• 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:
    The present invention concerns a fluorescence light microscope, comprising: - a target (6); - a support (10) to support a sample; - lighting means for projecting onto a sample a periodic lighting pattern in two transverse directions orthogonal to each other, from four point light sources; - a scanning system (7) to provide a relative displacement of the sample with respect to the illumination pattern; and - imaging means, comprising: - a light detector for sequentially detecting fluorescent light emitted by different portions of the sample, in respective scanning positions; and - means for forming an image from the light intensity existing in the fluorescent light detected by the light detector. The present invention also concerns a method adapted to carry out the operative steps of the microscope of the invention. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2768448A1
    申请号:ES201831272
    申请日:2018-12-21
    公开日:2020-06-22
    发明作者:Ortiga Emilio Sanchez；Tortosa Genaro Saavedra；Corral Manuel Martinez；Picabea Jorge Sola
    申请人:Universitat de Valencia；
    IPC主号:
    专利说明:

    [0002] Fluorescence light microscope and fluorescence light microscopy imaging method
    [0004] Technical sector
    [0006] The present invention concerns, in a first aspect, a fluorescence optical microscope, which combines a structured illumination according to a determined pattern with a scanning of the sample, to obtain optical sectioning and super-resolution without the need to apply any post-processing operation to the images obtained or rotate the lighting pattern.
    [0008] A second aspect of the present invention concerns a method for obtaining fluorescence light microscopy images adapted to perform the operating steps of the microscope proposed by the first aspect of the invention.
    [0010] State of the prior art
    [0012] The resolving power of optical systems in general and of microscopes in particular is limited by diffraction. In conditions of sample illumination and detection of light emitted or reflected by it that can be called conventional (uniform illumination and detection by means of a sensor located in the focal plane of the system image), the limit given by the diffraction is well known and can be expressed by the following equation:
    [0015] A r = 0.6 ----
    [0017] where l is the wavelength of the light emitted or reflected by the sample and NA is the numerical aperture of the microscope objective used. The distance Dr is the minimum separation at which two light emitting points could be in order to be distinguishable through the microscope.
    [0019] On the other hand, conventional microscopes are not capable of obtaining optical sections of three-dimensional samples, that is, for a focus position of the sample, the image will contain the corresponding image of the focal plane together with the unfocused light coming from the planes outside focus. This fact makes conventional images have little contrast, especially when high numerical aperture lenses are used since their depth of focus is very small.
    [0020] There are different systems capable of improving transverse resolution and obtaining optical sectioning. Optical sectioning, for example, can be obtained using a confocal scanning microscope. In this microscope, the sample is illuminated with a "spot" limited by diffraction and the light emitted or reflected by the sample is projected onto a pin of micrometer dimensions. The light that passes through the pin reaches a wide area detector and corresponds to one pixel of the confocal image. The light coming from the out-of-focus planes is blocked by the pin. To obtain an image it is necessary to sweep the sample. This type of microscope has optical sectioning capacity but does not improve the transverse resolution In addition, it has certain problems such as the bleaching of fluorescent dyes when having to concentrate light in a spot limited by diffraction.
    [0022] On the other hand, there are structured lighting systems that allow improvement in resolution and / or optical sectioning. To do this, they project an extensive illumination pattern onto the sample. Said pattern is typically a cosine generated by the interference of two plane waves. In order to obtain a super-resolved image, it is necessary to move the lighting pattern several times in addition to rotating it in different directions. In addition, it is necessary to apply a large number of post-processing computational operations in order to obtain the final image. These systems require a total of between 9 and 75 images that are combined by mathematical operations and require knowledge with high precision of the lighting pattern parameters. The results obtained with this type of systems depend highly on the post-processing operations, sometimes giving rise to artifacts in the image.
    [0024] In [1] and [2] microscopes are described that also allow obtaining super resolution and optical sectioning by complex post-processing operations of a large number of images of a sample (at least 15), also obtained by rotating (at least three times ) a periodic structured illumination pattern, only in a transverse direction and in the axial direction, that is applied to a sample. The images are obtained by means of a wide-field capture, that is to say that each of the images is a two-dimensional image composed of NxN pixels, and the post-processing operations involve the execution of a computational algorithm for the reconstruction that it needs, among other things. , detect the pattern frequency, phase shifts, the size of the optical transfer function and the direction of the illumination pattern. However, this method requires time to perform computational reconstruction since it is not obtained directly when capturing.
    [0025] Similarly, in [3] a microscope is proposed based on obtaining multiple two-dimensional images, also obtained by means of a wide-field capture, after rotating and laterally displacing an illumination pattern (periodic in a transverse direction and in the axial direction). ), and a complex image processing algorithm that can achieve high computation times.
    [0027] Therefore, it appears necessary to offer an alternative to the state of the art that covers the gaps found in it, through the proportion of a microscope that allows obtaining both a super-resolved image, that is to say of a high transverse resolution, and optical sectioning, without the need to apply any post processing operation or rotate the lighting pattern.
    [0029] Explanation of the invention
    [0031] To this end, the present invention concerns a fluorescence light microscope, comprising:
    [0033] - an optical microscope objective whose axis of symmetry, called the optical axis, defines a first direction or main direction of light propagation;
    [0035] - a support (such as a stage) to support a sample stained with a fluorescent dye, so that it is arranged in the focal plane object of the optical microscope objective, or in the axial range defined by the depth of field of said microscope objective optical;
    [0037] - structured illumination means configured and arranged to project onto a sample located on the support a periodic illumination pattern in two transverse directions orthogonal to each other, by generating and projecting on the aperture diaphragm of the optical microscope objective from at least four sources point light beams, spatially distanced from each other, that generate and provide at least four plane waves whose direction of propagation is equidistant angularly, or substantially equidistantly angularly (that is, with an angular deviation tolerance of ± 5%), of said first direction of the optical axis of the optical microscope objective, whose interference results in said illumination pattern;
    [0039] - a scanning system configured and arranged to provide a relative displacement of the support, and therefore of the sample supported by it, with respect to the illumination pattern, to adopt different scanning positions; and
    [0041] - imaging means, comprising:
    [0042] - at least one light detector configured and arranged on a conjugate plane of the target plane of the optical microscope objective, to sequentially detect fluorescent light emitted by at least two portions of the sample, in two respective of said scanning positions, by interacting with the light of the illumination pattern, collected through the optical microscope objective according to a second direction of the optical axis of the optical microscope objective, opposite to the first direction; and
    [0044] - imaging means configured and arranged to form an image of at least two pixels, from the existing light intensity in the fluorescent light detected by the light detector, which is at least one, for each of the at least two portions of the sample, one per pixel.
    [0046] For an exemplary embodiment, the light detector is an image sensor configured and arranged to sequentially detect, in a common detection area formed by one or more photodetector elements of the image sensor, the fluorescent light emitted by each of the two or more portions of the sample, the imaging means being configured and arranged to integrate the existing light intensity in the common detection area into an intensity value per scanning position, to form each pixel of said image. For an implementation of this exemplary embodiment, the image sensor is multipixel, where each pixel is made up of one or more of said photodetector elements.
    [0048] For an alternative embodiment, the light detector is a wide area detector, and the imaging means further comprise a pin configured and arranged, between the optical microscope objective and the wide area detector, so that the detector wide area sequentially detect the fluorescent light that passes through the pin and is emitted by each of the two or more portions of the sample, the imaging means being configured and arranged to form each pixel of the image with the respective light intensity values detected by the wide area detector for each scanning position.
    [0049] In any case, the detection carried out by means of the light detector of the microscope of the first aspect of the present invention is a detection that can be denominated as of the point type, either by means of a pin or using a few pixels of a image sensor, so that in each capture the image of only one point is registered, understanding as "point" the one associated with each value of light intensity obtained by scanning position, either by integrating the existing light intensity in the detection area common, for the example of realization that it includes an image sensor, or directly the light intensity value detected by the wide area detector for each scanning position, for the exemplary embodiment that includes such a wide area detector and a pin.
    [0051] According to a preferred embodiment, the structured lighting means are configured and arranged for the generation and projection on the aperture diaphragm of the optical microscope objective of the at least four point light sources, according to a coherent intensity distribution, forming respective Position vectors that start from the center of the aperture diaphragm of the optical microscope objective and that are angularly distanced from each other by relative angles of 90 °.
    [0053] A number of four point light sources is considered optimal, given the location of the resulting orders when interacting with the sample, which allows doubling the resolution on the X and Y axes, and, in turn, obtaining optical sectioning capacity.
    [0054] However, the present invention also contemplates some embodiment examples for which the number of point light sources is different to four, which would generate different geometries resulting from interacting with the sample. For example, you could increase the number of point light sources in the diagonal directions (that is, whose projections on the lens diaphragm form position vectors at 45 ° with respect to each of the other contiguous position vectors, and with a frequency close to the cutoff frequency) and thus achieve an increase in resolution not only in X and Y, but also in transverse directions.
    [0056] In order to optimize the results obtained in terms of transverse resolution, allowing to obtain twice that obtained with conventional microscopes, preferably, the modulus of each of the aforementioned position vectors is equal to or substantially equal to the radius of the diaphragm aperture of the optical microscope objective, thus extending the bandwidth of the microscope to the limit of the technique, that is, to twice the resolution of a conventional system.
    [0058] For an exemplary embodiment, the optical microscope objective and the support are configured and arranged so that, in use, the light emitted by each of the two or more portions of the sample, collected through the optical microscope objective, produces in the aperture diaphragm is an incoherent intensity distribution pattern made up of nine frequency components, which is proportional to the square modulus of the lighting pattern.
    [0059] According to an exemplary embodiment, the scanning system is configured and arranged so that said relative displacement is a transverse displacement to the optical axis of the microscope according to at least a first transverse direction.
    [0061] For another embodiment, the scanning system is configured and arranged so that the relative displacement also includes, in addition to the displacement according to a first transverse direction, an axial displacement along the optical axis of the microscope, to obtain an image, formed by part of the imaging means, corresponding to a southern section of the sample.
    [0063] Advantageously, the scanning system is configured and arranged so that the relative displacement is a displacement transverse to the optical axis of the microscope according to also a second transverse direction orthogonal to the first transverse direction.
    [0065] According to another embodiment, the scanning system is configured and arranged so that the relative displacement also includes, in addition to the displacement according to first and second transverse directions, an axial displacement along the optical axis of the microscope, to obtain a three-dimensional image of the sample, formed by the imaging means, from optical sections of the sample. When carrying out the aforementioned axial relative displacement, a different part of the sample is focused while other positions become out of focus. In conventional microscopy, on the plane of the image sensor the sum of the intensities of the plane of focus would be taken together with that of the other planes that are out of focus. However, since the microscope proposed by the first aspect of the present invention provides optical sectioning, once the scan has been carried out, the light from each of the focus planes is exclusively captured in the final image, eliminating the unfocused information.
    [0067] According to an embodiment, the microscope of the first aspect of the present invention further comprises a tube lens arranged between the optical microscope objective and the light detector (s) and configured to conjugate the focal plane object of the optical microscope objective to its focal length, providing the above mentioned conjugate plane.
    [0069] For another embodiment, the microscope further comprises at least one dichroic filter configured and arranged to direct, with the aforementioned first direction, light with an illumination wavelength, which generates the at least four point light sources, towards the aperture diaphragm of the optical microscope objective, and to direct towards the light detector or detectors, according to said second direction, exclusively the fluorescent light collected by the optical microscope objective, which has a wavelength greater than the illumination wavelength, blocking the passage of light with the illumination wavelength towards the light detector (s).
    [0070] Since the pixel size of the final image formed is given by the step chosen to carry out the sample sweep, in order to observe the improvement in resolution, this step must be less than a quarter of the resolution limit of the objective of optical microscope, so, for a preferred embodiment, the scanning system is configured and arranged to provide relative displacement with a scanning step less than a quarter of the resolution limit of the optical microscope objective, so that Do not overlap portions of the sample within the common detection area.
    [0072] The microscope proposed by the first aspect of the present invention allows lateral resolutions to be reached below the limit given by the diffraction, being able to double the transverse resolution of a conventional microscope. Simultaneously, the microscope has optical sectioning capacity, that is, it eliminates light from planes located out of focus in 3D samples.
    [0074] The present invention also concerns, in a second aspect, a method for obtaining fluorescence light microscopy images, comprising:
    [0076] - projecting onto a sample stained with a fluorescent dye and arranged in the focal plane object of an optical microscope objective, or in the axial range defined by the depth of field of said optical microscope objective, a periodic illumination pattern in two directions transverse orthogonal to each other, by generating and projecting on the aperture diaphragm of the optical microscope objective of at least four point light sources, spatially distanced from each other, that generate and provide at least four plane waves whose direction of propagation is equidistant angularly, or substantially angularly equidistant (ie, with an angular deviation tolerance of ± 5%), from a first direction of the optical axis of the optical microscope objective, the interference of which results in said illumination pattern;
    [0077] - providing a sweep that includes a relative displacement of the sample with respect to said illumination pattern, to adopt different sweep positions;
    [0078] - sequentially detecting, in a conjugate plane of the object plane of the optical microscope objective, fluorescent light emitted by at least two portions of the sample, in two respective of said scanning positions, when interacting with the light of the standard lighting, collecting it through the optical microscope objective according to a second direction opposite to the first direction; and
    [0080] - form an image of at least two pixels, from the light intensity existing in the detected fluorescent light for each of the at least two portions of the sample, one per pixel.
    [0082] The method of the second aspect of the present invention is adapted to carry out the different stages thereof using the microscope proposed by the first aspect of the invention.
    [0084] Brief description of the drawings
    [0086] The foregoing and other advantages and characteristics will be more fully understood from the following detailed description of some embodiment examples with reference to the attached drawings, which should be taken by way of illustration and not limitation, in which:
    [0087] Figure 1a shows a schematic of the microscope assembly proposed by the first aspect of the present invention, for an embodiment for which the light detector thereof comprises an image sensor or pixelated detector.
    [0089] Figure 1b shows a schematic of the microscope assembly proposed by the first aspect of the present invention, for an embodiment example for which the light detector thereof comprises a pin and a wide area detector.
    [0091] Figure 2 shows a diagram of the illumination used on the aperture diaphragm of the microscope objective proposed by the first aspect of the present invention, for an embodiment for which the module of each of the four position vectors of the Projection onto the aperture diaphragm of the four point light sources is equal to or substantially equal to the radius of the aperture diaphragm of the optical microscope objective.
    [0093] Figure 3. (a) Simulation of the pattern resulting from the interaction between the proposed lighting and a flat and uniform fluorescent sample, which is very similar to the interference produced between the four plane waves (which is the square root of said intensity distribution) . (b) Representation of the 9 collection orders on the aperture diaphragm.
    [0095] Figure 4. Illustration of the intensity distribution on the image sensor in the diagram of Figure 1a, for three scanning positions in one of the transverse directions. The white square represents the common detection area in which the intensity of the fluorescent light detected by each of the pixels contained in it is added to obtain a pixel of the final image.
    [0097] Figure 5 Calculation of the three-dimensional form of the optical transfer function of (left) a conventional microscope and (right) the microscope proposed by the present invention.
    [0099] Figure 6. Results obtained by simulating imaging using a conventional microscope (top) and the microscope proposed by the present invention (bottom). These results correspond to (left) the impulse response of the system, (center) the transverse OTF and (right) the image of a USAF 1951 test.
    [0101] Figure 7. Experimental results of the cross-section of a gold nanoparticle using a prototype of the microscope proposed by the present invention and a conventional microscope. As can be seen, the impulse response of the microscope proposed by the present invention (b) is twice as narrow as that of a conventional microscope (a).
    [0103] Figure 8 Measurements resulting from the axial scanning of a flat fluorescent sheet using a conventional microscope (a) and using the microscope proposed by the present invention (b). As can be seen, the microscope proposed by the present invention has optical sectioning capacity since it eliminates light from the planes of the sample that are not the plane of focus.
    [0105] Detailed description of some embodiment examples
    [0107] In Figures 1a and 1b two possible diagrams of the microscope proposed by the present invention are illustrated, for two corresponding embodiment examples, for which the microscope comprises the following components:
    [0109] (1) A point source of totally or partially coherent laser light. This point source can be obtained with a laser coupled to a fiber and use the fiber output as a point source or with a laser in the focused free space.
    [0111] (2) An optical element (diffractive or refractive) capable of generating four or more mutually coherent virtual point light sources from illumination with a single point source. Depending on the exemplary embodiment, a glass pyramid whose vertex coincides with the optical axis could be used, a square diffraction grating in which filter out higher orders or electronically controlled spatial light modulators, or any other optical element capable of fulfilling the described function.
    [0112] (3) A collimating lens located at its focal distance from the point source of laser light.
    [0114] (4) A projection lens that focuses the point source (s) on the diaphragm of an optical microscope objective (6).
    [0116] (5) A dichroic filter or filter set whose purpose is to illuminate using the wavelength of the laser, that is to say, to direct the aperture diaphragm of the optical objective (6), according to a first direction, the light emitted by the laser, and exclusively collect the light emitted by the sample, that is to say, direct towards the light detector, according to a second direction, exclusively the fluorescent light collected by optical objective 6, which has a wavelength greater than the illumination wavelength, blocking the passage of laser light.
    [0118] (6) Optical microscope objective used to illuminate the sample and collect the light emitted by it.
    [0120] (7) Sample scanning system: This system can be a mechanical scanning system, that is, an apparatus that allows the sample to be moved with high precision (below 50 nm). A beam scanning system could also be used in place of the sample, that is, a set of high-precision galvanometric mirrors (not shown). In the latter case, this element would be located between the dichroic filter (5) and the microscope objective (6).
    [0122] (8) Tube lens that allows the objective focal plane of the lens to be conjugated to its focal length (between 160 mm and 300 mm).
    [0124] (9a), (9b). Light detector that is part of an imaging means and that is configured and arranged on a conjugated plane of the object plane of the optical microscope objective (6), to sequentially detect fluorescent light emitted by different portions of said sample, in respective scanning positions, when interacting with the light of the illumination pattern, collected through the optical objective (6) according to said second direction.
    [0126] (10) Stage to support a sample stained with a fluorescent dye, so that it is arranged in the vicinity of the focal plane object of the optical microscope objective (6).
    [0127] For the exemplary embodiment of Figure 1a, the light detector (9a) is an image sensor (such as a digital camera) configured and arranged to detect sequentially, in a common detection area formed by one or more photodetector elements or pixels thereof, the fluorescent light emitted by each of the portions of the sample, the imaging means being configured and arranged to integrate the existing light intensity in a common detection area into an intensity value per scanning position, to form each pixel of the sample image.
    [0129] Figure 4 illustrates the distribution of intensities on the image sensor (9a) of the scheme of Figure 1a, for three scanning positions in one of the transverse directions, according to the direction indicated by the reference Db. The white square represents the one named above as the common detection area, in which the intensity of the fluorescent light detected by each of the pixels contained in it is added to obtain one pixel of the final image.
    [0131] For the exemplary embodiment of Figure 1b, the light detector (9b) is a wide area detector, and the imaging means further comprise a pin (11) configured and arranged, between the optical objective (6) and the wide area detector (9b), so that the wide area detector (9b) sequentially detects the fluorescent light that passes through the pin (11) and that is emitted by each of the different portions of the sample, the means being imaging channels configured and arranged to form each pixel of the sample image with the respective light intensity values detected by the wide area detector (9b) for each scanning position. The pin is preferably of micrometric dimensions (between 1-50 pm) and is located on the focal plane of the tube lens (8).
    [0133] In order for the microscope to double lateral resolution and have optical sectioning, instead of illuminating the sample with uniform light (as is typical of conventional microscopes) it is illuminated by the pattern resulting from the interference of four flat waves. These plane waves interfere in such a way that their wave vector forms an angle with the optical axis that will depend on the numerical aperture and whose transverse projection will be given by orthogonal vectors (each transverse projection of the wave vector is separated 90 degrees with respect to the vector adjacent). An easy way to achieve this illumination pattern is to image four point sources that are coherent with each other on the aperture diaphragm of the microscope objective. Said sources would be achieved by making the image of the virtual point sources generated with the element (2) of the assembly. With the purpose of To obtain the desired relative angles between the plane waves in the object plane, the position vectors with respect to the center of the diaphragm of each of the point light sources must form angles of 90, 180 and 270 ° with the rest, as illustrated in Figure 2, which shows the aperture diaphragm with four positional vectors that start from the center of the aperture diaphragm and end in respective circles that represent the projections of the four point light sources or lighting orders. The greater the modulus of the position vector of these sources with respect to the center of the diaphragm, the greater the angle that the plane waves form with respect to the optical axis in the object space. For the case illustrated in Figure 2, the modulus is almost equal to the radius of the aperture diaphragm.
    [0135] By means of the aforementioned structured pattern, a fluorescent sample is illuminated. Said sample interacts with the coherent light of the pattern emitting incoherently, as explained mathematically below.
    [0137] The plane waves generated in the object space represent a coherent distribution of intensities that can be represented mathematically in the following way:
    [0139] s ( X, y) = ejpAx e ~ pAx e ~ pAy e ~ jnAy,
    [0140] Where A is the vector module that defines the position of the point sources in the plane of the pupil. When interacting with a fluorescent sample with a spatial distribution of fluorophores O (x, y) , an inconsistent intensity distribution is generated that can be expressed as:
    [0142]
    [0144] As can be seen, the interference of the 4 coherent plane waves gives rise to an incoherent distribution of intensities with nine components and, therefore, in Fourier space there will be nine orders corresponding to each of these components.
    [0146] Therefore, when considering a thin, uniform fluorescent sample, the light resulting from the interaction between illumination and sample is nothing more than an intensity proportional to the square modulus of the coherent pattern. The objective (6) used for illumination collects the light emitted by the sample (wavelength greater than the illumination light). The light collected, being proportional to the square modulus of the illumination pattern, produces a pattern of 9 collection frequency components in the diaphragm of opening which will be called as collection orders, as illustrated in view (b) of Figure 3. These collection orders appear in addition to the same positions as the lighting orders (that is, from the projections of the four point light sources) and one positioned in the center of the aperture diaphragm, in intermediate positions at 45 ° (to be referred to as mixed orders) and with a position vector with respect to the center with a module equal to half than lighting. To optimize the microscope, the modulus of the position vectors of the vectors with the largest modulus must be equal to the radius of the aperture diaphragm. If the image of the flat and uniform fluorescent sample is made on a pixelated detector (9a), a pattern whose appearance is dotted mesh will be observed, as illustrated in view (a) of Figure 3. The interference produced between the 4 waves is very similar to that illustrated in said view (a) of Figure 3.
    [0148] The dotted mesh can be observed in the pixelated detector (9a) or it will be projected on a pin (11) located in front of a wide area detector (9b) (matching a maximum intensity of the mesh with the center of the pin (11 In the case of the pixelated detector (9a), a subregion with a relatively low number of pixels can be selected, so that the intensity of the pixels of said subregion is integrated into a single value. a sample with the appropriate fluorescent dye in the focal plane object of the optical microscope objective. (6) For a certain position of the sample, an intensity signal will be obtained, either by means of the mentioned pixel integration or by the wide area detector (9b). This intensity value corresponds to the relative position of the sample and the illumination. In order to obtain an image, the sample will be moved successively using, for example, a positioning system. or piezoelectric (7) connected to the stage (10), or lighting by means of a system of galvanometric mirrors (not illustrated). For each position of the sample or the illumination, which will be called the scanning position, an intensity value corresponding to one pixel of the final image will be obtained, duly positioned according to the scanning positions. Said scan can be (1) transverse: that is, perpendicular to the optical axis to obtain a plane of focus of the sample, (2) transverse and axial: sweeping the sample in a transverse direction and in the direction of the optical axis, or ( 3) three-dimensional: a succession of transverse sweeps 8 is carried out in two directions orthogonal to each other) for different axial positions of the sample that, using the appropriate software, can be compiled into a three-dimensional representation of intensities of the sample image.
    [0149] The microscope proposed by the present invention, by combining adequate structured illumination and scanning of the sample, allows the transverse resolution to be doubled with respect to a conventional microscope, as well as giving it optical sectioning capacity. This can be understood by studying the transmission of special frequencies from the object space to the image space. In a conventional microscope, said transmission occurs in a spatial frequency band limited by the three-dimensional function known as the optical transfer function (OTF). This function has a toroid shape and indicates that the spatial frequencies of the object that are inside it will pass from the object to its corresponding image (see Figure 5). On the one hand, the cross-sectional shape of this function is closely related to the size of the aperture diaphragm of the microscope objective and its focal distance (that is, by the numerical aperture). On the other hand, the fact that conventional microscopes lack optical sectioning is due to the shape of this function, since it presents an empty cone in the direction of spatial frequencies in the axial direction, which prevents the transmission of details in volume from the object to its corresponding image.
    [0150] The microscope proposed by the present invention, once an image has been captured by scanning the sample or lighting, has an OTF different from that of a conventional microscope. In fact, its OTF is given by 9 replicas of the conventional OTF placed on the positions of the space frequency space given by the collection orders in the aperture diaphragm of the microscope objective. If the point light sources are physically located at the ends of the aperture diaphragm, then the OTF bandwidth of the proposed microscope will be twice that of a conventional microscope, that is, it will have twice the resolution. In addition, the OTF replicas displaced to the positions given by the mixed orders allow the cone to be filled with losses, that is, they provide the microscope with optical sectioning capacity.
    [0152] Simulated results of the functionality of the microscope proposed by the present invention are presented in Figure 6. Said calculations correspond to the conventional image and to the image obtained using the proposed microscope by means of a single point transverse scan (commonly known as impulse response) and a USAF 1951 resolution test. The transverse impulse response is directly related to the resolution. of the microscope and its Fourier transform is the transverse OTF, also presented in Figure 6. As can be seen, the OTF of the proposed microscope doubles the bandwidth with respect to a conventional microscope. This can also be seen in the test image USAF 1951, in which the proposed microscope has twice the resolution of the conventional microscope (an improvement in resolution of one group, that is, six test elements).
    [0154] Furthermore, preliminary results obtained by means of a prototype of the microscope proposed by the first aspect of the present invention are presented. Figure 7 presents the image of a gold nanoparticle using a 50x NA = 0.6 objective both for the case of a conventional microscope (a) and for the one proposed by the present invention (b). As can be seen, the impulse response obtained by this measurement is approximately half as narrow in the proposed microscope, that is, the microscope proposed by the invention has twice the resolution of the conventional one.
    [0156] On the other hand, the optical sectioning capacity is tested by means of the axial scanning of a flat fluorescent sheet, both for the conventional case (a) and with the scanning structured illumination microscope (b), showing in Figure 8 the resulting measurements of such an axial scan of a flat fluorescent sheet.
    [0158] A person skilled in the art could introduce changes and modifications in the described embodiment examples without departing from the scope of the invention as defined in the attached claims.
    [0159] References
    [0161] [1] Mats G L Gustafsson; Lin Shao; Peter M Carlton; Rachel Wang C J; Inna N Golubovskaya; Zacheus Cande W; David A Agard; John W Sedat, "Three-Dimensional Resolution Doubling in Wide-Field Fluorescence Microscopy by Structured Illumination".
    [0163] [2] Yasuhiro Hirano; Atsushi Matsuda; Yasushi Hiraoka, "Recent advances in structured-illumination microscopy toward live-cell imaging".
    [0165] [3] Birk Udo; Hase Johann V; Cremer Christoph, "Super-resolution microscopy with very large working distance by means of distributed aperture illumination".
    权利要求:
    Claims (14)
    [1]
    1.- Fluorescence optical microscope, comprising:
    - an optical microscope objective (6) whose axis of symmetry, called the optical axis, defines a first direction or main direction of light propagation;
    - a support (10) to support a sample stained with a fluorescent dye, so that it is arranged in the focal plane object of said optical microscope objective (6), or in the axial range defined by the depth of field of said objective of light microscope (6);
    - structured illumination means configured and arranged to project onto a sample located in said support (10) a periodic illumination pattern in two transverse directions orthogonal to each other, by generating and projecting onto the aperture diaphragm of said optical microscope objective ( 6) from at least four point light sources, spatially distanced from each other, that generate and provide at least four plane waves whose direction of propagation is equidistant, or substantially equidistant, angularly from said first direction of the optical axis of the optical microscope objective (6 ), whose interference results in said lighting pattern;
    - a scanning system (7) configured and arranged to provide a relative displacement of said support (10), and therefore of the sample supported by it, with respect to said lighting pattern, to adopt different scanning positions; and
    - imaging means, comprising:
    - at least one light detector configured and arranged on a conjugated plane of the object plane of the optical microscope objective (6), to sequentially detect fluorescent light emitted by at least two portions of said sample, in two respective of said scanning positions, by interacting with the light of the illumination pattern, collected through said optical microscope objective (6) according to a second direction of the optical axis of the optical microscope objective (6), opposite to said first direction; and
    - imaging means configured and arranged to form an image of at least two pixels, from the existing light intensity in the fluorescent light detected by the light detector, which is at least one, for each of said at least two portions of the sample, one per pixel.
    [2]
    2. - Microscope according to claim 1, wherein said light detector, which is at least one, is an image sensor (9 (a)) configured and arranged to detect sequentially, in a common detection area formed by one or more photodetector elements of said image sensor (9 (a)), the fluorescent light emitted by each one of said at least two portions of the sample, said imaging means being configured and arranged to integrate the light intensity existing in said common detection area at an intensity value per scan position, to form each pixel of said image.
    [3]
    3. - Microscope according to claim 1, wherein said light detector is a wide area detector (9 (b)), and said imaging means further comprise a pin (11) configured and arranged, between the optical microscope objective (6) and said wide area detector (9 (b)), so that the wide area detector (9 (b)) sequentially detects the fluorescent light that passes through the pin (11) and which is emitted by each one of said at least two portions of the sample, said imaging means being configured and arranged to form each pixel of said image with the respective light intensity values detected by the wide area detector (9 (b) ) for each sweep position.
    [4]
    4. - Microscope according to any one of the preceding claims, wherein the structured lighting means are configured and arranged for the generation and projection on the aperture diaphragm of the optical microscope objective (6) of said at least four sources point light sources, according to a coherent intensity distribution, forming respective position vectors that start from the center of the aperture diaphragm of the optical microscope objective (6) and that are angularly spaced from each other by relative 90 ° angles.
    [5]
    5. - Microscope according to claim 4, wherein the modulus of each of said position vectors is equal to or substantially equal to the radius of the aperture diaphragm of the optical microscope objective (6).
    [6]
    6. - Microscope according to claim 4 or 5, in which the optical microscope objective (6) and the support (10) are configured and arranged so that, in use, the light emitted by each of the at least Two portions of the sample, collected through the optical microscope objective (6), produce in the opening diaphragm of the same an inconsistent intensity distribution pattern formed by nine frequency components, which is proportional to the square module of the illumination pattern.
    [7]
    7. - Microscope according to any one of the preceding claims, wherein said scanning system (7) is configured and arranged so that said relative displacement is a transverse displacement to the optical axis of the microscope according to at least a first transverse direction.
    [8]
    8. - Microscope according to claim 7, wherein said scanning system (7) is configured and arranged so that said relative displacement is a transverse displacement to the optical axis of the microscope according to also a second transverse direction orthogonal to said first direction cross.
    [9]
    9. - Microscope according to claim 7, wherein said scanning system (7) is configured and arranged so that said relative displacement also includes an axial displacement along the optical axis of the microscope, to obtain an image, formed by said imaging means, corresponding to a southern section of the sample.
    [10]
    10. - Microscope according to claim 8, wherein said scanning system (7) is configured and arranged so that said relative displacement also includes an axial displacement along the optical axis of the microscope, to obtain a three-dimensional image of the sample, formed by means of said imaging means, from optical sections thereof.
    [11]
    11. - Microscope according to any one of the preceding claims, further comprising a tube lens (8) arranged between said optical microscope objective (6) and said light detector, which is at least one, and configured to conjugate the focal plane object of the optical microscope objective (6) at its focal distance, providing said conjugate plane.
    [12]
    12. - Microscope according to any one of the preceding claims, further comprising at least one dichroic filter configured and arranged to direct, according to said first direction, light with an illumination wavelength, which is generating said at least four point light sources, towards the aperture diaphragm of the optical microscope objective (6), and to direct towards the light detector, which is at least one, according to said second direction, exclusively the fluorescent light collected by the optical microscope objective (6), which has a wavelength greater than said illumination wavelength, blocking the passage of light with said illumination wavelength towards the light detector.
    [13]
    13. Microscope according to any one of the preceding claims, wherein said scanning system (7) is configured and arranged to provide said relative displacement with a scanning step less than a quarter of the resolution limit of the optical microscope objective (6).
    [14]
    14.- Method for obtaining fluorescence light microscopy images, comprising:
    - projecting onto a sample stained with a fluorescent dye and arranged in the focal plane object of an optical microscope objective, or in the axial range defined by the depth of field of said optical microscope objective, a periodic illumination pattern in two directions transverse orthogonal to each other, by generating and projecting on the aperture diaphragm of said optical microscope objective of at least four point light sources, spatially distanced from each other, that generate and provide at least four plane waves whose equidistant direction of propagation, or substantially equidistant, angularly from a first direction of the optical axis of the optical microscope objective, whose interference results in said illumination pattern;
    - providing a sweep that includes a relative displacement of said sample with respect to said illumination pattern, to adopt different sweep positions;
    - sequentially detecting, in a conjugate plane of the object plane of the optical microscope objective, fluorescent light emitted by at least two portions of said sample, in two respective of said scanning positions, when interacting with the light of the illumination pattern, collecting it at through said optical microscope objective according to a second direction opposite to said first direction; and
    - forming an image of at least two pixels, from the light intensity existing in the fluorescent light detected for each of said at least two portions of the sample, one per pixel.
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    同族专利:
    公开号 | 公开日
    WO2020128130A1|2020-06-25|
    ES2768448B2|2021-08-12|
    EP3901684A1|2021-10-27|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    EP2801854A1|2013-05-10|2014-11-12|Ruprecht-Karls-Universität Heidelberg|Method and apparatus for combination of localization microscopy and structured illumination microscopy|
    WO2017177180A1|2016-04-08|2017-10-12|ARIZONA BOARD OF REGENTS on behalf of THE UNIVERSITY OF ARIZONA, A BODY CORPORATE|Systems and methods for extended depth-of-field microscopy|
    WO2018151599A1|2017-02-16|2018-08-23|Universiteit Van Amsterdam|Structured illumination scanning microscopy|
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    ES201831272A|ES2768448B2|2018-12-21|2018-12-21|OPTICAL MICROSCOPE BY FLUORESCENCE AND METHOD FOR OBTAINING IMAGES FROM OPTICAL MICROSCOPY BY FLUORESCENCE|ES201831272A| ES2768448B2|2018-12-21|2018-12-21|OPTICAL MICROSCOPE BY FLUORESCENCE AND METHOD FOR OBTAINING IMAGES FROM OPTICAL MICROSCOPY BY FLUORESCENCE|
    PCT/ES2019/070860| WO2020128130A1|2018-12-21|2019-12-18|Optical fluorescence microscope and method for the obtaining of optical fluorescence microscopy images|
    EP19898149.0A| EP3901684A1|2018-12-21|2019-12-18|Optical fluorescence microscope and method for the obtaining of optical fluorescence microscopy images|
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