WO2017152702A1 - Procédé et appareil d'imagerie pour microscope à éclairage structuré - Google Patents

Procédé et appareil d'imagerie pour microscope à éclairage structuré Download PDF

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WO2017152702A1
WO2017152702A1 PCT/CN2016/112637 CN2016112637W WO2017152702A1 WO 2017152702 A1 WO2017152702 A1 WO 2017152702A1 CN 2016112637 W CN2016112637 W CN 2016112637W WO 2017152702 A1 WO2017152702 A1 WO 2017152702A1
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original image
spatial
light illumination
structured light
original
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Chinese (zh)
Inventor
杨怀栋
刘国漩
张四纯
张新荣
金国藩
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Tsinghua University
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Tsinghua University
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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/364—Projection microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/365—Control or image processing arrangements for digital or video microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/365—Control or image processing arrangements for digital or video microscopes
    • G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison

Definitions

  • the present invention relates to the field of optical microscope technology, and in particular, to an imaging method and apparatus for a structured light illumination microscope.
  • Microscopy is an indispensable research tool in modern life science research.
  • the conventional optical microscope has a resolution limit.
  • SIM Structured Illumination Microscope
  • STORM Stochastic Optical Reconstruction Microscopy
  • PAM Photoactivated Localization Microscopy
  • STED Stimulated Emission Depletion Microscopy
  • the minimum number of original images required for SIM image reconstruction is the shortest.
  • SIM is the most suitable method for observing fast imaging in living cells or wide field of view in various super-resolution imaging methods.
  • Fig. 1 The basic structure of the existing structured light illumination microscope is shown in Fig. 1.
  • a coherent or incoherent collimated wide beam is used as the light source 1.
  • the incident light passes through the light modulating device 2, and is modulated by the lens 3 and the dichroic color.
  • An imaging system consisting of a beam 8 and an objective lens 9 is then projected onto the illumination sample 10 and shaped on the plane in which the illumination sample 10 is located A periodic light intensity distribution.
  • a spatial filtering system consisting of the lens 4, the spatial filter 5 and the lens 6 can also be selectively added to filter out the zero-order diffraction component, and the filtered light continues to pass through the dichroic color division.
  • An imaging system consisting of a beamer 8 and an objective lens 9 interferes with the formation of a structured light illumination pattern at the plane of the illumination sample 10.
  • the light of the illuminated sample is collected by the detector 13 through a microscope system consisting of the objective lens 9 and the lens barrel 11.
  • SIM-series time-lapse SIM
  • the imaging process is as follows:
  • each structured light illumination pattern captures an original image, and the original image is reconstructed to obtain a SIM super-resolution image.
  • the next SIM super-resolution image is reconstructed using the original image under the next set of structured light illumination. The above process is repeated to obtain a plurality of SIM super-resolution images, which constitute a time series of SIM super-resolution images.
  • SIMs the device used to modulate the structured light distribution was a grating. Modulation and switching of different structured light illumination patterns is achieved by translating or rotating the grating. Due to the presence of mechanical motion in the system, the imaging speed is relatively slow. Subsequent SIMs generally use a spatial light modulator (SLM) and a digital micro-mirror device (DMD) to modulate the structured light illumination pattern. Thanks to the fast response of SLM and DMD, coupled with high-sensitivity detectors such as Electron Multiplying CCD (EMCCD) and Scientific CMOS (sCMOS), the exposure time can be greatly reduced, which provides favorable conditions for SIM high-speed imaging.
  • SLM spatial light modulator
  • DMD digital micro-mirror device
  • the present invention provides an imaging method for a structured light illumination microscope, comprising:
  • the preset N pieces of structured light illumination patterns are cyclically switched in a preset order, where N is a preset constant;
  • the original image of the original image sequence and the N-1 original images after the image are reconstructed to obtain a super-resolution image sequence of the sample to be imaged.
  • the image is reconstructed by using the original image of the original image sequence and the N-1 original image after the original image to obtain a super-resolution image sequence of the sample to be imaged, which specifically includes:
  • Each spatial spectral component of the spatial frequency reduction in each spatial spectral component group is weighted and superimposed with each spatial spectral component whose spatial frequency is not changed, to obtain a super-resolution image sequence of the sample to be imaged.
  • the spatial frequency of each spatial spectral component whose spatial frequency is changed in each spatial spectral component group according to the spatial frequency of the preset N-structure light illumination pattern is restored, and the spatial spectral components of the spatial frequency reduction are obtained, which specifically include:
  • each spatial spectral component of the spatial frequency component change in each spatial spectral component group is multiplied by an exponential function corresponding to its spatial frequency change amount to obtain spatial spectral components of the spatial frequency reduction.
  • the photographing the original image of the sample to be imaged under each structured light illumination pattern comprises:
  • the original image of the sample to be imaged under each structured light illumination pattern is taken at an interval preset time.
  • the method further includes:
  • the spatial frequency of each spatial spectral component whose spatial frequency is changed in each spatial spectral component group is restored according to the spatial frequency of the preset N-structure light illumination pattern, and specifically includes:
  • the spatial frequency of each spatial frequency spectral component of the spatial frequency component of each of the deconvolved spatial spectral components is reduced according to the spatial frequency of the predetermined N-structured light illumination pattern.
  • the method before the periodically circulating the preset N pieces of structured light illumination patterns, the method further includes:
  • the method before the image reconstruction is performed on each of the original image sequence and the N-1 original images in the original image sequence, the method further includes:
  • the reconstructing each of the original image in the original image sequence and the N-1 original images after the image sequence comprises:
  • the method further includes:
  • the method before the image reconstruction is performed on each of the original image sequence and the N-1 original images in the original image sequence, the method further includes:
  • Performing image enlargement processing on each original image in the original image sequence may be generally performed by an image interpolation method or by performing zero-padding on the image spectrum and then transforming back into the spatial domain, thereby obtaining image enlargement.
  • the processing may be generally performed by an image interpolation method or by performing zero-padding on the image spectrum and then transforming back into the spatial domain, thereby obtaining image enlargement.
  • the reconstructing each of the original image in the original image sequence and the N-1 original images after the image sequence comprises:
  • the method further comprises: performing image enlargement processing on each super-resolution image in the super-resolution image sequence to obtain super-resolution after image enlargement Image sequence.
  • the method further includes:
  • the structured light illumination pattern is obtained by modulating incident light by a light modulating device disposed in an illumination light path of the structured light illumination microscope, and is obtained by projecting an image of the light modulation device disposed in the illumination light path of the microscope
  • the incident light is modulated, and then subjected to high-pass spatial filtering and projection imaging.
  • the structured light illumination pattern comprises one or more non-zero spatial frequencies.
  • the present invention provides an imaging device for a structured light illumination microscope, comprising:
  • a pattern switching unit configured to cyclically switch the preset N pieces of structured light illumination patterns in a preset order, where N is a preset constant;
  • An original image acquiring unit configured to acquire an original image of the sample to be imaged under each structured light illumination pattern, to obtain an original image sequence of the sample to be imaged;
  • an image reconstruction unit configured to reconstruct an image of each of the original image sequences and the N-1 original images after the original image sequence to obtain a super-resolution image sequence of the sample to be imaged.
  • a super-resolution image sequence of the sample to be imaged as a function of time is obtained, and the time interval of each of the two super-resolution images is equal to the time interval of capturing each of the two original images, compared with the prior art structured light illumination microscope imaging method.
  • the temporal resolution of the imaging method and apparatus of the present invention is greatly improved.
  • FIG. 1 is a schematic structural view of a conventional structured light illumination microscope
  • FIG. 2 is a schematic flow chart of an imaging method of a structured light illumination microscope according to an embodiment of the present invention
  • FIG. 3 is a schematic diagram of comparison between an imaging method of a structured light illumination microscope and an existing imaging method according to an embodiment of the present invention
  • FIG. 4 is a schematic light illumination of a non-zero spatial frequency according to an embodiment of the invention. Schematic diagram of the intensity distribution of the pattern;
  • FIG. 5 is a schematic diagram of light intensity distribution of a plurality of non-zero spatial frequency structured light illumination patterns according to an embodiment of the present invention
  • FIG. 6 is a schematic structural diagram of an image forming apparatus for a structured light illumination microscope according to an embodiment of the present invention.
  • FIG. 2 is a schematic flow chart showing an imaging method of a structured light illumination microscope according to an embodiment of the present invention. As shown in FIG. 2, the imaging method of the structured light illumination microscope of the present embodiment includes steps S21 to S23.
  • N is a preset constant
  • the preset N pieces of structured light illumination patterns are grouped, and the set of structured light illumination patterns are cyclically switched in a fixed order.
  • a set of structured light illumination patterns includes five structured light illumination patterns, respectively a b c d e.
  • the cyclic switching of the structured light illumination pattern is performed in the above a b c d e, and the order of the five sets of structured light illumination patterns of the first group can be arbitrarily set, but each structured light illumination pattern is required to be included, that is, the first
  • the order of the set of structured light illumination patterns may be a b c d e, d a c e b, e c a b d , and the like.
  • the order of the structured light illumination patterns of the first group is e d c b a
  • the order of the subsequent structured light illumination patterns is the same as the order of the first group of structured light illumination patterns, ie, during the user observation process, the structure
  • the switching order of the light illumination patterns is e d c b a e d c b a e d c b a...
  • each structured light illumination pattern illuminates the sample to be imaged
  • an original image of the sample to be imaged is taken, and the original image of the sample to be imaged is obtained within the observation time required by the user. sequence.
  • the order of the structured light illumination pattern is e d c b a e d c b a e d c b a... then the original image sequence of the sample to be imaged should be E D C B A E D C B A E D C B A...
  • the original image of the sample to be imaged described above constitutes a sequence of original images in chronological order of shooting.
  • S23 Perform image reconstruction on each original image in the original image sequence and the N-1 original images after the image to obtain a super-resolution image sequence of the sample to be imaged.
  • the first original image in the original image sequence of the obtained sample to be imaged and the N-1 original image after the image are taken, and a SIM super-resolution image is obtained through image reconstruction.
  • Each subsequent original image is reconstructed from the original N-1 original image to obtain a SIM super-resolution image.
  • All SIM super-resolution images are arranged in time to form a super-resolution image sequence.
  • the first original image E of the sample to be imaged and the subsequent 4 original images D C B A are reconstructed to obtain the first SIM super-resolution image.
  • the second original image D is grouped with the following four original images, and reconstructed to obtain a second SIM super-resolution image. That is, the original image E D C B A is reconstructed to obtain a first SIM super-resolution image S(1), and the original image D C B A E is reconstructed to obtain a second SIM super-resolution image S(2), the original image C B A E D reconstructs the third SIM super-resolution image S(3)...
  • the reconstructed SIM super-resolution image is arranged in order to form a super-resolution time series S(1) S(2) S(3) S(4 ) «
  • the reconstruction process of the original image may be performed after the original image sequence of the sample to be imaged is taken, or simultaneously with the original image of the sample to be imaged.
  • the imaging method of the structured light illumination microscope of the present embodiment can be used not only in the structured light illumination microscope SIM, but also in the following technologies: nonlinear structure light illumination microscope NL-SIM, total internal reflection fluorescence structure Light illumination microscope TIRF-SIM, 3D super-resolution imaging of a structured light illumination microscope 3D-SIM, a combination of a light microscope and a structured light illumination microscope Lattice-lightsheet-SIM.
  • nonlinear structure light illumination microscope NL-SIM total internal reflection fluorescence structure Light illumination microscope TIRF-SIM
  • 3D super-resolution imaging of a structured light illumination microscope 3D-SIM a combination of a light microscope and a structured light illumination microscope Lattice-lightsheet-SIM.
  • SIM structured light illumination microscope
  • the imaging method of the structured light illumination microscope of the embodiment can obtain the super-resolution image sequence of the sample to be imaged with time, and the time resolution is greatly improved.
  • step S23 specifically comprises sub-steps S231 to S233 which are not shown in FIG.
  • the N original images are composed of each original image in the original image sequence and N-1 original images thereafter.
  • a set of spatial spectral components can be calculated for every N original images. As described in the above embodiment, a plurality of N original images can be obtained in the original image sequence, and thus, a plurality of spatial spectral component groups can be obtained.
  • Each spatial spectral component group contains some spatial spectral components whose spatial frequencies have changed, and also some spatial spectral components whose frequencies have not changed.
  • each spatial spectral component whose spatial frequency is changed in each spatial spectral component group is restored to its original spatial frequency according to the spatial frequency of the preset N structured light illumination patterns to correctly reproduce the sample to be imaged.
  • the imaging method of the structured light illumination microscope of the embodiment can accurately obtain the super-resolution image sequence of the sample to be imaged, obtain a finer variation process of the sample to be tested, and improve The time resolution of the structured light illumination microscope SIM.
  • step S232 specifically includes sub-steps S2321 to S2323 not shown in FIG. 2:
  • S2321 Determine a spatial frequency change amount of each spatial spectral component of the spatial frequency component change in each spatial spectral component group according to the spatial frequency of the preset N-structure light illumination patterns.
  • the spatial frequency change of the spatial spectral component is determined prior to restoring the spatial frequency of the spatial spectral component of the spatial frequency change.
  • the amount of spatial frequency change is determined according to the spatial frequency of the preset N structured light illumination patterns.
  • S2322 Determine an exponential function corresponding to each spatial frequency change amount in each spatial spectral component group in the spatial domain according to the spatial frequency change amount.
  • Different spatial frequency changes correspond to different exponential functions, and the exponential function corresponding to each spatial spectral component is determined according to the spatial frequency change amount, so that the spatial spectral components of each spatial frequency change can be correctly restored.
  • each spatial spectral component of the spatial frequency component change in each spatial spectral component group by an exponential function corresponding to its spatial frequency change amount, the spatial frequency reduction of the frequency-changed spatial spectral component can be achieved.
  • the super-resolution image corresponding to each original image can be accurately obtained by restoring the spatial frequency of each spatial spectral component.
  • step S22 specifically includes the following steps not shown in FIG. 2:
  • the original time of the sample to be imaged under each structured light illumination pattern is taken at intervals of preset time image.
  • the time interval of two consecutive images taken continuously for reconstructing each super-resolution image should be basically the same.
  • time interval is the interval of the time at which the two original images are integrated.
  • the minimum time interval is greater than or equal to 0.5t, and the time interval is at least 2t.
  • FIG. 3 The specific imaging process of this embodiment is shown in FIG. 3 compared to the imaging method of the existing structured light illumination microscope.
  • the original image sequence of the captured sample to be imaged includes: original image 1, original image 2, ... original image n.
  • the imaging method of the existing structured light illumination microscope adopts 1-5 original images as one group, 6-10 original images as another group, and each set of original images is reconstructed to obtain a SIM super-resolution image.
  • the time interval between each of the two SIM super-resolution images obtained by continuous reconstruction is 5t.
  • the imaging method of the structured light illumination microscope of the embodiment adopts 1-5 original images as one group, 2-6 original images as another group, and 3-7 original images as a next group, each group original
  • the image is reconstructed to obtain a SIM super-resolution image.
  • the time interval between each of the two SIM super-resolution images obtained by continuous reconstruction is t. Equal to the time interval between the original image was taken.
  • the imaging method of the structured light illumination microscope of the embodiment can effectively shorten the super-resolution imaging time interval of the structured light illumination microscope, and effectively improve the time resolution of the structured light illumination microscope imaging.
  • step S231 the above method further The following steps are not shown in Figure 2:
  • Each spatial spectral component in each spatial spectral component group is deconvolved to obtain a deconvolved spatial spectral component group.
  • the deconvolution process can be added before the image reconstruction of the original image or during the image reconstruction process to compensate for the high frequency information in the spatial frequency domain during the imaging process. A decrease in the contrast of the original image caused by the attenuation.
  • the deconvolution process commonly used in SIM imaging methods is Wiener filtering: OTF * F(f) / (
  • the optical transfer function OTF can be obtained by theoretical calculation, which can be obtained by actual measurement, or it can be obtained by iterative calculation using relevant deconvolution software.
  • c is an empirical constant and can be adjusted according to the actual filtering effect.
  • Deconvolution algorithms such as iterative deconvolution and blind deconvolution can be applied to SIM imaging methods.
  • step S232 specifically includes:
  • the spatial frequency of each spatial frequency spectral component of the spatial frequency component of each of the deconvolved spatial spectral components is reduced according to the spatial frequency of the predetermined N-structured light illumination pattern.
  • the imaging method of the structured light illumination microscope of the embodiment can effectively improve the contrast of the sample to be imaged.
  • the method before the step S23, the method further includes the following steps not shown in FIG. 2:
  • the original image under each structured light illumination pattern of the sample to be imaged is subjected to noise reduction processing to obtain a sequence of the original image after noise reduction.
  • Denoising the original image can effectively improve the signal-to-noise ratio of the original image.
  • step S23 specifically includes:
  • the method further includes:
  • the imaging method of the structured light illumination microscope of the embodiment effectively reduces the signal-to-noise ratio of the original image or the super-resolution image by denoising the original image or the reconstructed super-resolution image.
  • the method before the step S23, the method further includes the following steps not shown in FIG. 2:
  • An image enlargement process is performed on each original image in the original image sequence to obtain each original image in which the image is enlarged.
  • the method further includes:
  • the image enlargement in this embodiment refers to expanding the number of pixels used to represent the entire image by using an interpolation method or an image spectrum zero-padding extension, for example, interpolating an image of N ⁇ N pixels to obtain (2N-1) ⁇ (2N). -1) An image of a pixel.
  • step S13 specifically includes:
  • An original image magnified by each of the original image sequences and an original image magnified by N-1 images subsequent thereto are subjected to image reconstruction.
  • the imaging method of the structured light illumination microscope of the present embodiment can make the actual size represented by the adjacent pixels smaller by the image enlargement processing, so that the fine reconstruction result can be expressed.
  • the method further includes the step S20 not shown in FIG. 2:
  • the structured light illumination pattern is obtained by modulating incident light by a light modulating device disposed in an illumination light path of the structured light illumination microscope, and then obtaining a light modulation in an illumination light path of the structured light illumination microscope;
  • the device modulates the incident light and performs high-pass spatial filtering and projection imaging.
  • any one of the spatial light modulator SLM, the digital micromirror device DMD, and the grating may be used to modulate the spatial distribution of the incident light, and the image is obtained by direct projection imaging or high-pass spatial filtering processing and then imaging.
  • a structured light illumination pattern is provided.
  • the structured light illumination pattern can be obtained in various ways, and the universality of the imaging method is improved.
  • the structured light illumination pattern has a periodic distribution of light intensity in a plane in which the sample to be imaged is located.
  • Each structured light illumination pattern may contain one or more non-zero spatial frequencies, wherein the structured light illumination pattern of a single non-zero spatial frequency is typically a series of parallel sinusoids, as shown in FIG.
  • a plurality of non-zero spatial frequency structured light illumination patterns are typically the result of superposition or multiplication of sinusoidal stripes in different directions, as shown in FIG.
  • the structured light illumination microscope shown in FIG. 1 is taken as an example.
  • the fiber coupled output laser, the collimator and the beam expander constitute the light source module 1 to obtain a collimated wide beam, and Use it as incident light.
  • the incident light is incident on the surface of the light modulating device 2 according to actual requirements, and is modulated by the light modulating device 2 to obtain a structured light illumination pattern.
  • the outgoing light transmitted through the light modulating device 2 is reflected by the lens 3 at the dichroic beam splitter 8, and the sample 10 to be imaged is illuminated by the objective lens 9.
  • a DMD is employed as a modulation device for incident light.
  • the image focal plane of the lens 3 coincides with the back focal plane of the objective lens 9, and the lens 3
  • the focal plane of the object coincides with the plane of the DMD.
  • the image to be imaged 10 is placed on the front focal plane of the objective lens, and the plane of the DMD and the sample 10 to be imaged is conjugated.
  • the image loaded on the DMD can be imaged by the projection image to be imaged. On sample 10, illumination of the structured light is achieved.
  • the imaging optical path of the sample 10 to be imaged is an existing microscope optical path, which is not described herein.
  • EMCCD is used as the detector 13.
  • the computer is used to simultaneously control the switching of the DMD loaded image and the detection of the EMCCD, and implement subsequent super-resolution image reconstruction.
  • the shooting integration time and the shooting interval time of the EMCCD are selected according to information such as the specific illumination light intensity and the moving speed of the sample to be imaged.
  • each time an image is captured the DMD-loaded image is translated in the x and y directions to change the phase of the structured light, and each structured light illumination pattern is in x and
  • the phase ( ⁇ x, ⁇ y) in the y direction is (0, 0), ( ⁇ /5, ⁇ /5), (2 ⁇ /5, 2 ⁇ /5), (3 ⁇ /5, 3 ⁇ /5), (4 ⁇ /). 5,4 ⁇ /5).
  • the main time spent on imaging is the time interval between the integration time taken by the EMCCD and the original image. Set the time for integrating each original image to 20ms. After the end of the integration, the interval is 10ms. Switch the DMD loading pattern (structured light illumination pattern) and start collecting the next original image. It can be seen from the calculation that with the existing imaging method, a SIM image can be obtained every 150 ms, that is, every five original images are acquired, and the motion of the sample within the 150 ms is not known.
  • image reconstruction can be performed every 30 ms, that is, every time an original image is acquired, and a SIM super-resolution image is obtained, and motion information of the sample to be tested in a shorter time can be obtained.
  • the time interval for obtaining the SIM image is significantly shortened compared with the existing imaging method, and the time resolution of the SIM imaging is remarkably improved.
  • FIG. 6 is a schematic structural diagram of an image forming apparatus of a structured light illumination microscope according to an embodiment of the present invention.
  • the imaging apparatus of the structured light illumination microscope of the present embodiment includes a pattern switching unit 601, an original image acquisition unit 602, and an image reconstruction unit 603.
  • the pattern switching unit 601 is configured to cyclically switch the preset N pieces of structured light illumination patterns according to a preset sequence, where N is a preset constant;
  • the original image obtaining unit 602 is configured to obtain an original image of the sample to be imaged under each structured light illumination pattern, to obtain an original image sequence of the sample to be imaged;
  • the image reconstruction unit 603 is configured to perform image reconstruction on each of the original image sequences and the N-1 original images after the original image sequence to obtain a super-resolution image sequence of the sample to be imaged.
  • the imaging device of the structured light illumination microscope of the present embodiment can effectively improve the temporal resolution of the super-resolution image of the structured light illumination microscope.
  • the invention provides an imaging method for a structured light illumination microscope, comprising: cyclically switching a preset N-structure light illumination pattern according to a preset sequence, N is a preset constant; and acquiring a sample to be imaged under each structured light illumination pattern Raw image, obtaining an original image sequence of the sample to be imaged; reconstructing each original image in the original image sequence and N-1 original images after the image to obtain a super-resolution image of the sample to be imaged sequence.
  • the super-resolution image sequence of the sample to be imaged changes with time, and the time resolution is greatly improved.
  • nonlinear structured light illumination microscope NL-SIM nonlinear structured light illumination microscope NL-SIM, total internal reflection fluorescence structure light illumination microscope TIRF-SIM, three-dimensional super-resolution imaging structured light illumination microscope 3D - Lattice-lightsheet-SIM combined with SIM, light microscopy and structured light illumination microscope.
  • TIRF-SIM total internal reflection fluorescence structure light illumination microscope
  • 3D three-dimensional super-resolution imaging structured light illumination microscope
  • 3D - Lattice-lightsheet-SIM combined with SIM
  • light microscopy and structured light illumination microscope light microscopy and structured light illumination microscope.
  • the super-resolution image sequence of the sample to be imaged can be accurately obtained, and the process of changing the sample to be tested can be obtained, and the time resolution of the structured light illumination microscope SIM can be improved.
  • An imaging device for a structured light illumination microscope comprising: a pattern switching unit, configured to cyclically switch a preset N-structure light illumination pattern according to a preset sequence, where N is a preset constant; and an original image acquisition unit configured to acquire a sample to be imaged An original image sequence of the sample to be imaged is obtained from an original image under each structured light illumination pattern; an image reconstruction unit for using each of the original image sequences and N-1 originals The image is subjected to image reconstruction to obtain a super-resolution image sequence of the sample to be imaged.
  • a super-resolution image sequence of the sample to be imaged as a function of time is obtained, and the time interval of each of the two super-resolution images is equal to the time interval of capturing each of the two original images, compared with the prior art structured light illumination microscope imaging method.
  • the temporal resolution of the imaging method and apparatus of the present invention is greatly improved.

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Abstract

L'invention concerne un procédé et un appareil d'imagerie pour un microscope à éclairage structuré. Le procédé d'imagerie comprend : la commutation cyclique de N motifs d'éclairage structuré prédéfinis selon une séquence prédéfinie, N étant une constante prédéfinie (S21); l'acquisition d'une image originale, sous chacun des motifs d'éclairage structuré, d'un échantillon à imager, et l'obtention d'une séquence d'images originales de l'échantillon à imager (S22); et la réalisation d'une reconstruction d'image sur chaque image originale et sur (N-1) des images originales suivantes dans la séquence d'images originales, et l'obtention d'une séquence d'images à super-résolution de l'échantillon à imager (S23). Selon le procédé et l'appareil, on peut obtenir une séquence d'images à super-résolution, qui varie dans le temps, d'un échantillon à imager et un intervalle de temps entre deux images à super-résolution est égal à un intervalle de temps de photographie de deux images originales, ce qui permet d'améliorer un rapport de résolution temporelle.
PCT/CN2016/112637 2016-03-10 2016-12-28 Procédé et appareil d'imagerie pour microscope à éclairage structuré Ceased WO2017152702A1 (fr)

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CN201610136330.2A CN105589188B (zh) 2016-03-10 2016-03-10 一种结构光照明显微镜的成像方法及装置

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CN109724951A (zh) * 2017-10-27 2019-05-07 黄晓淳 一种动态超分辨荧光成像技术
CN110223238A (zh) * 2019-04-30 2019-09-10 北京理工大学 编码光照成像重构方法及装置
CN110308125A (zh) * 2019-07-11 2019-10-08 清华大学 三维显微层析计算摄像方法及装置
CN111308682A (zh) * 2019-11-18 2020-06-19 天津大学 基于结构光照明的超分辨重构方法
CN112508791A (zh) * 2020-12-18 2021-03-16 中国工程物理研究院激光聚变研究中心 一种初始相位提取方法、装置、电子设备及存储介质
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CN116402678A (zh) * 2022-12-19 2023-07-07 中国科学院苏州生物医学工程技术研究所 超分辨结构光照明显微镜的频谱优化直接重建方法
CN116402678B (zh) * 2022-12-19 2023-10-20 中国科学院苏州生物医学工程技术研究所 超分辨结构光照明显微镜的频谱优化直接重建方法
CN118982461A (zh) * 2024-10-22 2024-11-19 中国科学院苏州生物医学工程技术研究所 一种空频域联合降噪的结构光照明超分辨图像重建方法
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