• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    An insert member for use in a corrugated sheet, wherein the insert member is provided with a mounting member for mounting to a recess of a corrugated plate, which ensures that the insert member in the inserted state protrudes into the recess. The insert member is further provided with a meniscus balance element which, when inserted, balances a meniscus on the surface of a liquid by displacing surface portions of the liquid.
    公开号:AT510898A1
    申请号:T1985/2008
    申请日:2008-12-19
    公开日:2012-07-15
    发明作者:
    申请人:Univ Graz Med；
    IPC主号:
    专利说明:

    .1
    .1 I
    At Medical University of Graz
    I
    Re: Number of business 4ΒΑ1985 / 2008 -1 j Graz, 5.11.09 r
    Submission of the translation according to §91a PatG.
    Based on the priority application EP 07025051.9-2113 MEDICAL UNIVERSITY OF GRAZ j
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    University Square 3 j j
    8010 Graz I
    I i Austria | ! i
    Meniscal Equilibration System for a Microtiter Plate
    I i
    The invention relates to an insert element for a | Well plate.
    The invention further relates to a Wellplat ^ en device.
    The invention further relates to an operating method for the corrugated plate device. j
    The invention also relates to a method of using corrugated plate devices for imaging live cells. !
    A microtiter plate, well plate or microplate is a flat plate with a certain number of wells that take the place of small test tubes. Microplates are standard equipment for analytical research and clinical diagnosis. One area of application of microtiter plates are live images of living cells. A microtiter plate may have 6r 24, 96, 384, 1536 or more wells arranged in a matrix. Each well usually contains a quantity of fluid from a few thousand to a few hundred
    Microliters or even less. ; | US Pat. No. 6,255,911 describes a cell culture vessel and microscope which permits a wide range of favorable observations of probes in several wells, even if a negative refractive index arises in the culture medium. Here, an annular aperture, a condenser, a lens array, a corrugated plate, a lens, and a phase contrast plate are arranged in this order from the light source side, and provide a phase contrast phase phase contrast recording device in association with the position of the projection aperture or the projection aperture of the objective. The cell culture vessel is constructed from the lens assembly and the corrugated plate such that there is a relative disposition in which the optical axis of each lens is coaxial with the central axis of the beam path of each well. The aperture of the annular opening is less than the aperture (the annular opening used when the cell culture vessel is not in the beam path.
    SUBSEQUENT
    Alfred Bahnson et al. (in: Automated measurement of cell motility and proliferation, BMC Cell Bio. 2005, 6, 19) report that the optical properties of inverted light microscopy of 384-well plates is problematic in the context of robust living cell segmentation. The meniscus prevents phase contact, bright field microscope images have low contrast values when meniscus is applied, and the processing complexity increases considerably. As a result, a set of filters is used to measure the differences (signal-to-noise ratio) between the background and the foreground! (cell-like objects) to enlarge, and steps are taken to separate zeliähnliche objects clearly from the background. Heterogeneous illumination of the image is achieved by means of local histogram equalization. Brightness deviations between individual images (in terms of time) are compensated by means of histogram matching. Deviations in the area of the background are reduced by means of anisotropic filters and median fittering, whereby details and texture of the cells are preserved. Finally, in bright field microscopy through the cytoplasmic membrane a fusion of the cell boundary with the background results, whereby a series of elaborate gradation variants and texture filters must be used to make the cell boundaries visible. i
    I, Basics of Light Microscopy & Imaging, Special Edition of Light Microscopy & Imaging 'published by Olympus, Git Publishing (download at http://www.eitverlae.com/media/downloads/Qljfmpus Special Issue.pdf) reported on page 25 that when observing cells in a chamber, for example a 24 -Well plate, the use of special Olympus PHC contrast inserts was needed to achieve a better contrast in multiwell plates with meniscus problem.
    Despite the use of such balancing operations, the readout of individual wells of a corrugated plate remains cumbersome, especially when mesenteric problems occur.
    The aim of the invention is a corrugated plate system, which can be read out with sufficient accuracy even when meniscus problems occur.
    In order to achieve the above object, there are used a corrugated plate insert element, a corrugated plate device, a corrugated plate device assembling method, and a corrugated plate advancing method for living cell imaging. i
    With reference to an exemplary device of the invention, it will be apparent that an insert element
    I (or adapter) is provided for retraction into the recess of a corrugated plate, wherein the insert element has a mounting element (eg a Ve ¬ climbing element), with which the insert element can be secured to the corrugated plate, that the insert element after mounting in the Recesses protrudes and the mejlosis balancing system, which after insertion of the insert element ensures displacement (reduction or complete equalization) of a meniscus at the surface of a liquid in a well by displacing surface portions of the liquid, becomes effective. j
    According to another device of the invention, it can be seen that a corrugated plate assembly consisting of a corrugated plate (eg a microtiter plate) with at least one
    POSSIBLE 4 · * *
    4 * * * I
    t i
    I
    Recess {e.g., a plurality of recesses) and an insert having the above-mentioned properties for insertion into corrugated sheets is possible.
    Another device of the invention provides a method of using wafer plate devices. According to this method, an insert element is lowered into a depression of a corrugated plate, a mounting element is mounted on the insert element in the region of the depression such that after attachment to the depression a meniscus on the surface of a liquid is displaced by the use of the meniscal balancing system in that a surface portion of the liquid is displaced by the insertion of the system.
    In another device of the invention, the use of the corrugated plate device for imaging living cells becomes possible.
    According to another device of the invention, there is the possibility of inserting a recess into a well plate of a microtiter plate which, when placed in a sample-filled well, displaces a surface portion of the liquid and thereby forms a meniscus (ie the surface curvature of a liquid which results from the interaction with the container surface or other object adjacent to the liquid, which curvature may be convex or concave) on the surface of a liquid, or compensates entirely. Between the bottom surface of the insert element and the surface of the liquid, a planar surface portion is formed, which generates defined conditions without optical impairments due to scattering or refractive effects due to the meniscus. Thus, accurate readout of biochemical experiments in one well is possible, and this essentially applies to the entire well range. In addition to the meniscus balancing function, by covering the sample surface, the insert enables evaporation of the sample liquid to be avoided, allowing sample analysis over a long period of time. In addition, defined measurement conditions for parallel measurements can be determined on several samples in individual wells of the well plate. Measurement errors resulting from different sample volumes in different wells can be avoided because the insert elements can shift significant sample volumes, resulting in identical effective sample volumes for the measurements. In addition, the fair is also possible at very small sample volumes, since the f
    Distance between the bottom of the Einsatzelemerjtes and the bottom of the recess can be chosen very small, while a flat surface is ensured even with very small sample volumes. j i
    In addition to the possibility of reducing or avoiding meniscus effects, devices of the invention have the advantage of ensuring defined measurement conditions. For example, different measurement heights in the different wells of the corrugated plate can be compensated. Thus, different sample quantities in the different wells can be compensated. By covering the surface of the liquid samples with the insert, evaporation effects on the samples can be prevented thereby increasing the possible observation time. Another advantage of possible devices of the invention is that it is possible to observe / measure even very small amounts of sample since the i
    Distance of the bottom of the insert to the bottom of which recess can be reduced to very low heights. For this purpose, the corrugated board insert elements can be provided with a transparent underside, which represents a mechanical barrier.
    FOLLOW-UP ι:% · In the following, further combination possibilities of the insert elements are explained. These combinations can also be used with the corrugated plate devices and the methods. ι
    The mounting member (which may act as a flange and / or anchor) may include a round member (e.g., circular) provided so as to be mounted on a flat surface of the corrugated sheet directly around a depression. By means of supports between the flat surface of the ring of the mounting element and the annular flat surface of the corrugated plate, an exact horizontal orientation of the insert element or an orientation relative to the recess can be produced. This results in a sophisticated spatial alignment system without much effort.
    The mounting element can be provided with a latching element (which is also called anchoring element) which mechanically couples with a latching counterpart in the region of a depression on the corrugated plate. With such a combination, the mounting member can be fixed (but removable) by two correspondingly shaped latching mechanisms on insert element and corrugated plate, whereby an ekakte geometric alignment between insert element and corrugated plate can be defined.
    In particular, the latching element may include at least one pin which engages in at least one corresponding complementary groove around a recess in a corrugated plate. Alternatively, the latching element can be equipped with at least one groove into which at least one pin engages in the region of a depression of a corrugated plate. As an alternative to pin and groove constructions, magnetic fasteners, snaps or other snap-in elements may also be used. With the use of pin-and-groove constructions, multiple pins, for example, three pins equidistant from each other along the snap-fit construction of an insert member with e.g. be arranged in a circular cross section to achieve increased precision of the anchoring or latching mechanism.
    The mounting member for mounting the insert member to a recess of a corrugated plate may be adapted to prevent rotational or rotational movement of the insert member relative to the corrugated plate when inserted. By defining a precise angle geometry, unwanted rotation between the insert element and the corrugated plate can be prevented. This is advantageous in combinations where a particular alignment between insert member and corrugated sheet is required, for example, when vertically aligned polarizing filters are mounted in the bottom plate of an insert member and in the corrugated sheet to provide a suitable contrast ratio for the observation of one inserted into the recess To get a sample.
    Likewise, the mounting element can be designed so that it allows rotational or rotational movements of the insert element relative to the corrugated plate. Allowing a rotation of the insert relative to the corrugated plate fm inserted state allows to adapt the spatial characteristics of a corrugated plate assembly in a user-specific manner.
    The meniscus balancing system may have a pliant bottom surface fraction associated with a liquid (or other) sample in the well. Through such a planar surface can
    SUBSEQUENT
    optical artifacts resulting from the curvature of surface portions of a liquid are suppressed. One or more optical / optoelectronic elements may be added to the meniscal compensation system. These may be filters (eg wavelength filters or polarizing filters), electromagnetic radiation sources (eg light emitting diodes, laser diodes, etc.), electromagnetic radiation detectors (z, eg a photodiode), electromagnetic radiation amplifiers (eg pigments of fluorescent particles around the electromagnetic radiation in one) to amplify or generate a specific wavelength), optical fibers (to guide externally generated electromagnetic radiation into the starting element), and / or opening properties (eg, by an optically transparent portion surrounded by an optically nontransparent portion). In particular, optical elements (which diffuse the optical properties of a beam of light) through the insert element) or optoelectronic elements (which convert electrical signals into optical or vice versa) can be integrated into the meniscus balancing parts of the insert element which simultaneously displace meniscus and optical fibers / have optoelectronic functions.
    The meniscal compensation part may be partially or entirely optically transparent. To perform the observation, light can be directed to the upper part of the meniscus-outgrowth steeply when it is inserted into the recesses. The light may pass through the optically transparent portion of the meniscal compensation tab and the bottom of the corrugated plate and be observed below the corrugated plate by means of an electromagnetic radiation detector. i
    The insert member may include a connector with which the mounting member is connected (or connected) to the meniscal compensation member. Such a connecting element may have vertical walls and a tubular geometry to connect an annular anchoring element with disk-shaped meniscus balancing parts.
    The connecting element can be perpendicular to the fastening element and the meniscus compensation part; be aligned. For this purpose, a recess-like insert element of smaller dimensions than the recess element in which the insert element is to be used is used.
    The connecting element may contain an optically opaque part. Thus, e.g. an inner or outer wall of the connecting elements are executed in black color or provided with an opaque cover to exclude unwanted effects of ambient electromagnetic radiation on the operations in the field of use and depression. Likewise, the wall of the connector may be made partially or entirely of an optically diffused material (either by appropriate coating or surface treatment of the wall, e.g., by sandblasting or scavenging).
    The insert element can be produced in particular from polycarbonate and / or polymethylmethacrylate (PMMA). These or other polymeric materials may be advantageous because they can be produced inexpensively, have favorable optical transparency values and are mechanically and chemically resistant. However, other materials such as e.g. Glass to be used.
    The insert element can be produced by injection molding. As a result, the
    Insert element of one piece or be made entirely of a material. Thus is
    NACHGERBCHT ensures that the insert elements can be produced economically. The insert elements can also be composed of different components. It may be an insert, e.g. be made of a disc with the function of meniscus compensation and a tubular body with annular mounting ring as fastening and connecting part. These two components (each with their own optimal properties) can then be glued together (e.g., by means of UV glue), welded or soldered. The bottom plate of the insert element may e.g. glued to a tubular side wall element. The insert element may also be made of a single material. In the sequel further device possibilities of the corrugated plate application are explained. These combination options relate equally to the inserts and the methods.
    The smallest recess amount of a corrugated plate may be one. In such a case, a recess and an insert member may form a mounting block. It can also be arranged more wells linearly in a Weliplatte. Thus, the corrugated plate may have a strip-shaped grid in which all depressions are arranged along a line. Furthermore, a plurality of wells in a matrix-like arrangement may take the form of a two-dimensional microtiter plate. Thus, 6, 12, 24, 96, 384, 1536 or more depressions can be arranged in such a one- or two-dimensional matrix.
    Likewise, one- or two-dimensional recess plates can be produced, which have perforations or other predetermined break points between the individual depressions and enable the user to cancel precisely the size of the strip or matrix desired for the particular test situation. Two-dimensional arrays can be divided into small two-dimensional or zero-dimensional arrays (i.e., a single indentation) to custom-separate, split, or isolate the required corrugated sheet assembly, just as a chocolate bar can be divided into individual ribs and pieces.
    In particular, the corrugated plate may take the form of a microtiter plate. By microtiter plate is meant a sample vessel as used in combinatorial chemistry, biochemistry, or high throughput assay methods. A microtiter plate or microplate may take the form of a flat plate having a plurality of wells that function as small test tubes.
    The one or more recesses may be shaped and dimensioned to allow insertion of that element into the recesses, matched to the shape and dimension of the insert. In this case, the size, dimensions and shape of the recesses and the inserts can be adapted to each other and matched. In this case, the insert element can be dimensioned so that ei either with play or strictly fits into the depression and a well-defined distance between recess wall and insert element complies, or have a distance-support, which determines the distance between the insert element and a wall of Weliplatte.
    The cross-section of the one (or more) recesses (s) can be chosen taking into account manufacturing tolerances that correspond to the cross-section of the insert element. In such a combination, when inserting the insert element remains in the
    SUBSEQUENT
    Recess no distance between the inner wall of the recess and the outer wall of the insert element.
    The corrugated plate device may include an electromagnetic radiation source (eg, a light emitting diode) directing electromagnetic radiation directly to the insert element, and may include an electromagnetic radiation detector (eg, a photodiode, charge coupled device (CCD) or CMOS camera) for observing electromagnetic beam (eg, an optical beam) after its irradiation of the insert element, the liquid sample between insert element and recess bottom and the bottom of the recess.
    The term "electromagnetic radiation " may refer in particular to a photon beam of a corresponding wavelength. This may refer to the optical spectrum (eg the range between 400nm and SOOnm) as well as the electromagnetic radiation of other wavelengths such as UV, infrared or even X-radiation. In other possible combinations of the invention, such electromagnetic radiation may be used as a measuring instrument as this electromagnetic radiation is radiated through the insert member inserted into a well of a corrugated plate, through the liquid in the well and through the corrugated plate and observed under the corrugated plate. The direction of the radiation can also be reversed. Thus, the readout of a corrugated plate analysis by suppression of undesirable meniscus effects can be made possible.
    The corrugated plate may be equipped with a first polarizing filter in the bottom of at least one recess. The meniscal compensation element of the insert element may be equipped with a second polarization filter. Thus, these filters of these two polarizing filters can e.g. In consequence, in the absence of any effects in the sample, no (polarized) light will penetrate the two polarizing filters arranged perpendicular to each other.
    Only in the case of optical activity in the sample: is the polarization of the light rotated or shifted so that bright portions become visible in the observation region of such an optically active sample. Thus, a desired bright-dark contrast can be generated.
    Any polymeric material can be used to make corrugated sheets, e.g. Polystyrene and / or poly-methyl methacrylate (PMMA). Such materials have good optical transmission properties, are environmentally compatible and cost-effective.
    In accordance with an exemplary apparatus, a well plate system for living cell observation can be created that can reduce or eliminate the difficulties usually associated with meniscus formation on the surface of liquid samples due to surface tensions of the liquids. This problem increases with decreasing scale size. Thus, it may be of particular concern, especially in high throughput observations (e.g., in pharmaceutical science), to address the meniscal problem. The invention offers possibilities for simple and at the same time highly efficient methods for avoiding the meniscal problem. Thereby, the observation of the total cross-section of all wells of a corrugated plate can be made possible because no unexplainable portions of the corrugated plate remain.
    SUBSEQUENT
    Observation in transmission is possible, while a light source above (or below) the Miktotiterplatte and the detector below (or above) be arranged- It is also possible to drive this system in reflection geometry, both light source and the detector on the same side of Well plate and insert element are arranged (ie both above or both below). Detection methods for fluorescent light are also possible.
    The observation of living cells and spectrophotometry (MTT, XTT), but also other applications in the field of biochemistry and other fields, can be carried out by means of the methods made possible by this invention. Another example of the use of devices of the invention is cell mechanics. In the process, signals can be observed that become visible through the effect of cellular forces on a surface. This can be done by two vertical polarizing filters in the bottom of a corrugated plate and in the meniscal compensation element of the insert plate. The measurement of Krafterhustern can be done by observing interference patterns that occur when cell forces affect the optical properties of the surface of a well.
    It is possible to perform surface functionalization at the bottom of the insert element and / or the bottom of a corrugated plate (e.g., functionalizing the surface with collagen to facilitate the association of cells in a liquid).
    The insert can be delivered sterile packaged to be unpacked just prior to use.
    The corrugated sheet and insert may be used as a mating set (e.g., as a kit), i. be attuned to each other.
    The bottom of the insert element may be optically transparent, while the sidewalls may be diffractively refractive or opaque. This may be achieved by coating, sandblasting, coloring / brushing O.a. respectively.
    In a 6 well plate, the sample volume can be 4ml to 5ml. In a 96 well plate the sample volume can be 300μΙ to 400pl. Other volumes in the picoliter, nanoliter, microliter or milliliter range are also possible.
    The thickness of the bottom plate of the insert element can be 170pm. These dimensions can be adapted to the lens properties of an optical system.
    A device option of the invention effects the correction of optical artifacts resulting from the refraction of light at menisci in phase-contrast microscopy. In phase contrast microscopy, anomalies are usually visible in experimental situations where menisci occur in a curve at the air-medium interface. Refractions at this interface result from diffraction of the light away from the phase ring and lead to poor contrast or loss of image in phase-contrast microscopy. The compensation of this diffraction requires a change at the interface in such a way that it has to be aligned parallel to the sample carrier. A design consisting of a coverslip in parallel alignment with the sample holder viewing window allows observation of the sample from a greater distance from the center of the well without causing distortion or loss of the image.
    POSSIBILITY In an exemplary device, a multiwell plate optical insert is used. Thus, the field of view in the microscopic evaluation of multiwell plates is increased and meniscus distortions are effectively corrected.
    An apparatus of the invention is designed for studies that require the observation of short-lived cells that allows observations over a period of three or more days. For this, the use of chambers with carbon dioxide atmosphere may be necessary. Such a device can be manufactured for six, two or more chambers.
    An insert element may, according to an exemplary device, create an improved field of view in a well such that microscopy (e.g., by phase contrast) may occur in monitoring experiments without the troublesome effects of meniscal curvature. The meniscus effect can be suppressed or prevented by optical plates in a plane parallel to the bottom of the well. In this case, very small depressions can be used and thus a large number of fields of view on a microplate or in a single recess are made possible. This can have the advantage that higher information can be obtained from a disk. By changing the optical properties of the optical disk, it is possible to ensure defined experimental conditions, e.g. in terms of refractive index, gas permeability, filter properties.
    Specific devices of the invention may be used for the observation of living cells, e.g. when using twelve or more culture wells. Insert members according to exemplary devices of the invention are inexpensive, simple, and readily adaptable to virtually any system or experiment.
    Devices of the invention may be combined with robotic systems and provide top or bottom-level observations.
    The above-mentioned aspects and further aspects of the invention will be apparent from the examples of possible devices and will be described and explained below in accordance with these device examples.
    The invention will be explained in more detail below with reference to the exemplary devices, but the invention is not limited to these examples.
    Figs. 1 to 3 illustrate a corrugated plate device according to exemplary devices of the invention.
    Fig. 4 shows photographs of observations on living cells.
    Fig. 5 is a diagram for increasing the undisturbed observation field using exemplary devices of the invention.
    Figs. 6-10 show various views of the harvesting element according to an exemplary apparatus of the invention.
    Fig. 11 shows a microtiter plate system according to an exemplary device of the invention.
    Fig. 12 shows various views of an insert according to an exemplary apparatus of the invention. REPLACED |
    Fig. 13 shows various views during observation of living cells in a conventional arrangement and arrangement according to an exemplary apparatus of the invention.
    14 shows a typical well plate situation,
    The illustration in the figures is schematic. In different illustrations, similar or identical elements are given the same reference characters.
    Figure 1 illustrates a corrugated plate device 100 corresponding to an exemplary device of the invention.
    The corrugated plate device 100 consists of a corrugated plate 102 having a number of wells 104.106 μSW of ordinary plasticized substrate. The corrugated plate 102 may be made of polystyrene.
    Each recess 104, 106 has its own insert element 108, 110, which is inserted into the corresponding recess 104, 106 of the corrugated plate 100.
    The corrugated plate 102 is shaped as a microtiter plate. As can be seen in FIG. 1, the recesses 104, 106 are shaped and dimensioned to correspond to the shape and dimension of the insert elements 10S, H0, thus enabling insertion of the insert elements 108, 110 in the corresponding recesses 104, 106.
    A movable (for interrogating the corrugated sheet 100 see arrows 112) light source 114, e.g. a light-emitting diode or a laser causes electromagnetic radiation directed to the insert elements 108, HO. A CCD detector 1106 is mounted below the corrugated sheet 102 for observing the electromagnetic radiation after its propagation through the corresponding insert member 108, 110, through the recess 104, 106, through the liquid sample 110B in each recess 104, 106 and the bottom plate of the bottom plate 102. A tuned detector element / pixel of the CDD detector 116 observes the electromagnetic radiation beam.
    In the following, the tongue member 108 will be described in more detail. The type of insert HO is similar or the same.
    The insert 108 has been adapted for insertion into the recess 104 in the wafer plate 102 and consists of a fastener 120. in the form of a ring with protruding portions 122 for mounting in correspondingly shaped grooves in the surface of the corrugated sheet 102. The latching devices 122 with the Corresponding Nutrillen the rotation of the insert member 108 is prevented and ensures a fixed spatial arrangement of the two parts to each other. The fastener 120 is adapted to allow the insert member 108 to be mounted to the recess 104 so as to project into the recess 104 after mounting to the recess 104.
    A meniscus displacement member 124 is part of the bottom of the insert member 108 and is designed to displace a meniscus at the surface of a liquid H8 in a depression 104 by displacing a surface portion of the liquid 124 once inserted into the depression 104. From Fig. 1 it can be seen that the Bodenfiäche 126 of
    SUBSEQUENT
    Meniscus displacement element 118 is planar. The meniscal displacement element 124 is optically transparent. A vertical connecting wall 130 connects the meniscus displacing element 124 to the fastener 120 and may be optically diffused or blackened to avoid crosstalk with the adjacent recesses 104, 106.
    The insert 108 is made by injection molding of a part and is made of polycarbonate.
    Fig. 2 shows a corrugated plate assembly 200 according to another exemplary apparatus of the invention.
    FIG. 2 shows a depression 104 with a meniscus compensation element 206. From FIG. 2, a useful observation field 202 can be seen that extends over almost the entire cross section of the depression 104. On the sides, an unusable observation field 204 necessary for gas exchange surfaces can be seen, which is smaller compared to conventional corrugated plate systems. FIG. 3 illustrates a corrugated plate assembly 300 according to another exemplary apparatus of the invention.
    Figure 3 illustrates a corrugated plate assembly 300 having a meniscal compensation element 306. On the sides, the useful field of view 202 is larger than in conventional systems. The arrows 204 show the unusable field of observation in the area of the sidewalls of the insert element 306. In the device of Fig. 3, the connecting wall 130 closes close to vertical walls of the corrugated plate 202. Fig. 3, with reference numeral 302, shows a curved surface of a gel 304 in which cells grow and move. In Fig. 4, the illustrations 400 can be seen.
    Cells cultured in and on gel in phase contrast are difficult to analyze in 96 well plates (see A). The depression is small and the meniscus has a disturbing effect on the phase contrast over the entire observation field (see A, C). Using the device from FIG. 3, an expansion of the observation field can be effected, whereby almost the entire depression cross-section for phase contrast Studies can be used (see B, D).
    5 is a plot 500 with an abscissa 502 along which the number of pits per plate is plotted. Along the ordinate 504 an increase in percent is plotted. The first curve 506 corresponds to the properties of the device of FIG. 2. The second curve 508 shows the characteristics of the device of FIG. 3.
    Multiwell plates can be of various sizes, e.g. 6,12,24,96 etc. Due to the meniscus formation on the surface of liquids, the readable area of the field of view, e.g. reduced in phase-contrast microscopy.
    Table 1 represents the maximum useful diameters of observation screens in relation to pit diameters.
    Groove Diameter BF normal BF Fig. 2 Rise (%) BF Fig. 3 Rise (%) of the depression later12 12mm 12,6 18 43 20 59 24 15,6mm 6,2 11,6 87 13,6 119 96 6,1 mm 2.2 220 3.7 370
    Table 1; Increase in the useful field of observation for different well sizes.
    Two devices (Figures 2, 3) and the rise in the field of view (BF) are shown for different pit sizes, the device of Figure 3 significantly increasing the field of view and resulting in a 24 well plate doubling the field of view. Both devices Fig. 2 and Fig. 3 allow by their construction the gas exchange with the culture medium.
    FIGS. 6 to 10 show various views of an insert 600 according to an exemplary apparatus of the invention;
    FIG. 6 shows a plan view of the insert element 600, FIG. 7 shows a three-dimensional representation 700. FIG. 8 shows a section 800. FIG. 9 shows a further section 900. FIG. 10 shows the dimensions of the insert element 600.
    11 shows image 1100 of a 6-well microtitre plate 1102 1104. FIG. 11 further shows the insert 1106 for use in the corresponding wells 1102, 1104. As can be seen from Fig. 11, the illustrated construction can be applied to all dimensions of multi-well plates or other containers which require correction of the optical interface if the reduction in refractive anomalies is necessary or desired. In this case, the loss of the phase contrast can be corrected by correcting the curvature of the meniscus with a resulting optical section parallel to the observed surface. This is described in more detail in FIG.
    Fig. 12 shows various views of an insert element 1200 with tubular plastic body 1202, an optical plates ll204 and mounting pins 1206. The construction consists of the plastic tube 1202 with the optical disc (cover glass) 1204 closed on one side. The tube 1202 stands on three feet 1206 to ensure parallel alignment of the coverslip with the observation surface. The construction is smaller than the well in which it is used to ensure the gas exchange between the liquid medium and the surrounding gas atmosphere for the growth of the cells in the medium.
    Fig. 13 shows conventional phase contrast microscopy 1300. In addition, Fig. 13 shows phase scanning microscopy using a device of the invention with reference number 1310.
    Fig. 13 shows a multiwell plate 1302, a liquid medium 1304, a microscope objective 1306, and a surface 1308 to be observed. The left half of Fig. 13 shows a diffraction interface 1310 in the region of the meniscus and a resulting diffraction direction 1312-The right half FIG. 13 shows the insert element 1200 with support legs 1203 placed on a recess base.
    SUBSEQUENT
    Fig. 13 illustrates the effect of the device of the invention. The figures provided with the reference number 1300 show the results without the use of a device of the invention. The left bank of figures of Fig. 13 along the ascending direction is provided with corresponding drawings representing the increasing deviation from the central axis. An increasing deviation from the central axis results in an increase of the relative angle between the surface of the meniscus and the surface in the region of the central axis, in row 3 a difference due to the diffraction at meniscus 1310 is visible. This can lead to an increase in intensity, since parts of the surrounding light can not pass through the phase ring diaphragm. In row 2, a complete loss of image and phase contrast has occurred in the left image, since no light at all could pass through the phase ring diaphragm, whereas in the right imaging column, only a small change can be seen. The change results from a restricted by the construction passage of the ambient luminescence and a subsequent decrease in light intensity and thus to a reduction in the mode of action of the phase contrast. The south on the right row of images is still readable, but the intensity has decreased because the construction depicts the light outside the field of observation.
    The images of Figure 13 thus show that the device can significantly increase the number of images obtained from a single experiment, and for this the use of a plurality of wells is not necessary. This also reduces the need for medium, lines and test reagents. This simple, cost-effective and easily adjustable device can simplify volt-automated analysis without the need for complex optical adjustments.
    Figure 14 shows a conventional wafer 1400 having a liquid 1402 and a meniscus 1404. The field of view 1406 is small and the unusable portions 1408 are large.
    It is noted that the term includes' does not exclude other elements or components and the indefinite article 'a' or 'an' does not exclude that a plurality of elements or components may be meant. Furthermore, the devices described may also occur in conjunction with other various devices. It should also be noted that the recited reference numbers in the claims do not affect the scope of the claims. FOLLOW-UP: T {
    权利要求:
    Claims (25)
    [1]
    Claims: 1. An insert element for use in a recess of a corrugated sheet, the insert member includes a mounting member for mounting the insert member to a recess of a corrugated plate so that the insert member in the inserted state protrudes into the recess, a meniscus Ausgeichs part for balancing a meniscus on the surface of a liquid in a well by displacing surface areas of the liquid when the seed is inserted into a depression.
    [2]
    2. The insert element of claim 1, wherein the mounting member has an annular portion which is directed for attachment to a planar portion of the corrugated plate around a recess.
    [3]
    3. The insert of claim 1 or 2, wherein the mounting member includes a latching device for mounting to the corresponding latching device on the corrugated plate around a recess.
    [4]
    4. The insert of claim 3, wherein the latching device includes at least one pin that can be guided in the corresponding Nutrille a corrugated plate in the area around a recess.
    [5]
    5. The insert member of claim 1 or any preceding claim, wherein the mounting member is directed to the mounting of the insert member to the recess of a corrugated plate, that rotational movements of the insert element are prevented relative to the corrugated plate in the inserted state.
    [6]
    6. The insert member of claim 1 or any preceding claim, wherein the mounting member is so directed to the mounting of the insert member to the recess of a corrugated plate, that rotational movements of the insert element are made possible relative to the corrugated plate in the inserted state.
    [7]
    7. The insert member of claim 1 or any preceding claim, wherein the meniscus balancing member has a planar bottom surface facing the liquid in the recess.
    [8]
    8. The insert member of claim 1 or any preceding claim, wherein at least one optical and one opto-electronic element are integrated into the meniscus balancing member.
    [9]
    The insert element of claim 8, wherein the optical or opto-electronic element comprises one of: a filter, a wavelength filter, a polarizing filter, an electromagnetic radiation source, an electromagnetic radiation detector, an electromagnetic radiation amplifier, an optical fiber, an aperture , a photodiode, a light-emitting diode, a wire, a laser diode. SUBSEQUENT
    [10]
    The insert of claim 1 or any preceding claim, wherein the meniscus displacer is optically transparent.
    [11]
    11. The insert element of claim 1 or any preceding claim, in connection with a connection part for the connection of the mounting member with the meniscus displacement Elemeni.
    [12]
    12. The insert of claim 11, wherein the connector is aligned perpendicular to the mounting element and perpendicular to the meniscal compensating element.
    [13]
    13. The insert element of claim 11 or 12, wherein the connection element contains one of the following: an optically opaque portion or a diffusely scattering portion.
    [14]
    14. The insert of claim 1 or any preceding claim, wherein it contains one of the following materials: polycarbonate or polymethyl methacrylate
    [15]
    15. The insert element of claim 1 or any preceding claim made by injection molding.
    [16]
    16. A world plate assembly, wherein the corrugated plate assembly consists of a whiteboard having at least one recess and an insert member for insertion into the recess of a corrugated plate according to claim 1 or any preceding claim.
    [17]
    17. A corrugated plate assembly as claimed in claim 16, wherein the whiteboard is or consists of the following: a single well, a plurality of wells in a linear array, a plurality of matrix wells, 6 wells, 12 wells, 24 wells, 96 wells, 384 wells and 1536 wells.
    [18]
    18. A WeKplattenanordnung of claim 16 or 17, wherein the Weiipiatte is a microtiter plate.
    [19]
    19. A corrugated plate assembly as claimed in claim 16 or any preceding claim, wherein at least one recess is shaped and dimensioned to conform in shape and dimension to an insert member so that the insert member can be inserted into the recess.
    [20]
    20. A corrugated plate assembly according to claim 16 or any preceding claim, wherein the cross section of the at least one recess corresponds to the cross section of the insert element.
    [21]
    21. A corrugated plate assembly according to claim 16 or any preceding claim, comprising an electromagnetic radiation source by means of which an electromagnetic beam can be directed onto the insert element; REPLACED consisting of a detector for electromagnetic radiation, by means of which an electromagnetic beam can be observed after its propagation through the insert element and the at least one depression.
    [22]
    22. A corrugated plate assembly as claimed in claim 16 or any preceding claim, wherein the corrugated plate includes a polarizing filter in at least one recess; wherein the corrugated plate includes a first polarizing filter in at least one recess; wherein the meniscal compensation element of the insert element includes a second polarizing filter.
    [23]
    23. A corrugated plate assembly according to claim 22, wherein a first polarizing filter and a second polarizing filter are arranged or arranged so as to be perpendicular to each other.
    [24]
    24. A corrugated plate assembly according to claim 16 or any preceding claim, wherein the corrugated sheet consists of one of the following materials: polystyrene and Polym ethoy I methycry lat. 2s. An operation method of a corrugated plate assembly, the method including an insert member for a well of a corrugated plate. a mounting element of the insert element on the recess by means of which the insert element protrudes into the recess of a corrugated plate after assembly, in which the meniscus is compensated on the surface of a liquid, by the meniscus compensation element of the insert element a Oberflächenantei! the fluid displaced in a recess when it is inserted into the recess.
    [25]
    26. An operation method of a corrugated plate assembly of claim 16 or any preceding claim for living cell imaging. REPLACED [1/5

    III IH | l ΠΠΙΠΎΠΤΠ /

    fao ί

    • J.



    SUBSEQUENT





    SUBSEQUENT

    REQUIRED f Claims: 1. An insert element for use in a well of a corrugated plate for living cell observation, in which a permanent gas exchange is ensured, the insert element includes a mounting member for mounting the insert element to a recess of a corrugated plate so that the insert element in the inserted state protrudes into the depression, a meniscus-balancing part for the compensation of a meniscus on the surface of a liquid in a recess by the displacement of surface portions of the liquid when the insert element is inserted into a recess. 2. The insert element of claim 1, wherein the mounting member has an annular portion which is directed for attachment to a planar portion of the corrugated plate around a recess. 3. The insert element of claim 1 or 2, wherein the mounting member includes a latching device for mounting to the corresponding latching device on the corrugated plate around a recess. 4. The insert element of claim 3, wherein the latching device comprises at least one pin which can be guided in the corresponding Nutrille a corrugated plate in the area around a recess. 5. The insert element of claim 1 -4, wherein the mounting member is directed so for the mounting of the insert member to the recess of a corrugated plate, that rotational movements of the insert element are prevented relative to the corrugated plate in the inserted state. 6. The insert element of claim 1 -4, wherein the mounting member is directed so for the mounting of the insert member to the recess of a corrugated plate, that rotational movements of the insert element are made possible relative to the corrugated plate in the inserted state. The insert member of claim 1 or any preceding claim, in connection with a connector for connecting the mounting member to the meniscal displacement member. 8. The insert element of claim 7, wherein the connecting wall contains one of the following: an optically opaque portion or a diffusely scattering portion. 9. The insert element of claim X to 8, produced by injection molding, in which the optical element is already cast in the first manufacturing step and thus the entire device consists of a material. 10. A corrugated plate assembly wherein the corrugated plate assembly consists of a corrugated plate having at least one recess and an insert member for insertion into the well of a corrugated plate according to claim 1 or any preceding claim. 11. A corrugated plate assembly as claimed in claim 10, wherein the corrugated sheet consists of or includes: a single well, a plurality of wells in a linear array, a plurality of wells in a matrix arrangement, six wells, twelve wells, twenty-four wells; 96 wells, 384 wells and 1536 wells, 12. A corrugated plate assembly of claim 10 or 11, wherein the world plate is a microtiter plate. 13. A corrugated plate assembly as claimed in claim 10 or any preceding claim, wherein at least one recess is shaped and dimensioned to conform in shape and dimension to an insert member so that the insert member can be inserted into the recess. 14. A corrugated plate assembly according to claim 10 or any preceding claim, wherein the cross section of the at least one recess corresponds to the cross section of the insert element. 15. A corrugated plate assembly as claimed in claim 10 or any preceding claim, comprising an electromagnetic radiation source (a light source, preferably a lamp) by means of which an electromagnetic beam (eg visible light) can be directed onto the insert element; and consisting of a detector (for example a camera) for electromagnetic radiation, by means of which an electromagnetic beam can be observed after its propagation through the insert element and the at least one depression. 16. A corrugated plate assembly as claimed in claim 10 or any preceding claim, wherein the corrugated plate includes a polarizing filter in at least one recess; wherein the corrugated plate includes a first polarizing filter in at least one recess; wherein the meniscal compensation element of the insert element includes a second polarizing filter. 17. A corrugated plate assembly according to claim 16, wherein a first polarizing filter and a second polarizing filter are arranged or can be arranged to be perpendicular to each other. 18. A corrugated plate assembly according to claim 10 or any preceding claim, wherein the corrugated plate is made of one of the following materials: polystyrene and polymethyl methacrylate. 19. A method of handling a corrugated plate assembly, the method including an insert member for a well of a corrugated plate. rrr: m -IT a mounting member of the Einsauelementes on the recess by means of which the insert element after installation projects into the recess of a corrugated plate in which the meniscus is compensated on the surface of a liquid by the meniscus-balancing element of the insert element has a surface portion of the Fluid displaced in a well when inserted into the well. A method for handling a corrugated plate assembly of claim 10 or any of the preceding claims for live cell imaging. i FOLLOWED > - *. ·
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    同族专利:
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    法律状态:
    2016-08-15| MM01| Lapse because of not paying annual fees|Effective date: 20151219 |
    优先权:
    申请号 | 申请日 | 专利标题
    EP07025051|2007-12-21|
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