to an imaging sensor that captures images of a flow cytometer flow stream in a detection field and generates a data signal that corresponds to the physical dimensions of the flow stream with based on captured images. Where the flow current is a direct current, in some cases the processor is configured to take the captured images and generate a data signal that corresponds to the width of the flow current. In detection fields where the flow current is made up of different droplets, in some cases the processor is configured to generate a data signal that corresponds to the droplet diameter.
[0063] In certain embodiments, the processor can be configured to compare the physical dimensions of the flow current determined from the captured images with expected dimensions based on empirical characteristics of the flow cytometer (such as size of the cell nozzle orifice) coating fluid flow and pressure) and parameters entered by the user. In these modalities, the processor is configured to generate a data signal that corresponds to the ratio of the physical dimensions of the flow current as compared to the expected flow current dimensions based on the empirical characteristics of the flow cytometer and parameters entered by the user. For example, the processor can be configured to generate a data signal that indicates that the flow current is 99% or less of the expected flow current size based on the empirical characteristics of the flow cytometer and parameters entered by the user, such as 95% or less, such as 90% or less, such as 85% or less, such as 80% or less, such as 75% or less, such as 50% or less, such as 25% or less and including 10% or less of the expected size of the flow stream. In other embodiments, the processor can be configured to generate a data signal that indicates that the flow current is greater than the expected size based on the empirical characteristics of the flow cytometer and parameters entered by the user, such as 105% or greater than the size of the flow stream, such as 110% or greater, such as 125% or greater and including 150% or greater. In these modalities, the processor can be configured to automate adjustments to one or more parameters of the flow cytometer based on the data signal that corresponds to the ratio of the flow current size from the captured images and the expected flow current size with based on empirical characteristics of the flow cytometer and user input. For example, the processor can be configured to automatically adjust the pumping rate, hydrostatic pressure and drop conduction frequency in response to the given ratio.
[0064] In certain cases, the processor is configured to determine a flow cell nozzle opening diameter. In these modalities, the processor is operationally coupled to an imaging sensor that captures images of the flow current in the orifice of the flow cell nozzle and generates a data signal that corresponds to the physical dimensions of the flow current. Based on the data signal that corresponds to the physical dimensions of the flow stream, the processor is configured to determine the flow cell nozzle opening diameter. In some cases, based on the data signal corresponding to the physical dimensions of the flow stream, the processor may determine that the flow cell nozzle opening diameter is 25 pm or greater, such as 35 pm or greater, such as 45 pm or greater, such as 50 pm or greater, such as 60 pm or greater, such as 75 pm or greater, such as 100 pm or greater and including 150 pm or greater. For example, the system can be configured to determine a flow cell nozzle opening diameter of the physical dimensions of the flow stream in the range of 25 pm to 200 pm, such as from 35 pm to 175 pm, such as from 50 pm at 150 pm and including from 75 pm to 100 pm.
[0065] In certain cases, the nozzle opening diameter is determined based on the width of the flow stream. In certain cases, the nozzle opening diameter is determined based on droplet volume.
[0066] The processor can, in certain cases, be configured to automatically adjust one or more parameters based on the determined nozzle opening diameter, such as, for example, hydrostatic pressure, coating fluid pressure, drop charge , deviation voltage, load correction value, drop delay, drop conduction frequency, load phase drop amplitude and any combinations thereof, as discussed above.
[0067] In some embodiments, the processor can be configured to automatically adjust the drop conduction frequency in response to the data signal that corresponds to the flow cell nozzle orifice size determined using the images captured from the flow stream . For example, the drop conduction frequency can be increased by 0.01 Hz or more, such as 0.05 Hz or more, such as 0.1 Hz or more, such as 0.25 Hz or more, such as 0.5 Hz or more, such as 1 Hz or more, such as 2.5 Hz or more, such as 5 Hz or more, such as 10 Hz or more and including 25 Hz or more. In other cases, the processor is configured to automatically reduce the drop conduction frequency in response to the flow cell nozzle orifice size determined using images captured from the flow current, such as at 0.01 Hz or more , such as 0.05 Hz or more, such as 0.1 Hz or more, such as 0.25 Hz or more, such as 0.5 Hz or more, such as 1 Hz or more, such such as 2.5 Hz or more, such as 5 Hz or more, such as 10 Hz or more and including 25 Hz or more.
[0068] In other embodiments, the processor can be configured to adjust the coating fluid pressure in response to the data signal that corresponds to the flow cell nozzle orifice size determined using the captured images of the flow stream. For example, the coating fluid pressure can be increased by 6.89 Pa (0.001 psi) or more, such as 34.47 Pa (0.005 psi) or more, such as 68.94 Pa (0.01 psi) or more, such as at 344.73 Pa (0.05 psi) or more, such as at 689.47 Pa (0.1 psi) or more, such as 3,447.37 Pa (0.5 psi) or more, such as 6,894.74 Pa (1 psi) or more, such as 34,473.72 Pa (5 psi) or more, such as 68,947.44 Pa (10 psi) or more, such as 172,368.62 Pa ( 25 psi) or more, such as at 344,737.24 Pa (50 psi) or more, such as at 517,105.86 Pa (75 psi) or more and including increasing the coating fluid pressure by 689,474.48 Pa ( 100 psi) or more. In other cases, the processor is configured to automatically reduce the coating fluid pressure in response to the flow cell nozzle orifice size determined using images captured from the flow current, such as at 689.47 Pa (0 , 1 psi) or more, such as 0.5 psi or more, such as at 6,894.74 Pa (1 psi) or more, such as at 34,473.72 Pa (5 psi) or more, as at 68,947.44 Pa (10 psi) or more, as in 172,368.62 Pa (25 psi) or more, as in 344,737.24 Pa (50 psi) or more, as in 517,105.86 Pa (75 psi) or more and including reducing the coating fluid pressure by 689,474.48 Pa (100 psi) or more.
[0069] In some modalities, systems of interest include an imaging sensor configured to capture images in a detection field at the point of interruption of the flow current. The term "breakpoint" is used in this document in its conventional sense to refer to the point at which the continuous flow current begins to form droplets. In these modalities, the systems in question include a processor operationally coupled to the imaging sensor and configured to generate a data signal that corresponds to the volume of droplet drops downstream of the breakpoint. The processor takes the captured images of the flow current droplets and measures the drop volume. The data signal corresponding to the drop volume can be used by the processor to automatically adjust one or more parameters of the flow cytometer.
[0070] In some embodiments, the data signal corresponding to a drop volume is used by the processor to automatically adjust the drop conduction frequency of the flow current. For example, the processor can be configured to automatically reduce the drop conduction frequency, such as by 5% or more, such as by 10% or more, such as by 15% or more, such as by 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, such as 95% or more and including 99% or more. In other cases, the processor is configured to automatically reduce the drop conduction frequency 2 times or more in response to the data signal that corresponds to the determined drop volume, such as 3 times or more, such as 4 times or more, such as like 5 times or more and including 10 times or more. In still other cases, the processor is configured to automatically increase the drop conduction frequency, such as by 5% or more in response to the data signal corresponding to a drop volume, such as by 10% or more, such as 15% or more, such as 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, such as 95% or more and including 99% or more. In still other cases, the processor is configured to automatically increase the drop conduction frequency 2 times or more, such as 3 times or more, such as 4 times or more, such as 5 times or more and including 10 times or more.
[0071] In other modalities, the data signal corresponding to the drop volume is used by the processor to automate the volume of sample collection during cell classification. For example, the desired volume for each sample collected can be inserted into the processor and based on the data signal that corresponds to the drop volume, the flow cytometer can be automated to stop sample collection after a predetermined amount of time, such as removing the collection vessel or ceasing the flow current through the flow cytometer.
[0072] In some embodiments, the processor can be configured to determine the presence or absence of a flow current in a detection field. Systems of interest may include an imaging sensor configured to capture images of the flow cytometer flow current flowing out of the flow cell nozzle orifice and a processor operationally coupled to the imaging sensor configured to evaluate captured images to determine how much whether a flow current is present or not present in the detection field. For example, determining whether a flow stream is present or not present in captured images of the flow cell nozzle orifice can be used to determine whether the flow cell has a clogged nozzle. In these modalities, images captured by the imaging sensors are evaluated by the processor and if a flow current is detected in the images by the processor, the processor is configured to generate a signal that indicates the presence of a flow current. On the other hand, if after evaluating the captured images, the processor determines that the flow current is absent in the captured images, the processor can be configured to generate a signal that indicates the absence of a flow current.
[0073] Where the processor determines that no flow current is present in the captured images, in certain modalities, the systems in question are configured to automatically alert a user that the absence of flow current is a result of faulty operation of a flow cytometer. flow, such as a clogged nozzle. In these modalities, the processor correlates the data signal that corresponds to the absence of a flow current with user input according to whether a flow current is to be expected. In some embodiments, a user can configure the system to have a “closed loop” configuration where the flow stream from the nozzle is directed to a waste receptacle without forming a flow stream. In these modalities, the flow cytometer does not alert the user of a malfunction (for example, clogged nozzle) since a flow current is not expected. However, where a flow current is expected (such as during normal use), the processor is automated to alert the user of malfunction if, after evaluating the captured images, no flow current is detected.
[0074] In certain modalities, after the processor has generated a data signal that corresponds to one or more properties of the flow current based on the captured images, an output module that can communicate the parameters of the flow cytometer can be adjusted in response to the data signal. In some cases, the output module communicates an output in conjunction with the systems in question that adjust flow cytometer parameters. In other cases, the output module communicates the parameters before adjustment and may require confirmation of adjustment by the user. A processor output can be communicated to the user by any convenient protocol, such as, for example, displaying on a monitor or printing a report.
[0075] As discussed above, systems in some modalities include one or more support platforms operationally coupled to the processors. Suitable support platforms can be any convenient mounting device configured to hold one or more components of the systems in question, such as flat substrate, contoured mounting devices, cylindrical or tubular support structures, laser or LED retainers, among other types support structures. In some cases, the support platform is a support for a lighting device, such as a laser or an LED. In other cases, systems include a support structure for holding one or more containers to collect particles from the flow stream. For example, the support platform can be configured to hold containers that include, but are not limited to test tubes, conical tubes, multi-compartment containers such as microtiter plates (for example, 96-well plates), tubes centrifuge, culture tubes, microtubes, lids, cuvettes, bottles, straight polymeric containers, among other types of containers.
[0076] Systems of interest may include one or more support decks, as desired, such as two or more, such as three or more, such as four or more and including five or more support decks. For example, the number of support decks can be in the range of 1 to 10 support decks, such as 2 to 7 support decks and including 3 to 5 support decks. In certain embodiments, systems of interest include a support platform. In other modalities, systems include two support platforms. In one example, the systems in question include a support platform that has a container for collecting droplets from the flow stream. In another example, the In systems in question include a support platform that has a laser mounted. In yet another example, the system in question includes a first support frame that has a laser mounted and a second support frame that has a container for collecting droplets from the flow stream.
[0077] In some modalities, support platforms are mobile. For example, in one example the support platform can be moved to adjust the position of collection containers on the support platform so that they are aligned with the flow stream. In another example, the support platform can be moved to adjust the position of a laser. In some cases, the support platform is moved in two dimensions, such as in an X-Y plane orthogonal to the geometric axis of the flow stream. In other cases, the support structure is moved in three dimensions. Where the support platform is configured to move, the support platform can be moved continuously or at different intervals. In some embodiments, the support platform is moved in a continuous motion. In other embodiments, the support platform is moved at discrete intervals, such as, for example, by 0.01 microns or greater increments, such as 0.05 microns or greater, such as 0.1 microns or greater, such as 0 , 5 microns or greater, such as 1 micron or greater, such as 10 microns or greater, such as 100 microns or greater, such as 500 microns or greater, such as 1 mm or greater, such as 5 mm or greater, such as 10 mm or greater and including 25 mm or greater increments.
[0078] Any displacement protocol can be used to move the support structures, such as moving the support platforms with a motor-driven translation platform, lead screw translation assembly, geared translation device, such as those that they employ a stepper motor, servomotor, brushless electric motor, brushed DC motor, micro stepper motor, high resolution stepper motor, among other types of motors.
[0079] Certain embodiments of the present disclosure can be described with reference to Figure 1. A flow cytometer 100 employing a embodiment of the present invention is illustrated in Figure 1. As discussed above, flow cytometer 100 includes flow cell 104, a sample reservoir 106 to supply a fluid sample (e.g., blood sample) to the flow cell and a coating reservoir 108 to supply a coating fluid to the flow cell. Flow cytometer 100 is configured to carry fluid sample that has cells in a flow stream to flow cell 104 in conjunction with a coating fluid lamination flow. An analysis of the flow current in an interrogation zone 103 within the flow cell 104 can be used to determine properties of a sample and control the classification parameters (as described in this document). Sample interrogation protocols may include a light source (for example, laser) 112 to illuminate the flow stream and one or more detectors 109 (for example, photomultiplier tubes (PMTs), charged coupled device (CCD)) or any other suitable type of light detection device. Where light from the light source crosses the sample stream in interrogation zone 103, the laser light is dispersed by the sample stream fluid and, in particular, by any cells present in the sample stream. A first portion of the scattered laser light will propagate in the direction prior to crossing the sample stream (referred to herein as the forward scattering light). A second portion of the laser light crosses the question mark will be scattered at an angle other than the direction of propagation (referred to herein as the side scatter light). Within the flow cell 104, the coating fluid surrounds the cell stream and the combined coating fluid and cell stream exits the flow cell 104 through a nozzle 102 which has an orifice 110 as flow stream 111. The stream flow can be a continuous flow of fluid or a series of droplets depending on the action of a droplet generator.
The flow current 111 exits the nozzle 102 in the nozzle orifice 110 which can have any diameter, for example, 50 pm, 70 pm, 100 pm, or any other suitable diameter. The nozzle diameter will affect the properties of a flow stream, such as the current dimensions, droplet breakpoint and drop volume. To view flow stream 111, a light source 112, such as an LED strobe, laser, or any other lighting device, can be optionally used and positioned in the region of the sample fluid stream 111. A camera 113 or other Image collection device can be positioned to capture an image of the flow stream in a first detection field. In some embodiments, the flow stream may comprise a direct current or a series of droplets. If the flow stream is a continuous flow of liquid, the image captured by the camera in the detection field can provide a user or controller with sufficient information to determine the position and / or dimensions of the flow stream.
[0081] In some aspects of this invention the camera 113 or other detection device can affect some action on the flow cytometer 100 based on the image collected by the camera 113. An organized controller 114 that comprises a computer algorithm can receive the image of the current flow and determine some action to be taken by the flow cytometer, which advantageously frees the user from manual organization tasks. In some embodiments, the diameter of the nozzle opening 110 can be determined based on an image analysis of the dimensions of the flow current captured by the camera 113. In some embodiments, an organized controller 114 can be operationally connected to the flow cytometer 100 and start automatically the adjustment of a series of parameters in the flow cytometer based on the nozzle diameter determine from the image received by the camera. The parameters can include any flow cytometric parameter such as hydrostatic pressure, drop load, deflection stress, load correction value, drop delay, drop frequency, drop amplitude and loading phase.
[0082] The organized controller 114 can be operationally connected to a fluidic system 115 that can control the rate of the flow current 111 in the flow cytometer 100, The organized controller 114 can initiate a pause in the flow current based on a received image of the camera 113.
[0083] The image collected from camera 113 of flow current 111 in the detection field can provide additional information about the position of the flow current in an XY plane. The camera can be operationally connected to one or more platforms 116, 119 and the position of the platform or platforms can be moved in response to a signal from the camera or organized controller connected to the camera. A collection device or light emitting device such as a laser 117 can be attached to a master bed and beneficially aligned to intercept flow stream 111 in response to the image from camera 113. The emission device can be aligned to maximize the amount of light received by the flow stream. A collection device 118 can be attached to the first frame or to a second frame 119 and be aligned to maximize the collection of a flow stream or orient the flow stream with respect to an "initial position" on the collection device. The improved automatic alignment of the laser or collection device with the flow current beneficially reduces the user's manual adjustment of the platform.
[0084] A second camera 120 or data collection device can be positioned below the first camera 113 or data collection device and configured to collect an image in a second detection field. The second camera can be positioned orthogonally in an XY plane in relation to the first camera, or optionally a series of lenses can be positioned in an XY plane such that the first and second detection fields are oriented orthogonally. The second camera 120 can also be operationally connected to one or more mobile stages 116, 119 either directly or via the organized controller 114. Collection or analysis devices can be attached to the platforms. The second detection field can be oriented by motorcycle orthogonal to the first detection field. Images from the second camera can be used to refine the position of the flow stream determined from the first camera and provide improved positioning of one or more stages associated with the flow stream. Although cameras 113 and 120 are shown as individual detectors for exemplary purposes, a plurality of cameras can be used to detect the flow current in a plurality of detection fields. An additional light source 123 can be used to provide sufficient illumination to capture the image of the flow stream in that position. Alternatively, the 117 laser can provide sufficient illumination. In addition, filters and other lenses 121 and 122 can be positioned in front of the light receiving areas of cameras 113 and 120, respectively, to filter any light or to adjust the resolution or direction of the detection field.
[0085] Images from the first and / or second camera 113, 120 can be analyzed by an organized controller 114 to determine any number of properties of the flow current, such as position of the flow current in a detection field or dimensions of the current flow. In some embodiments, a signal corresponding to the location of the flow current in the detection field can be transmitted to an organized controller 114 or directly to a mobile platform 116, 119 and initiates the automatic alignment of devices or vessels fixed on the platform in relation to the current. flow.
The flow stream can be a series of droplets that are partially deflected by a pair of deflection plates 124 and become a plurality of currents 125, 126, 127. As further illustrated, the flow cytometer can include a plurality collection vessels 118, 128 and 129 to collect the plurality of flow currents. The collection vessels can be a single vessel with multiple wells such as a 96 or 364 well plate or a series of vessels. In the example shown in Figure 1, droplets 127 that have been negatively charged in the interrogation zone will be directed by the potentials applied to the deflection plates 124 towards the collection vessel 128. Droplets 126 that have not been positively or negatively charged will not be deflected by the potentials applied to deflection plates 124 and therefore continue along their original paths to the central collection vessel 118. The droplets 125 that have been positively charged will be deflected by the potentials applied to deflection plates 124 towards the collection vessel 129. The alignment of the collection vessels in relation to deflected flow currents is essential to maximize collections of classified cells.
[0087] The collection vessel or vessels can be automatically aligned by collecting data from the first and / or second camera 113, 120 to determine the position of flow currents in an XY plane. The collection vessel (s) can be attached to a mobile platform 119 in communication with the controller 114 or directly with the first or second camera 113, 120. The cameras can determine the current position in the detection field, and generate a signal to the controller. Controller 114 can automatically control the position of a collection vessel 118 disposed under the flow current in order to optimize the position of the collection vessel in relation to the flow current. In some embodiments, the controller can also control the magnitude of the electrical charge received by a portion of the droplets. The magnitude of the electrical charge can affect the degree of deflection experienced by the droplets and, therefore, the position of the droplets in the XY plane.
[0088] The organization controller 118 can perform additional action depending on the input of parameters in the device. One aspect of the invention is the application of an input value for drop volume in the organization controller 114. The drop volume can be determined by any means such as empirical measurements of a volume after a defined number of drops of a nozzle diameter. particular have been collected over a defined period of time. The drop volume can then be inserted into the organization controller 114. In some embodiments, the controller can cause the fluidic system 115 to stop after a defined volume is dispensed into a collection vessel 118. This method improves in a way beneficial, a collection protocol because the use of a calibrated drop volume can provide a more accurate determination of collection volume than conventional methods that rely on drop counting to control collection times. Using the methods of this invention and the available classified fluid volume information, an additional “interruption rule” for the classification process can be implemented.
[0089] In some aspects of this invention, the organization controller can be used to distinguish between a clogged nozzle and a closed loop nozzle specifically designed not to generate a flow current. The “closed loop” nozzle has an outlet that is connected to a piping system that goes directly to the trash. It does not create an open classifiable chain, and is used for analysis only. It is important to have the ability to discern that nozzle from a clogged rating nozzle that should create an open classifiable chain, but is disabled for any reason. In some embodiments, the organization controller picks up electrically when the closed loop nozzle is installed. The electrical pickup can take any form such as an inserted closed-loop nozzle that provides a floor for an “energized” resistor circuit. If the closed-loop nozzle is captured in this way, an image of a current is not expected by the camera, then a clogged nozzle is not misreported when a current image is not seen. For rating nozzles, a current image is expected, and using the area value of that image, a nozzle size is determined and the appropriate instrument definition values for the nozzle are performed. If a current image is not seen and an electrical signal that means the presence of a closed loop nozzle is not detected, a rating nozzle is determined to be installed and clogged. The organization controller can initiate a series of actions at that event. For example, the user may be notified of a clogged nozzle, the fluidic system may be interrupted, or any other action may be initiated. METHODS FOR ADJUSTING PARAMETERS OF A FLOW CYTOMETER
[0090] Development aspects also include methods for adjusting one or more parameters of a flow cytometer. The methods, according to certain modalities, include capturing one or more images of a flow current from the flow cytometer in a detection field, determining one or more properties of the flow current in the detection field, generating a data signal that corresponds to one or more properties of the flow current and adjust one or more parameters of the flow cytometer in response to the data signal.
[0091] As discussed above, the term "adjust" refers to changing one or more functional parameters of the flow cytometer. The desired adjustment can vary in terms of objective, in which in some cases the desired adjustments are adjustments that ultimately result in improved efficiency of some desirable parameter, for example, improved cell classification accuracy, improved particle collection, identification component malfunction (eg, flow clogged cell nozzle), energy consumption, particle charge efficiency, more accurate particle charge, improved particle deflection during cell classification, among other settings. In modalities, the methods in question totally reduce or eliminate the need for user input or manual adjustment during sample analysis with a flow cytometer. In certain embodiments, the methods of interest can be fully automated, so that adjustments made in response to data signals that correspond to one or more parameters of the flow current require little or no human intervention or manual input by the user. In certain embodiments, methods include adjusting one or more parameters of the flow cytometer based on the data signals that correspond to one or more parameters of the flow stream without any human intervention, such as two or more parameters, such as three or more parameters, such as four or more parameters and including five or more parameters. In some embodiments, methods may include adjusting hydrostatic pressure, coating fluid pressure, drop load, deflection stress, load correction value, drop delay, drop conduction frequency, load amplitude phase drop and any combinations thereof.
[0092] When practicing the methods, according to certain modalities, one or more images of a flow cytometer flow stream are captured in a detection field. As discussed above, the detection fields can vary depending on the properties of the flow stream to be interrogated. In embodiments, methods may include capturing a detection field in an image that extends 0.001 mm or more of the flow current, such as 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mm or more , such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 2 mm or more, such as 5 mm or more and including 10 mm or more of the flow stream. The interrogated detection field may vary. In some embodiments, the detection field includes the flow cell nozzle orifice. In other embodiments, the detection field includes the location of the flow stream into which the droplets containing the particles of interest are charged (that is, the "interruption" point at which the continuous flow stream begins to form distinct droplets). In still other embodiments, the detection field includes the region where charged particles are deflected by deflector plates during cell classification.
[0093] When capturing one or more images of the flow current, a detection field is illuminated with a light source. In some embodiments, the flow current is illuminated with a broadband light source or a narrow band of light (as described above). The appropriate broadband light source protocol may include, but is not limited to, a halogen lamp, deuterium arc lamp, xenon arc lamp, stabilized fiber coupled broadband light source, a broadband LED with continuous spectrum, superluminescent emitting diode, semiconductor light emitting diode, broad spectrum white LED light source, an integrated multiple LED white light source, among other broadband light sources or any combination thereof. Suitable narrowband light sources include, but are not limited to, a narrow wavelength LED, laser diode or a wide band light source coupled to one or more optical bandpass filters, diffraction grids, monochromators or any combination thereof.
[0094] In certain modalities, the light source is a strobe light source in which the flow current is illuminated with periodic flashes of light. For example, the strobe frequency can be 0.01 kHz or more, such as 0.05 kHz or more, such as 0.1 kHz or more, such as 0.5 kHz or more, such as 1 kHz or more , such as 2.5 kHz or more, such as 5 kHz or more, such as 10 kHz or more, such as 25 kHz or more, such as 50 kHz or more and including 100 kHz or more. In some cases, the strobe frequency is synchronized with the droplet conduction frequency. In other cases, the strobe frequency is synchronized with image capture.
[0095] Capturing one or more images of the flow stream may include illuminating the flow stream with a combination of light sources, such as with two or more light sources, such as three or more light sources, such as four or more light sources and including five or more light sources. When more than one light source is used, the flow current can be illuminated with the light sources simultaneously or sequentially, or a combination of them. For example, when images of the flow stream are captured by lighting with two light sources, the methods in question may include simultaneously illuminating the flow stream with both light sources. In other embodiments, capturing images of the flow stream may include lighting sequentially with two light sources. When two light sources are illuminated sequentially, the time that each light source illuminates the flow stream can be independently 0.001 seconds or more, such as 0.01 seconds or more, such as 0.1 seconds or more, such as such as 1 second or more, such as 5 seconds or more, such as 10 seconds or more, such as 30 seconds or more and including 60 seconds or more. In modalities in which the images of the flow current are captured by lighting sequentially with two or more light sources, the duration of the flow current that is illuminated by each light source can be the same or different.
[0096] The images of the flow stream can be captured continuously or at different intervals. In some cases, methods include capturing images continuously. In other cases, methods include capturing images at different intervals, such as capturing an image of the flow current every 0.001 millisecond, each 0.01 millisecond, each 0.1 millisecond, each 1 millisecond, each 10 milliseconds, each 100 milliseconds and including every 1,000 milliseconds, or some other interval.
[0097] One or more images can be captured in each detection field, such as 2 or more images of the flow current in each detection field, such as 3 or more images, such as 4 or more images, such as 5 or more images, such as 10 or more images, such as 15 or more images and including 25 or more images. When more than one image is captured in each detection field, the plurality of images can be automatically stitched together using a processor that has a digital image processing algorithm.
[0098] Images of the flow current in each detection field can be captured at any suitable distance from the flow current as long as a usable image of the flow current is captured. For example, images in each detection field can be captured 0.01 mm or more from the flow stream, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 2.5 mm or more, such as 5 mm or more, such as 10 mm or more, such as 15 mm or more, such as 25 mm or more and including 50 mm or more from the flow cytometer flow stream. The images of the flow current in each detection field can also be captured at any angle from the flow current. For example, images in each detection field can be captured at an angle to the geometric axis of the flow current that ranges from 10 ° to 90 °, such as from 15 ° to 85 °, such as from 20 ° to 80 ° °, such as 25 ° to 75 ° and including 30 ° to 60 °. In certain modes, the images in each detection field can be captured at an angle of 90 ° to the geometric axis of the flow stream.
[0099] In some modalities, capturing the images of the flow current includes moving one or more imaging sensors along the path of the flow current. For example, the imaging sensor can be moved upstream or downstream along the flow current capture images in a plurality of detection fields. For example, methods may include capturing images of the flow stream in two or more different detection fields, such as 3 or more detection fields, such as 4 or more detection fields and including 5 or more detection fields. The imaging sensor can be moved continuously or at discrete intervals. In some embodiments, the imaging sensor is moved continuously. In other embodiments, the imaging sensor can be moved along the flow current path at different intervals, such as, for example, 1 mm or more increments, such as 2 mm or more increments and including 5 mm or more increments.
[00100] As summarized above, the methods include generating a data signal that corresponds to one or more properties of the flow current from the captured images. In detection fields where the flow current is continuous, methods may include generating a data signal that corresponds to the spatial position of the flow current, the dimensions of the flow current such as the width of the flow current, as well as the rate of flow. flow and flow turbulence based on captured images. In detection fields where the flow current is made up of distinct droplets, methods may include generating a data signal that corresponds to the spatial position of the flow current, drop size including volume and drop diameter, frequency of drop conduction , droplet amplitude as well as uniformity of frequency and droplet size. In certain embodiments, the methods include generating a data signal that corresponds to the ratio of the flow current size when compared to the expected flow current size based on empirical characteristics of the flow cytometer and user input data. In other modalities, methods include estimating captured images to determine whether a current flow is present or absent in a particular detection field. In still other embodiments, the methods include estimating the captured images of the flow stream to determine the size of the flow cell nozzle orifice.
[00101] In some embodiments, the methods include capturing one or more images of a flow current from the flow cytometer in a detection field, determining the spatial position of the flow current in the detection field based on the captured images and generating a data signal corresponding to the spatial position of the flow stream. For example, methods may include capturing images of the flow current in a detection field and mapping the spatial position of the flow current in an X-Y plane. In some cases, the position of the flow stream in the X-Y plane is compared to the vertical geometric axis of the flow cell nozzle to determine the position of the flow current in relation to the vertical geometric axis formed by the flow cell nozzle. When the spatial position of the flow current is determined in more than one detection field, the spatial position of the flow current can be mapped on an XY plane in each detection field and compared to regulating the precise spatial position of the flow current in the XY plane. Based on the determined spatial position of the flow current in the detection field, methods may include generating a data signal that corresponds to the spatial position of the flow current.
[00102] In modalities, according to the methods in question, one or more parameters of the flow cytometer are adjusted in response to the data signal that corresponds to the spatial position of the flow current. In some cases, the data signal is used to adjust the position of a support platform that has containers for collecting particles, such as during cell classification. In certain embodiments, methods include generating a data signal that corresponds to the spatial position of the flow stream and automatically adjusting the position of a support bed so that the collection containers on the support bed are aligned with the path of the bed stream. flow. For example, methods may include mapping the position of the flow stream in each detection field on an XY plane, mapping the position of the container on the XY plane and combining the position of the container on the XY plane with the position of the flow stream on the plane XY to align the collection container with the flow stream. In some cases, methods include mapping the position of the flow stream in two detection fields. In these cases, the spatial position of the flow current is mapped to the first detection field on an X-Y plane and the spatial position of the flow current is mapped to the second detection field on the X-Y plane. Based on the mapped positions of the flow current in the first and second detection fields, a data signal is generated that corresponds to the spatial position of the flow current in the flow cytometer.
[00103] In these modalities, a data signal is generated that corresponds to the spatial position of the flow current and automatically adjust the position of a support platform in an X-Y plane in order to optimize the flow current collection. For example, optimizing particle collection may include reducing the number of particles not collected by the containers on the support bed due to misalignment of the flow stream with the collection containers, such as by 5% or more when compared to collecting the flow current. flow in a container on a support platform not adjusted in response to the data signal, such as 10% or more, such as 15% or more, such as 20% or more, such as 25% or more, such as 35 % or more, such as 50% or more, such as 75% or more, such as 90% or more, such as 95% or more and including 99% or more.
[00104] As described above, the support platforms can be positioned anywhere along the flow stream as desired when collecting particles from the flow stream. In some cases, the particles are collected in containers on a support platform positioned downstream of baffle plates in which the droplets of flow stream have been separated based on charge (for example, positive, negative and neutral). In such cases, the methods include capturing images of a flow stream in a detection field downstream of the deflector plates and generating a data signal that corresponds to the spatial positions of the flow currents of the positive, negative and neutral particles. Based on the spatial positions determined for the flow currents from the captured images, the position of a support bed that has a multi-compartment container (or three separate containers) can be automatically adjusted to optimize the collection of each flow stream. For example, methods may include adjusting the position of the support platform so that the collection of flow currents is improved by 5% or more when compared to collecting flow currents on an unadjusted support platform in response to the data, such as 10% or more, such as 15% or more, such as 20% or more, such as 25% or more, such as 35% or more, such as 50% or more, such as 75% or more, such as 90% or more, such as 95% or more and including 99% or more.
[00105] The methods, according to certain modalities, are outlined in the combination of steps shown in Figure 2. The steps of this invention can occur in any order or in any combination. For example, in Figure 2, an experimental organization may include the installation of an appropriate nozzle for the desired classification task. The flow current can be started and an image collected from the flow current by camera 1. The nozzle opening can be determined from the flow current image and any number of parameters can be determined automatically and set based on this value. The laser can be aligned automatically and roughly according to the signal from the first camera. As the flow current flows through camera 2, a second image can be captured. The two images from camera 1 and camera 2 can provide precise location of the flow current in an XY plane. A laser can be positioned automatically and finely based on this information and a collection vessel can be positioned automatically based on this information. In some embodiments, the fine alignment of the laser can facilitate the alignment of the collection vessel.
[00106] As summarized above, the methods, in accordance with the modalities of the present disclosure, include adjusting one or more parameters of the flow cytometer in response to data signals derived from images captured in one or more fields of detection of a current of flow cytometer flow. In certain embodiments, methods include adjusting the coating fluid pressure, droplet charge voltage, deflection plate voltage, charge correction value, drop delay, drop conduction frequency, drop amplitude and charge phase or a combination of them.
[00107] In some embodiments, the methods include adjusting the coating fluid pressure in response to a data signal that corresponds to one or more properties of the flow current determined based on the captured images. In some cases, the coating fluid pressure may be increased such as 0.000006 MPa (0.001 psi) or more, such as 0.00003 MPa (0.005 psi) or more, such as 0.00006 MPa (0, 01 psi) or more, such as 0.003 MPa (0.05 psi) or more, such as 0.0006 MPa (0.1 psi) or more, such as 0.003 MPa (0.5 psi) or more, such as in 0.006 MPa (1 psi) or more, as in 0.03 MPa (5 psi) or more, as in 0.06 MPa (10 psi) or more, as in 0.17 MPa (25 psi) or more, such as 0.34 MPa (50 psi) or more, such as 0.51 MPa (75 psi) or more and including increasing the hydrostatic pressure by 0.68 MPa (100 psi) or more. In other cases, the coating fluid pressure is reduced, such as 0.000006 MPa (0.001 psi) or more, such as 0.00003 MPa (0.005 psi) or more, such as 0.00006 MPa (0, 01 psi) or more, such as 0.003 MPa (0.05 psi) or more, such as 0.0006 MPa (0.1 psi) or more, such as 0.003 MPa (0.5 psi) or more, such as in 0.006 MPa (1 psi) or more, as in 0.05 MPa (5 psi) or more, as in 0.06 MPa (10 psi) or more, as in 0.17 MPa (25 psi) or more, such as 0.34 MPa (50 psi) or more, such as 0.51 MPa (75 psi) or more and includes reducing the hydrostatic pressure by 0.68 MPa (100 psi) or more.
[00108] In still other embodiments, the methods include adjusting the drop charge voltage in response to a data signal that corresponds to one or more properties of the flow current determined based on the captured images. In some cases, the drop charge voltage is increased, such as 0.01 V or more, such as 0.05 V or more, such as 0.1 V or more, such as 0.5 V or more , such as 1 V or more, such as 5 V or more, such as 10 V or more, such as 15 V or more, such as 25 V or more, such as 50 V or more and includes increase the drop charge voltage by 75 V or more. In other cases, the drop charge voltage is reduced, such as 0.01 V or more, such as 0.05 V or more, such as 0.1 V or more, such as 0.5 V or more , such as 1 V or more, such as 5 V or more, such as 10 V or more, such as 15 V or more, such as 25 V or more, such as 50 V or more and includes reduce the drop charge voltage by 75 V or more.
[00109] In still other embodiments, the methods include adjusting the deflection plate voltage in response to a data signal that corresponds to one or more properties of the flow current determined based on the captured images. In some cases, the deflection plate voltage is increased, such as 5 V or more, such as 10 V or more, such as 50 V or more, such as 100 V or more, such as 250 V or more, such as 500 V or more, such as 1,000 V or more and includes increasing the deflection plate voltage by 2,000 V or more. In other cases, the drop charge voltage is reduced, such as at 5 V or more, such as at 10 V or more, such as at 50 V or more, such as at 100 V or more, such as at 250 V or more, such as 500 V or more, such as 1,000 V or more and including reducing the deflection plate voltage by 2,000 V or more.
[00110] In still other embodiments, the methods include adjusting the drop conduction frequency in response to a data signal that corresponds to one or more properties of the flow current determined based on the captured images. In some cases, the drop conduction frequency is increased, such as 0.01 Hz or more, such as 0.05 Hz or more, such as 0.1 Hz or more, such as 0.25 Hz or more, such as 0.5 Hz or more, such as 1 Hz or more, such as 2.5 Hz or more, such as 5 Hz or more, such as 10 Hz or more and including 25 Hz or more. In other cases, the drop frequency is reduced, such as 0.01 Hz or more, such as 0.05 Hz or more, such as 0.1 Hz or more, such as 0.25 Hz or more , such as 0.5 Hz or more, such as 1 Hz or more, such as 2.5 Hz or more, such as 5 Hz or more, such as 10 Hz or more and including 25 Hz or more.
[00111] In still other embodiments, the methods include adjusting the drop delay in response to a data signal that corresponds to one or more properties of the flow current determined based on the captured images. In some cases, the drop delay is increased, such as 0.01 microseconds or more, such as 0.05 microseconds or more, such as 0.1 microseconds or more, such as 0.3 microseconds or more , such as 0.5 microseconds or more, such as 1 microseconds or more, such as 2.5 microseconds or more, such as 5 microseconds or more, such as 7.5 microseconds or more and including increasing the drop delay by 10 microseconds or more. In other cases, the drop frequency is reduced, such as 0.01 microsecond or more, such as 0.05 microsecond or more, such as 0.1 microsecond or more, such as 0.3 microseconds or more , such as 0.5 microseconds or more, such as 1 microsecond or more, such as 2.5 microseconds or more, such as 5 microseconds or more, such as 7.5 microseconds or more and including reducing the drop delay by 10 microseconds or more.
[00112] In still other embodiments, the methods include adjusting the drop amplitude in response to a data signal that corresponds to one or more properties of the flow current determined based on the captured images. In some cases, the drop amplitude is increased, such as 0.01 volts or more, such as 0.025 volts or more, such as 0.05 volts or more, such as 0.1 volts or more, such such as 0.25 volts or more, such as 0.5 volts or more and includes increasing the drop amplitude by 1 volt or more. In other cases, the drop amplitude is reduced, such as 0.01 volts or more, such as 0.025 volts or more, such as 0.05 volts or more, such as 0.075 volts or more, such as 0.1 volts or more, such as 0.25 volts or more and includes reducing the drop amplitude by 1 volt or more.
[00113] In some modalities, the methods include capturing one or more images of a flow current from the flow cytometer in a detection field, characterizing the physical dimensions of the flow current in the detection field based on the captured images and generating a data signal corresponding to the physical dimensions of the flow stream. In detection fields where the flow current is a direct current, methods may include taking the captured images and generating a data signal that matches the width of the flow current. In detection fields where the flow stream is made up of distinct droplets, methods may include obtaining the captured images and generating data signals that correspond to the droplet size, such as droplet diameter.
[00114] In certain cases, methods may include determining a flow cell nozzle orifice diameter based on the captured images. In such cases, methods may include capturing images of the flow stream through the orifice of the flow cell nozzle and generating a data signal that corresponds to the physical dimensions of the flow stream. Based on the data signal corresponding to the physical dimensions of the flow stream, the flow cell nozzle opening diameter is determined. In certain cases, methods include determining the flow cell nozzle opening diameter using the width of the flow stream. In other cases, the methods include determining the flow cell nozzle opening diameter using the droplet diameter. In these embodiments, the methods may additionally include automating adjustments to one or more flow cytometer parameters based on the determined flow cell nozzle opening diameter. For example, methods may include automatically adjusting the coating fluid pressure, drop conduction frequency, drop charge, deflection voltage, charge correction value, drop delay, drop frequency, load amplitude phase drop and or a combination thereof, as discussed above.
[00115] In certain embodiments, the methods may include automating adjustments to the drop conduction frequency in response to the determined flow cell nozzle orifice diameter. For example, the drop conduction frequency can be increased by 0.01 Hz or more, such as 0.05 Hz or more, such as 0.1 Hz or more, such as 0.25 Hz or more, such as 0.5 Hz or more, such as 1 Hz or more, such as 2.5 Hz or more, such as 5 Hz or more, such as 10 Hz or more and including 25 Hz or more . In other cases, the methods include reducing the frequency of droplet conduction in response to the determined flow cell nozzle orifice diameter, such as at 0.01 Hz or more, such as at 0.05 Hz or more, such as 0.1 Hz or more, such as 0.25 Hz or more, such as 0.5 Hz or more, such as 1 Hz or more, such as 2.5 Hz or more, such as 5 Hz or more, such as 10 Hz or more and including 25 Hz or more.
[00116] In other embodiments, the methods may include automating the adjustments to the coating fluid pressure in response to the determined flow cell nozzle orifice diameter. For example, the coating fluid pressure can be increased by 0.000006 MPa (0.001 psi) or more, such as 0.00003 MPa (0.005 psi) or more, such as 0.00006 MPa (0.01 psi) or more, such as 0.003 MPa (0.05 psi) or more, such as 0.0006 MPa (0.1 psi) or more, such as 0.003 MPa (0.5 psi) or more, such as 0 .0068 MPa (1 psi) or more, such as 0.03 MPa (5 psi) or more, such as 0.068 MPa (10 psi) or more, such as 0.17 MPa (25 psi) or more, such as 0.35 MPa (50 psi) or more, such as 0.51 MPa (75 psi) or more and including increasing the coating fluid pressure by 0.68 MPa (100 psi) or more. In other cases, methods include reducing the coating fluid pressure in response to the determined flow cell nozzle orifice diameter, such as 0.000006 MPa (0.001 psi) or more, such as 0.00003 MPa (0.005 psi) or more, such as 0.00006 MPa (0.01 psi) or more, such as 0.003 MPa (0.05 psi) or more, such as 0.0006 MPa (0.1 psi) or more , such as 0.003 MPa (0.5 psi) or more, such as 0.0068 MPa (1 psi) or more, such as 0.03 MPa (5 psi) or more, such as 0.068 MPa (10 psi) ) or more, such as 0.17 MPa (25 psi) or more, such as 0.34 MPa (50 psi) or more, such as 0.51 MPa (75 psi) or more and including reducing pressure coating fluid at 0.68 MPa (100 psi) or more.
[00117] In some embodiments, methods may include comparing the physical dimensions of the flow current determined from the captured images with expected dimensions based on empirical characteristics of the flow cytometer (such as flow cell nozzle orifice size and coating fluid pressure) as well as parameters entered by the user. In such cases, the methods include generating a data signal that corresponds to the ratio of the physical dimensions of the flow current determined from the captured images when compared to the expected flow current dimensions based on the empirical characteristics of the flow cytometer and parameters entered by the user. For example, methods may include generating a data signal that indicates that the flow current as determined from the captured images is 99% or less than the expected size of the flow current, such as 95% or less, such as 90% or less, such as 85% or less, such as 80% or less, such as 75% or less, such as 50% or less, such as 25% or less and including 10% or less of the expected flow stream size . In other embodiments, the methods may include generating a data signal that indicates that the flow current, as determined from the captured images, is greater than the expected size, such as being 105% or greater of the flow current size, such as 110% or more, such as 125% or more and including 150% or more. In these modalities, the methods may include additionally automating adjustments to one or more parameters of the flow cytometer based on the generated data signal. For example, methods may include automatically adjusting the coating fluid pumping rate, the coating fluid pressure or drop conduction frequency.
[00118] The methods of interest may also include capturing images in a detection field at the interruption point of the current flow, generating a data signal corresponding to the droplet drop volume downstream of the interruption point and adjusting one or more flow cytometer properties in response to the determined drop volume. In some embodiments, the methods include automatically adjusting the drop conduction frequency of the flow stream in response to the determined drop volume. For example, methods may include reducing the frequency of gout conduction, such as by 5% or more, such as by 10% or more, such as by 15% or more, such as by 25% or more, as in 50% or more, such as 75% or more, such as 90% or more, such as 95% or more and including 99% or more. In other cases, the methods may include increasing the frequency of gout conduction in response to the determined drop volume, such as by 5% or more, such as by 10% or more, such as by 15% or more, as in 25% or more, such as 50% or more, such as 75% or more, such as 90% or more, such as 95% or more and including 99% or more.
[00119] In other modalities, the methods include automatically regulating the volume of sample collection during cell classification based on the determined drop volume. For example, the desired volume for each sample collected can be inserted into a processor and based on the data signal that corresponds to the drop volume, the flow cytometer can be automated to stop sample collection after a predetermined amount of time, such as removing the collection vessel or ceasing the flow current through the flow cytometer.
[00120] In still other modalities, the methods may include capturing images of a flow current in a detection field, determining the presence or absence of a flow current in the detection field and adjusting one or more parameters of the flow cytometer in response to the determined presence or absence of the flow current. As discussed above, the methods in question to determine whether a flow stream is present or not in captured images of the flow cell nozzle orifice can be used to determine whether the flow cell has a clogged nozzle. In these modalities, the images captured by the imaging sensors are estimated and if a flow current is detected in the images, a data signal is generated indicating the presence of a flow current. On the other hand, if after estimating the captured images, it is determined that the flow current is absent in the captured images, a data signal is generated indicating the absence of a flow current.
[00121] When no flow current is present in the captured images, in certain modalities, the methods may include automatically alerting a user that the absence of flow current is a result of malfunction of the flow cytometer, such as a clogged nozzle. In these modalities, the data signal that corresponds to the absence of a flow current is correlated with user input as to the fact that a flow current must be expected. In some embodiments, when a user has configured the system to have a “closed loop” configuration, no flow current is expected. In these modalities, the flow cytometer does not alert the user of a malfunction (for example, clogged nozzle) as a flow current is not expected.
[00122] Figure 3 shows a flow chart that illustrates methods for adjusting one or more parameters of a flow cytometer according to certain modalities of the present disclosure. As summarized above, methods include capturing one or more images in a flow cytometer flow detection field. Images can, in certain cases, be captured in two or more detection fields, such as 3 or more and including 4 or more detection fields. In some embodiments, methods include determining whether a flow current is present or absent. When a flow current is determined to be absent and a flow current is expected (such as during normal use), an alert can be given to the user of a possible instrument malfunction (eg, clogged mouthpiece). In other embodiments, methods include determining the spatial position of the flow current or determining the physical dimensions of the flow current. In certain cases, methods include initially determining that a flow current is present in one or more captured images, followed by determining the spatial position of the flow current. In other cases, the methods include determining initially that a flow current is present in one or more captured images, followed by determining the physical dimensions of the flow current. In some embodiments, the methods include determining a physical property of the flow cytometer based on the physical dimensions of the flow current from the captured images. For example, the flow cell nozzle orifice can be determined based on the physical dimensions of the flow of captured images.
[00123] The methods also include automatically adjusting one or more flow cytometer parameters in response to data signals derived from captured images, such as adjusting coating fluid pressure, droplet charge voltage, deflection plate voltage, value load correction, drop delay, drop conduction frequency, drop amplitude and loading phase or a combination thereof. In certain embodiments, the one or more parameters includes adjusting the position of one or more support platforms, for example, a support platform that has a container for collecting flow stream particles during cell classification.
[00124] As discussed above, the methods in question can be completely automated, so that adjustments are made in response to data signals that correspond to one or more parameters of the flow current with little, if any, human intervention or input user manual. COMPUTER CONTROLLED SYSTEMS
[00125] Aspects of the present disclosure additionally include computer-controlled systems for practicing the methods in question, wherein the systems additionally include one or more computers for complete automation or partial automation of a system for practicing the methods described in this document. In some embodiments, the systems include a computer that has a computer-readable storage medium with a computer program stored on it, where the computer program when loaded on the computer includes instructions for capturing one or more images from a stream. flow cytometer in a detection field; algorithm to determine the spatial position of the flow current in the detection field; algorithm to generate a data signal that corresponds to the spatial position of the flow stream; and instructions for adjusting one or more flow cytometer parameters in response to the data signal. In certain cases, systems include a computer that has a computer-readable storage medium with a computer program stored on it, where the computer program, when loaded on the computer, includes instructions for capturing one or more images from a chain flow from the flow cytometer in a detection field; algorithm to determine the physical dimensions of the flow current in the detection field; algorithm to generate a data signal that corresponds to the physical dimensions of the flow stream; and instructions for adjusting one or more flow cytometer parameters in response to the data signal.
[00126] In modalities, the system includes an input module, a processing module and an output module. The processing modules of interest can include one or more processors that are configured and automated to adjust one or more parameters of a flow cytometer as described above. For example, processing modules can include two or more processors that are configured and automated to adjust one or more parameters of a flow cytometer as described above, such as three or more processors, such as four or more processors and including five or more more processors.
[00127] In some embodiments, the systems in question may include an input module so that parameters or information about the fluid sample, coating fluid pressure, hydrostatic pressure, flow current load, deflection voltage, value of load correction, drop delay, drop conduction frequency, drop amplitude and load phase, orifice flow cell nozzle, support bed position, imaging sensors, light sources, optical adjustment protocols, well amplifiers how properties, resolution and sensitivity of imaging sensors can be entered before practicing the methods in question.
[00128] As described above, each processor includes memory that has a plurality of instructions for performing the steps of the methods in question, such as capturing one or more images of a flow stream from the flow cytometer in a detection field; determine one or more properties of the flow current in the detection field; generating a data signal that corresponds to one or more properties of the flow stream; and adjusting one or more flow cytometer parameters in response to the data signal. After the processor has performed one or more of the steps of the methods in question, the processor can be automated to make adjustments to flow cytometer parameters, such as adjustments as described above.
[00129] The systems in question can include both hardware and software components, in which the hardware components can take the form of one or more platforms, for example, in the form of a server, so that the functional elements, ie , those elements of the system that perform specific tasks (such as managing the input and output of information, processing information, etc.) of the system can be accomplished by running software applications on and through one or more computer platforms represented of the system.
[00130] The systems may include an operator input and display device. The operator input devices can be, for example, a keyboard, a mouse, or the like. The processing module includes a processor that has access to a memory that has instructions stored in it to perform the steps of the methods in question. The processing module can include an operating system, a graphical user interface (GUI) controller, a system memory, memory storage devices and input-output controllers, cache memory, a data backup unit and many other devices. The processor may be a commercially available processor, or it may be one among other processors that are available or that will be available. The processor runs the operating system and the operating system interfaces with firmware and hardware in a well-known manner and facilitates the processor to coordinate and execute the functions of various computer programs that can be written in a variety of programming languages, such as, Java, Perl, C ++, other high-level or low-level languages, as well as combinations of them, as known in the art. The operating system, typically in cooperation with the processor, coordinates and performs the functions of the other components of the computer. The operating system also provides scheduling, input-output control, file and data management, memory management and communication control and related services, all in accordance with known techniques.
[00131] System memory can be any of a variety of known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media, such as a hard drive or resident tape, optical media, such as a compact read and write disk, flash memory devices, or other memory storage device. The memory storage device can be any of a variety of known or future devices, including a compact disk drive, a tape drive, a removable hard drive, a floppy drive. Such types of memory storage devices typically read, and / or written to, program storage media (not shown), such as, respectively, a compact disk, magnetic tape, removable hard disk, or floppy disk. Any of these program storage media, or others now in use or that can be developed later, can be considered a computer program product. As will be noted, these program storage media typically store a program and / or computer software data. Computer software programs, also called computer control logic, are typically stored in system memory and / or the program storage device used in combination with the memory storage device.
[00132] In some embodiments, a computer program product is described comprising a media usable by computer that has a control logic (computer software program, which includes program code) stored therein. The control logic, when executed by the computer's processor, causes the processor to perform the functions described in this document. In other modalities, some functions are implanted primarily in hardware using, for example, a hardware state machine. The implantation of the hardware status machine in order to carry out the functions described in this document is apparent to people skilled in the relevant techniques.
[00133] Memory can be any suitable device on which the processor can store and retrieve data, such as magnetic, optical or solid state storage devices (including magnetic or optical disks or tape or RAM, or any other suitable device, as well fixed as portable). The processor may include a general purpose digital microprocessor programmed appropriately from computer-readable media that carries the required program code. Programming can be provided remotely to a processor via a communication channel or previously saved to a computer program product, such as memory or other portable or fixed computer-readable storage media that uses any of these devices in connection with a memory. For example, an optical or magnetic disc can carry programming and can be read by a disc reader / writer. The systems of the invention also include programming, for example, in the form of computer program products, algorithms for use in the practice of methods, as described above. Programming in accordance with the present invention can be recorded on computer-readable media, for example, any media that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy disks, hard disk storage media, and magnetic tape; optical storage media, such as CD-ROM; electrical storage media, such as RAM and ROM; portable flash drive; and hybrids of these categories, such as magnetic / optical storage media.
[00134] The processor may also have access to a communication channel to communicate with a user at a remote location. Remote location means that the user is not in direct contact with the system and relays input information to an input manager from an external device, such as a computer connected to a Wide Area Network (“WAN”) by relay , telephone network, satellite network, or any other suitable communication channel, including a mobile phone (ie, smart phone).
[00135] In some embodiments, the systems according to the present disclosure can be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or transmitter to communicate with a network and / or other device. The communication interface can be configured for wired or wireless communication, including, but not limited to, radio frequency (RF) communication (for example, Radio Frequency Identification (RFID), communication protocols by Zigbee, WiFi, infrared, Serial Bus Wireless Universal (USB), Ultra-Wide Band (UWB), Bluetooth® communication protocols and cellular communication, such as code division multiple access (CDMA) or Global System for Mobile Communications (GSM).
[00136] In one embodiment, the communication interface is configured to include one or more communication ports, for example, physical ports or interfaces, such as a USB port, an RS-232 port, or any other electrical connection port suitable to allow data communication between the systems in question and other external devices, such as a computer terminal (for example, in a doctor's office or in a hospital environment) that is configured for similar complementary data communication.
[00137] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol to enable the systems in question to communicate with other devices, such as terminals. computer and / or networks, communication enabled by mobile phones, personal digital assistants, or any other communication devices that the user can use in combination.
[00138] In one embodiment, the communication interface is configured to provide a connection for data transfer that using an Internet Protocol (IP) over a cell phone network, Short Message Service (SMS), connection without wire to a personal computer (PC) on a Local Area Network (LAN) that is connected to the internet, or a WiFi connection to the internet at a WiFi access point.
[00139] In one embodiment, the systems in question are configured to communicate wirelessly with a server device via the communication interface, for example, using a common standard, such as the 802.11 RF protocol or Bluetooth®, or an IrDA infrared protocol. The server device can be another portable device, such as a smart phone, Personal Digital Assistant (PDA) or notebook computer; or a larger device, such as a desktop computer, utensils, etc. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), as well as an input device, such as buttons, a keyboard, mouse, or a touchscreen.
[00140] In some modalities, the communication interface is configured to automatically or semi-automatically communicate data stored in the systems in question, for example, in an optional data storage unit, with a network or server device using one or more of the communication protocols and / or mechanisms described above.
[00141] Output controllers can include controllers for any of a variety of known display devices to present information to a user, whether human or machine, whether local or remote. If one of the display devices provides visual information, that information can typically be organized in a logical and / or physical way as an arrangement of photo elements. A graphical user interface (GUI) controller can include any of a variety of known or future software programs to provide graphical input and output interfaces between the system and a user, and to process user input. The functional elements of the computer can communicate with each other via a system bus. Some of these communications can be accomplished in alternative ways using a network or other types of remote communications. The output manager can also provide information generated by the processing module to a user at a remote location, for example, via the Internet, telephone or satellite network, in accordance with known techniques. The presentation of data by the output manager can be implemented in accordance with a variety of known techniques. According to some examples, the data may include SQL, HTML or XML documents, email or other files, or data in other forms. The data can include Internet URL addresses so that a user can retrieve additional SQL, HTML, XML, or other documents or data from remote sources. The one or more platforms present in the systems in question can be any type of known computer platform or a type to be developed in the future, although they typically belong to a class of computer commonly known as servers. However, the media can be a main computer, a workstation, or another type of computer. They can be connected through any known or future type of cabling or other communication system that includes wireless systems, both in a network and otherwise. They can be located or they can be physically separated. Various operating systems can be used on any of the computer platforms, possibly depending on the type and / or production of the chosen computer platform. Suitable operating systems include Windows NT®, Windows XP, Windows 7, Windows 8, iOS, Sun Solaris, Linux, OS / 400, Compaq Tru64 Unix, SGI IRIX, Siemens Reliant Unix, among others. UTILITY
[00142] The systems, methods and computer systems in question are useful in a variety of different applications in which it is desirable to automate adjustments to one or more parameters of a flow cytometer to provide fast, reliable systems for characterizing and classifying cells to from a biological sample. The modalities of the present disclosure are useful when minimizing how much human input and adjustment to the system is trusted is desired, for example, in research and high throughput in laboratory testing. The present disclosure is also useful when it is desirable to provide a flow cytometer with improved cell classification accuracy, enhanced particle collection, systems that provide alerts regarding component malfunction (e.g., clogged flow cell nozzle), reduced energy consumption, particle loading efficiency, more accurate particle loading and increased particle deviation during cell sorting. In the modalities, the present disclosure reduces the need for user input or manual adjustment during sample analysis with a flow cytometer. In certain embodiments, the systems in question provide fully automated protocols so that adjustments to a flow cytometer during use require little human input, if any.
[00143] The present disclosure is also useful in applications where cells prepared from a biological sample may be desired for research, laboratory testing or for use in therapy. In some embodiments, the methods in question and devices may facilitate obtaining individual cells prepared from a biological fluid or tissue target sample. For example, the methods and systems in question make it easy to obtain cells from fluid or tissue samples to be used as a research or diagnostic specimen for diseases such as cancer. Likewise, the methods and systems in question make it easier to obtain cells from fluid or tissue samples to be used in therapy. The methods and devices of the present disclosure allow to separate and collect cells from a biological sample (for example, organ, tissue, tissue fragment, fluid) with enhanced efficiency and low cost, compared to traditional flow cytometry systems.
[00144] Despite the attached clauses, the disclosure presented in this document is also defined by the following clauses: 1. A system comprising:
[00145] an imaging sensor configured to capture one or more images of a flow current in a flow cytometer detection field; and
[00146] a processor comprising a memory operatively coupled to the processor, wherein the memory includes instructions stored therein to determine one or more properties of the flow stream and generate a data signal corresponding to one or more properties of the flow stream flow,
[00147] in which the processor is configured to automatically adjust one or more flow cytometer parameters in response to the data signal. 2. The system, according to clause 1, in which the processor is configured to determine the spatial position of the flow stream and generate a data signal corresponding to the spatial position of the flow stream from the one or more images. 3. The system, according to any of clauses 1 to 2, in which the system additionally comprises a support platform positioned downstream from the detection field. 4. The system, according to clause 3, in which the system is configured to automatically adjust the position of the support platform in response to the data signal corresponding to the spatial position of the flow current in the detection field. 5. The system, according to clause 4, in which the system is configured to adjust the position of the support platform in two dimensions. 6. The system according to any of clauses 3 to 5, in which the support platform comprises a laser. 7. The system according to any of clauses 3 to 5, wherein the support platform comprises a container. 8. The system according to clause 7, in which the system is configured to automatically align the container to the determined spatial position of the flow stream. 9. The system, according to clause 8, in which the automatic alignment of the container to the flow stream comprises:
[00148] map the position of the flow current in the detection field in an X-Y plane;
[00149] map the position of the container in the X-Y plane; and
[00150] make the position of the container compatible with the position of the flow current in the X-Y plane. 10. The system, according to any of clauses 1 to 9, in which the system additionally comprises:
[00151] a second imaging sensor configured to capture one or more images of the flow current in a second detection field; and
[00152] a processor comprising a memory operatively coupled to the processor, wherein the memory includes instructions stored therein to determine one or more properties of the flow stream in the second detection field and generate a second corresponding data signal for the one or more properties of the flow current in the second detection field. 11.0 system, according to clause 10, in which the processor is configured to determine the spatial position of the flow current in the second detection field and generate a second data signal corresponding to the spatial position of the flow current in the second detection field . 12. The system, according to clause 11, in which the system additionally comprises a second support platform positioned downstream from the first support platform. 13. The system, according to clause 12, in which the system is configured to automatically adjust the position of the second support platform in response to the first and second data signals. 14. The system according to clause 13, in which the system is configured to adjust the position of the second support platform in two dimensions. 15. The system, according to clause 13, in which the second support platform comprises a container to collect the flow current. 16. The system, according to clause 15, in which the system is configured to automatically align the container to the determined spatial position of the flow current in the second detection field. 17. The system, according to clause 16, in which the automatic alignment of the container comprises:
[00153] map the position of the flow current in the second detection field in an X-Y plane;
[00154] map the position of the container in the X-Y plane; and
[00155] make the position of the container compatible with the position of the flow current in the X-Y plane. 18. The system, according to clause 1, in which the processor is configured to determine the physical dimensions of the flow current from one or more images and generate a data signal corresponding to the physical dimensions of the flow current. 19. The system, according to clause 18, in which the processor is configured to determine the width of the flow stream from one or more images and generate a data signal corresponding to the width of the flow stream. 20. The system, according to clause 19, in which the processor is configured to determine the flow cell nozzle orifice diameter and generate a data signal corresponding to the flow cell nozzle orifice diameter based on determined width of the flow stream. 21. The system, according to clause 20, in which the processor is configured to automatically adjust one or more flow cytometer parameters based on the determined flow cell nozzle orifice diameter. 22. The system, according to clause 21, in which the flow cytometer parameters are selected from the group consisting of hydrostatic pressure, coating fluid pressure, flow current load, bypass voltage, frequency of oscillator driving, load correction value, drop delay, drop frequency, drop amplitude and load phase. 23. The system according to clause 22, where the processor is configured to automatically adjust the coating fluid pressure based on the determined flow cell nozzle orifice diameter. 24. The system, according to clause 22, in which the processor is configured to automatically adjust the oscillator driving frequency based on the determined flow cell nozzle orifice diameter. 25. The system, according to any of clauses 1-24, in which the imaging sensor is a CCD camera. 26. A system for automatically locating a current position in a liquid flow from a flow cytometer that comprises;
[00156] a first camera, adapted to detect a current position in a first detection field and to generate a first signal representative of the current position; and
[00157] a first stage in which the first platform is operationally connected to the first camera and configured to move in an XY plane in response to the first signal. 27. The clause 26 system, which further comprises a second camera adapted to detect a current position in a second detection field and to generate a second signal representative of the current position;
[00158] in which the first and second detection fields of the first and second cameras are oriented substantially orthogonal in the XY plane; and
[00159] in which the first platform is operationally connected to the second camera and configured to move the XY plane in response to the second signal in addition to the first signal. 28. The system, according to any of clauses 26 to 27, in which a laser is mounted on the first pallet. 29. The system, according to any of clauses 26 to 27, in which a collection device is mounted on the first pallet. 30. The system, according to clause 26, which additionally comprises a second platform in which a collection device is mounted on the second platform, and the second platform is configured to move in the XY plane in response to the first signal. 31. The system, according to clause 30, which additionally comprises a second platform in which a collection device is mounted on the second platform, the second platform being configured to move in the XY plane in response to the second signal beyond the first sign. 32. The system, according to clause 30, which additionally comprises an electrical system configured to adjust an electrical charge in the flow current in response to the second signal from the second camera. 33. The system, according to clause 30, in which the operational connection is mediated by a controller connected to the first camera and to the first and second cameras and to the first stage and in which the controller is configured to receive the signals from the first and second cameras and to calculate an ideal position for the first stage. 34. The system, according to clause 33, in which the operational connection is mediated by a controller connected to the first and second cameras and to the second stage and configured to receive the signals from the first and second cameras and to calculate a position ideal for the second platform. 35. The system, according to any of clauses 26 to 34, in which the current is comprised of a series of drops. 36. A system for automatically determining a nozzle opening diameter that comprises
[00160] a first camera, adapted to detect a current dimension in a first detection field and to generate a first signal representative of the current dimension;
[00161] a controller that comprises a computer algorithm configured to determine a value for the nozzle opening diameter from the current dimension and transmit the value to a flow cytometer. 37. The system, according to clause 36, in which the chain dimension is the width of the chain. 38. The system, according to any of clauses 36 to 37, in which the flow cytometer is configured to automatically adjust a series of parameters after receiving the transmitted value. 39. The system, according to clause 38, in which the series of parameters is selected from the group comprising hydrostatic pressure, drop load, deflection voltage, load correction value, drop delay, conduction frequency of drop, drop amplitude and loading phase. 40. A method for adjusting one or more parameters of a flow cytometer, the method comprising:
[00162] capture one or more images of a flow cytometer flow stream in a detection field;
[00163] determine one or more properties of the flow current in the detection field;
[00164] generating a data signal corresponding to one or more properties of the flow current; and
[00165] adjust one or more parameters of the flow cytometer in response to the data signal. 41. The method, according to clause 40, wherein the method comprises determining the spatial position of the flow current in the detection field and generating a data signal corresponding to the spatial position of the flow current. 42. The method, according to any of clauses 40 to 41, in which the flow current in the detection field is continuous. 43. The method, according to clause 40, in which the detection field comprises the upstream flow current from the flow current interruption point. 44. The method, according to clause 43, in which determining the spatial position of the flow current comprises mapping the position of the flow current in an X-Y plane. 45. The method, according to clause 44, which further comprises adjusting the position of a support platform in response to the data signal corresponding to the spatial position of the flow stream. 46. The method, according to clause 45, in which the support platform comprises a laser. 47. The method, according to clause 45, in which the support platform comprises a collection container. 48. The method, according to clause 47, wherein the method comprises aligning the container to the determined spatial position of the flow stream. 49. The method according to clause 48, in which aligning the container to the flow stream comprises:
[00166] map the position of the flow current in the detection field in an X-Y plane;
[00167] map the position of the container in the X-Y plane; and
[00168] make the position of the container compatible with the position of the flow current in the X-Y plane. 50. The method, in accordance with any of clauses 40 to 49, wherein the method additionally comprises:
[00169] capture one or more images of a flow cytometer flow stream in a second detection field;
[00170] determine one or more properties of the flow current in the second detection field; and
[00171] generate a data signal corresponding to one or more properties of the flow current in the second detection field. 51. The method according to clause 50, wherein the method comprises determining the spatial position of the flow current in the second detection field and generating a second data signal corresponding to the spatial position of the flow current in the second detection field. 52. The method, according to clause 50, in which the flow current in the second detection field comprises different droplets. 53. The method, according to clause 50, in which the second detection field comprises the downstream flow current from the flow current interruption point. 54. The method, according to clause 50, which further comprises adjusting the position of a second support platform in response to the second data signal corresponding to the spatial position of the flow current in the second detection field. 55. The method, according to clause 50, wherein the method comprises adjusting the position of the second support platform in response to the first and second data signals. 56. The method, according to clause 55, in which the second support platform comprises a collection container. 57. The method, according to clause 56, wherein the method comprises aligning the container to the determined spatial position of the flow current in the second detection field. 58. The method, according to clause 57, in which the alignment of the container comprises:
[00172] map the position of the flow current in the second detection field in an X-Y plane;
[00173] map the position of the container on the X-Y piano; and
[00174] make the position of the container compatible with the position of the flow current in the X-Y piano. 59. The method, according to clause 40, wherein the method comprises determining the physical dimensions of the flow current in the detection field and generating a data signal corresponding to the physical dimensions of the flow current. 60. The method, according to clause 59, wherein the method comprises determining the width of the flow stream from one or more images and generating a data signal corresponding to the width of the flow stream. 61. The method, according to clause 60, which further comprises determining the flow cell orifice diameter and generating a data signal corresponding to the flow cell nozzle orifice diameter based on the determined width of the flow stream . 62. The method, according to clause 61, which further comprises adjusting one or more parameters of the flow cytometer based on the determined diameter of the flow cell nozzle orifice. 63. The method, according to clause 62, in which the flow cytometer parameters are selected from the group consisting of hydrostatic pressure, coating fluid pressure, flow current charge, bypass voltage, frequency of oscillator driving, load correction value, drop delay, drop driving frequency, drop amplitude and loading phase. 64. The method according to clause 63, which further comprises adjusting the drop conduction frequency in response to the determined flow cell nozzle orifice diameter. 65. The method according to clause 64, which further comprises adjusting the pressure of coating fluid in response to the determined diameter of the flow cell nozzle orifice. 66. A method that comprises:
[00175] capture one or more images of a flow cytometer flow stream in a detection field;
[00176] determine that the flow current is not present in the captured image;
[00177] evaluate the flow cytometer parameters entered by a user to determine if the flow current is expected to be present in the captured image; and
[00178] generate an alert to the user that indicates a malfunction of the flow cytometer. 67. The method, according to clause 66, in which the malfunction is a clogged mouthpiece. 68. The method according to clause 66, wherein the detection field comprises an upstream flow current from the flow current interruption point. 69. The method, according to clause 66, in which the flow current in the detection field is continuous. 70. The method, according to clause 66, which further comprises inserting the flow cytometer which comprises an open flow cell nozzle orifice. 71. A method for adjusting one or more parameters of a flow cytometer, the method comprising:
[00179] injecting a sample into the sample port of a flow cytometer, in which the flow cytometer comprises a system comprising a processor with a memory operationally coupled to the processor in which the system is automated to:
[00180] capture one or more images of a flow cytometer flow stream comprising the sample in a detection field;
[00181] determine one or more properties of the flow current in the detection field;
[00182] generating a data signal corresponding to one or more properties of the flow current; and
[00183] adjust one or more parameters of the flow cytometer in response to the data signal. 72. The method, according to clause 71, wherein the method comprises determining the spatial position of the flow current in the detection field and generating a data signal corresponding to the spatial position of the flow current. 73. The method, according to clause 71, in which the flow current in the detection field is continuous. 74. The method, according to clause 71, in which the detection field comprises the upstream flow current from the flow current interruption point. 75. The method, according to clause 74, in which the determination of the spatial position of the flow current comprises mapping the position of the flow current in an X-Y plane. 76. The method, according to clause 71, which further comprises adjusting the position of a support platform in response to the data signal corresponding to the spatial position of the flow stream. 77. The method, according to clause 76, in which the support platform comprises a laser. 78. The method, according to clause 76, in which the support platform comprises a collection container. 79. The method, according to clause 78, wherein the method comprises aligning the container to the determined spatial position of the flow stream. 80. The method, according to clause 79, in which the alignment of the container to the flow stream comprises:
[00184] map the position of the flow current in the detection field in an X-Y plane;
[00185] map the position of the container in the X-Y plane; and
[00186] make the position of the container compatible with the position of the flow current in the X-Y plane. 81. The method, according to clause 71, in which the method additionally comprises:
[00187] capture one or more images of a flow cytometer flow stream in a second detection field;
[00188] determine one or more properties of the flow current in the second detection field; and
[00189] generate a data signal corresponding to one or more properties of the flow current in the second detection field. 82. The method, according to clause 81, in which the method comprises determining the spatial position of the flow current in the second detection field and generating a second data signal corresponding to the spatial position of the flow current in the second detection field . 83. The method, according to clause 82, in which the flow current in the second detection field comprises different droplets. 84. The method, according to clause 82, wherein the second detection field comprises the flow stream downstream from the point of flow current interruption. 85. The method, according to clause 82, which further comprises adjusting the position of a second support platform in response to the second data signal corresponding to the spatial position of the flow current in the second detection field. 86. The method, according to clause 81, in which the method comprises adjusting the position of the second support platform in response to the first and second data signals. 87. The method, according to clause 86, in which the second support platform comprises a collection container. 88. The method, according to clause 87, wherein the method comprises aligning the container to the determined spatial position of the flow current in the second detection field. 89. The method, according to clause 88, in which the alignment to the container comprises:
[00190] map the position of the flow current in the second detection field in an X-Y plane;
[00191] map the position of the container in the X-Y plane; and
[00192] make the position of the container compatible with the position of the flow current in the X-Y plane. 90. The method, according to clause 71, in which the method comprises determining the physical dimensions of the flow current in the detection field and generating a data signal corresponding to the physical dimensions of the flow current. 91. The method, according to clause 90, wherein the method comprises determining the width of the flow stream from one or more images and generating a data signal corresponding to the width of the flow stream. 92. The method, according to clause 91, which further comprises determining the flow cell orifice diameter and generating a data signal corresponding to the flow cell nozzle orifice diameter based on the determined width of the flow stream . 93. The method, according to clause 92, which further comprises adjusting one or more parameters of the flow cytometer based on the determined diameter of the flow cell nozzle orifice. 94. The method, according to clause 93, in which the parameters of the flow cytometer are selected from the group consisting of hydrostatic pressure, coating fluid pressure, flow current load, bypass voltage, frequency of oscillator driving, load correction value, drop delay, drop frequency, drop amplitude and load phase. 95. The method according to clause 94, which further comprises adjusting the oscillator conduction frequency in response to the determined flow cell nozzle orifice diameter. 96. The method, according to clause 94, which further comprises adjusting the pressure of coating fluid in response to the determined diameter of the flow cell nozzle orifice. 97. A system for configuring a flow cytometer, the system comprising:
[00193] a processor comprising a memory operationally coupled to the processor, in which the memory includes instructions stored therein, the instructions comprising:
[00194] instructions to capture one or more images of a flow cytometer flow stream in a detection field;
[00195] algorithm to determine one or more properties of the flow current in the detection field;
[00196] algorithm to generate a data signal corresponding to one or more properties of the flow current; and
[00197] instructions to adjust one or more parameters of the flow cytometer in response to the data signal. 98. The system, according to clause 97, in which the memory comprises an algorithm that determines the spatial position of the flow current in the detection field and that generates a data signal corresponding to the spatial position of the flow current. 99. The system according to clause 97, in which the flow current in the detection field is continuous. 100. The system, according to clause 97, in which the detection field comprises the upstream flow current from the flow current interruption point. 101. The system, according to clause 97, in which the memory comprises an algorithm to determine the spatial position of the flow current, comprises a mapping of the position of the flow current in an X-Y plane. 102. The system, according to clause 97, in which the memory comprises an algorithm to adjust the position of a support platform in response to the data signal corresponding to the spatial position of the flow stream. 103. The system, according to clause 102, in which the support platform comprises a collection container. 104. The system, according to clause 103, in which the memory comprises an algorithm to align the container to the determined spatial position of the flow stream. 105. The system, according to clause 104, in which the alignment of the container to the flow stream comprises:
[00198] map the position of the flow current in the detection field in an X-Y plane;
[00199] map the position of the container in the X-Y plane; and
[00200] make the position of the container compatible with the position of the flow current in the X-Y plane. 106. The system, according to clause 97, in which the memory additionally comprises:
[00201] instructions for capturing one or more images of a flow cytometer flow stream in a second detection field;
[00202] algorithm to determine one or more properties of the flow current in the second detection field; and
[00203] algorithm to generate a data signal corresponding to one or more properties of the flow current in the second detection field. 107. The system, according to clause 106, in which the memory comprises an algorithm to determine the spatial position of the flow current in the second detection field and to generate a second data signal corresponding to the spatial position of the flow current in the second detection field. 108. The system, according to clause 106, in which the flow current in the second detection field comprises different droplets. 109. The system, according to clause 106, in which the second detection field comprises the downstream flow current from the flow current interruption point. 110. The system, according to clause 106, in which the memory additionally comprises an algorithm to adjust the position of a second support platform in response to the second data signal corresponding to the spatial position of the flow current in the second detection field. . 1. 1.0 system, according to clause 106, in which the memory comprises an algorithm to align a container on the second support platform to the determined spatial position of the flow current in the second detection field. 112. The system, according to clause 111, in which the alignment of the container comprises:
[00204] map the position of the flow current in the second detection field in an X-Y plane;
[00205] map the position of the container in the X-Y plane; and
[00206] make the position of the container compatible with the position of the flow current in the X-Y plane. 113. The system, according to clause 97, in which the memory comprises an algorithm to determine the physical dimensions of the flow current in the detection field and to generate a data signal corresponding to the physical dimensions of the flow current. 114. The system, according to clause 113, in which the physical dimension is the width of the flow stream. 115. The system according to clause 113, wherein the memory additionally comprises an algorithm for determining the flow cell orifice diameter and for generating a data signal corresponding to the flow cell nozzle orifice diameter based on the determined width of the flow stream. 116. The system, according to clause 115, wherein the memory additionally comprises an algorithm for adjusting one or more parameters of the flow cytometer based on the determined diameter of the flow cell nozzle orifice. 117. The system, according to clause 116, in which the one or more parameters are selected from the group consisting of hydrostatic pressure, coating fluid pressure, flow current load, bypass voltage, conduction frequency oscillator, load correction value, drop delay, drop frequency, drop amplitude and load phase. 118. A system for configuring a flow cytometer, the system comprising:
[00207] a processor comprising a memory operationally coupled to the processor, in which the memory includes instructions stored therein, the instructions comprising:
[00208] instructions for capturing one or more images of a flow cytometer flow stream in a detection field;
[00209] algorithm to determine that the flow current is not present in the captured image;
[00210] algorithm to evaluate flow cytometer parameters entered by a user to determine if the flow current is expected to be present in the captured image; and
[00211] instructions to generate an alert to the user that indicates a flow cytometer malfunction. 119. The system, according to clause 118, in which the malfunction is a clogged nozzle. 120. The system, according to clause 118, in which the detection field comprises an upstream flow current from the flow current interruption point. 121. The system, according to clause 118, in which the flow current in the detection field is continuous. 122. The system, according to clause 118, in which the inserted parameter comprises the indication that the flow cytometer comprises an open flow cell nozzle orifice.
[00212] Although the aforementioned invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it is readily apparent to people of ordinary skill in the art in light of the teachings of the present disclosure that certain changes and modifications can be made to it without departing from the spirit and scope of the attached claims.
[00213] Consequently, the above just illustrates the principles of the invention. It will be appreciated that persons skilled in the art may develop several provisions which, although not explicitly described or shown in this document, incorporate the principles of the invention and are included within the spirit and scope of the same. Furthermore, all examples and conditional language recited in this document are primarily intended to assist the reader in understanding the principles of the invention that are not limited to such examples and conditions recited explicitly. In addition, all statements that recite principles, aspects and modalities of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents. In addition, such equivalents are intended to include both currently known equivalents and future developed equivalents, that is, any developed elements that perform the same function, regardless of structure. Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described in this document. Preferably, the scope and spirit of the present invention are incorporated by the appended claims.

权利要求:
Claims (11)
[0001]
1.System characterized by the fact that it comprises: an imaging sensor configured to capture one or more images of a flow current in a detection field of a flow cytometer; and a processor comprising a memory operably coupled to the processor, wherein the memory includes instructions stored therein to determine one or more properties of the flow stream and generate a data signal corresponding to one or more properties of the flow stream, where the processor is configured to automatically adjust one or more flow cytometer parameters in response to the data signal, and where the processor is configured to perform one of the procedures listed below: a) determine the spatial position of the flow current flow and generate a data signal corresponding to the spatial position of the flow current based on data obtained from a system that further comprises: a second imaging sensor configured to detect the position of the flow current in a second detection field which is substantially orthogonally oriented in an XY plane from the first detection field and generate a second signal In view of the current position in the second detection field, a first support frame comprising a laser and operationally coupled to the first and second imaging sensors, where the first frame is configured to automatically adjust the positions in the XY plane in response to the second signal, in addition to a first sign that is representative of Petition 870200028595, of 03/03/2020, p. 108/120 flow in the detection field of the first imaging sensor; and a second support platform comprising a collection container and configured to adjust positions on the XY plane in response to the first signal; b) determine the width of the flow stream from one or more images and generate a data signal corresponding to the width of the flow stream, compare the determined width of the flow stream with the width of the flow stream that is expected based on the size of the orifice of the flow cell nozzle and generate a data signal corresponding to a ratio between the determined width of the flow current and the width of the flow current which is expected based on the size of the orifice of the flow cell nozzle flow; and c) determine the presence or absence of the flow current in the captured images, evaluate a flow cytometer parameter that is inserted by a user who understands a data signal that the flow current is expected to be absent in the captured images and generate a indicative alert, in case the parameter entered by the user and the captured image are not compatible.
[0002]
2. System according to claim 1, characterized by the fact that it additionally comprises a support platform.
[0003]
3. System, according to claim 2, characterized by the fact that the system is configured to automatically align the second support platform with the determined spatial position of the flow current.
[0004]
4. System according to claim 3, characterized by the fact that the automatic alignment of the container to the flow stream comprises: mapping the position of the flow stream in the detection field in an XY plane; mapping the position of the container in the plane XY; and make the position of the container compatible with the position of the flow stream in the X-Y plane.
[0005]
5. System according to claim 1, characterized by the fact that the processor is configured to automatically adjust one or more parameters of the flow cytometer based on the determined diameter of the flow cell nozzle orifice, in which the parameters of the flow cell flow cytometers are selected from the group consisting of hydrostatic pressure, coating fluid pressure, flow current charge, bypass voltage, drop conduction frequency, charge correction value, drop delay, drop amplitude and charging phase.
[0006]
6. System according to claim 1, characterized by the fact that the parameter entered by the user comprises a data signal that the flow current is directed to a waste receptacle without forming a flow current in the detection field.
[0007]
7. Method for adjusting one or more parameters of a flow cytometer, characterized by the fact that it comprises: capturing one or more images of a flow current of flow cytometer in a detection field; determine one or more properties of the flow current in the detection field; generating a data signal corresponding to one or more properties of the flow stream; and adjust one or more parameters of the flow cytometer in response to the data signal, where the method comprises any of the following steps: a) determine the spatial position of the flow current in the detection field and generate a data signal corresponding to the spatial position of the flow current based on data obtained from a system comprising: a first imaging sensor configured to detect a flow current position in a first detection field and generate a first signal representative of the current position in the first field detection; a second imaging sensor configured to detect the position of the flow current in a second detection field that is substantially orthogonally oriented in an XY plane from the first detection field and generate a second signal representative of the current position in the second field of detection detection; a first support platform comprising a laser and operationally coupled to the first and second imaging sensors, in which the first platform is configured to automatically adjust positions in the X-Y plane in response to the second signal, in addition to a first signal; and a second support platform comprising a collection container and configured to adjust positions on the XY plane in response to the first signal; b) determine the width of the flow stream from one or more images and generate a data signal corresponding to the width of the flow stream, compare the determined width of the flow stream with the width of the flow stream that is expected based on the size of the orifice of the flow cell nozzle and generate a data signal corresponding to a ratio between the determined width of the flow current and the width of the flow current which is expected based on the size of the orifice of the flow cell nozzle flow; and c) determine the presence or absence of the flow current in the captured images, evaluate a flow cytometer parameter that is inserted by a user who understands a data signal that the flow current is expected to be absent in the captured images and generate a indicative alert, in case the parameter entered by the user and the captured image are not compatible.
[0008]
8. Method according to claim 7, characterized in that the method additionally comprises automatically aligning the second support platform based on the determined spatial position of the flow stream.
[0009]
9. Method, according to claim 8, characterized by the fact that the alignment of the support platform to the spatial position of the flow current comprises: mapping the position of the flow current in the detection field in an X-Y plane; map the position of the container in the X-Y plane; and make the position of the container compatible with the position of the flow stream in the X-Y plane.
[0010]
10. Method according to claim 7, characterized by the fact that the parameter entered by the user comprises a data signal that the flow current is directed to a waste receptacle without forming a flow current in the detection field.
[0011]
11. Method for adjusting one or more parameters of a flow cytometer, characterized by the fact that it comprises: injecting a sample into the sample port of a flow cytometer, in which the flow cytometer comprises a system comprising a processor with memory operationally coupled to the processor in which the system is automated to: capture one or more images of a flow cytometer flow stream comprising the sample in a detection field; determine one or more properties of the flow current in the detection field; generating a data signal corresponding to one or more properties of the flow stream; and adjusting one or more flow cytometer parameters in response to the data signal, where the processor is configured to automatically adjust one or more flow cytometer parameters in response to the data signal, and where the method comprises any of the following steps: a) determine the spatial position of the flow current and generate a data signal corresponding to the spatial position of the flow current based on data obtained from a system comprising: a first imaging sensor configured to detect a current position flow in a first detection field and generate a first signal representative of the current position in the first detection field; a second imaging sensor configured to detect the position of the flow current in a second detection field that is substantially orthogonally oriented in an XY plane from the first detection field and generate a second signal representative of the current position in the second field of detection detection; a first support platform comprising a laser and operationally coupled to the first and second imaging sensors, in which the first platform is configured to automatically adjust positions in the X-Y plane in response to the second signal, in addition to a first signal; and a second support platform comprising a collection container and configured to adjust positions on the XY plane in response to the first signal; b) determine the width of the flow stream from one or more images and generate a data signal corresponding to the width of the flow stream, compare the determined width of the flow stream with the width of the flow stream that is expected based on the size of the orifice of the flow cell nozzle and generate a data signal corresponding to a ratio between the determined width of the flow current and the width of the flow current which is expected based on the size of the orifice of the flow cell nozzle flow; and c) determine the presence or absence of the flow current in the captured images, evaluate a flow cytometer parameter that is inserted by a user who understands a data signal that the flow current is expected to be absent in the captured images and generate a indicative alert, in case the parameter entered by the user and the captured image are not compatible.

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法律状态:
2018-11-13| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-12-10| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-04-22| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-10-06| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 11/04/2014, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201361811465P| true| 2013-04-12|2013-04-12|

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US61/811,465|2013-04-12|

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PCT/US2014/033835|WO2014169231A1|2013-04-12|2014-04-11|Automated set-up for cell sorting|

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