CN115420721A - Three-dimensional optical sheet super-resolution imaging method and device based on fluorescence radial fluctuation algorithm - Google Patents
Three-dimensional optical sheet super-resolution imaging method and device based on fluorescence radial fluctuation algorithm Download PDFInfo
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Abstract
The invention discloses a three-dimensional optical sheet super-resolution imaging method and device based on a fluorescence radial fluctuation algorithm. The light beam modulation module generates exciting light and vortex hollow loss light respectively, then the exciting light and the vortex hollow loss light are coupled and coaxial, a light sheet is formed through a cylindrical lens and an illumination objective lens, only fluorescence at the hollow position of the loss light is excited, axial super-resolution imaging is realized, the imaging module collects and records a series of sample fluorescence images on an xy plane of a sample, a computer analyzes fluorescence fluctuation signals of the series of fluorescence images, and an image sequence is calculated and reconstructed by utilizing a fluorescence radial fluctuation algorithm to obtain a transverse resolution (-100 nm) super-resolution image; and moving the objective table to realize the step-by-step scanning of the sample in the z direction, completing the step of S2 at each layer, and after collecting the super-resolution images at all positions, performing three-dimensional reconstruction on all transverse super-resolution images to obtain the final three-dimensional super-resolution (-100 nm) imaging.
Description
Technical Field
The invention relates to the technical field of optical microscopy, in particular to a three-dimensional optical sheet super-resolution imaging method and device based on a fluorescence radial fluctuation algorithm.
Background
In the biological science research, a microscope is used as an essential tool for researching the micro world, and an optical microscope is also used as a necessary tool for observing living cells. In an optical microscope, the light sheet microscopic imaging technology is a three-dimensional microscopic imaging technology, which forms a light beam into a thin sheet to scan in one direction of a sample, receives fluorescence in a direction perpendicular to the scanning direction, and takes an image. The imaging mode has the advantages of high penetration depth, low bleaching, high imaging speed and the like. However, the presence of diffraction limits in optical microscopy has prevented the search for smaller cell structures. Super-resolution imaging techniques refer to several types of methods for breaking through the diffraction limit of optics, one of which is called stimulated emission depletion (STED), which is a method for breaking through the diffraction limit by reducing the point spread function. Specifically, one beam of excitation light excites the fluorescent substance in the range of the light spot to emit a small range of fluorescence. Meanwhile, a beam of high-power-loss hollow light with another wavelength is emitted along the optical axis direction of the light beam, the fluorescent molecules which generate fluorescence near the fluorescent center are subjected to stimulated radiation, and the fluorescence emission is stopped, so that the fluorescent range is reduced, and the imaging resolution is improved. Another super-resolution technique, called fluorescence radial fluctuation algorithm (SRRF), is to calculate the radial gradient change of the fluorescence signal of the fluorescent protein according to the characteristic of the fluctuation (flicker) of the signal along with time so as to distinguish two close fluorescent molecules, thereby realizing super-resolution imaging. In light sheet microscopy, the diffraction limit is limited in all three dimensions: the xy plane resolution is about 250nm to 300nm, the z direction resolution is 500nm-1 μm, the three-dimensional resolution is limited while the resolution is not uniform in the xz (yz) direction. Such resolution makes it difficult to observe details of biological cellular structures and subcellular organelles. In order to improve the resolution, researchers have proposed lattice structure optical sheet imaging based on interference, which realizes super-resolution imaging in a certain direction, but cannot realize three-dimensional balanced super-resolution imaging of all the same properties. Therefore, the realization of three-dimensional balanced super-resolution imaging with the resolution ratio superior to 100nm has extremely important significance.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a three-dimensional optical sheet super-resolution imaging method and device based on a fluorescence radial fluctuation algorithm.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for realizing imaging of a three-dimensional optical sheet super-resolution imaging device based on a fluorescence radial fluctuation algorithm is characterized by comprising the following steps:
the method comprises the following steps that S1, a light beam modulation module is utilized to respectively generate exciting light and vortex hollow loss light, the exciting light and the vortex hollow loss light are coupled and coaxial, a light sheet is formed through a cylindrical lens and an illumination objective lens, only fluorescence at the hollow position of the loss light is excited, and axial super-resolution imaging (about 100 nm) is achieved;
s2, the imaging module acquires and records a series of sample fluorescence images on a sample xy plane, the computer analyzes fluorescence fluctuation signals of the series of fluorescence images, and an image sequence is calculated and reconstructed by utilizing a fluorescence radial fluctuation algorithm to obtain a super-resolution image with transverse resolution (100 nm);
and S3, moving the objective table to realize the step-by-step scanning of the sample in the z direction, completing the step S2 at each layer, and after collecting the super-resolution images at all positions, performing three-dimensional reconstruction on all transverse super-resolution images to obtain the final three-dimensional super-resolution (-100 nm) imaging.
In step S1, a fluorescent protein, a commercial organic dye probe, a fluorescent quantum dot, an upconversion nanoprobe, or the like is used, and the fluorescent probe has the characteristic of fluorescence fluctuation, so as to mark the sample to be detected.
It should be noted that the present invention utilizes the basic principle of stimulated radiation loss to generate super-resolution in the axial direction:
the vortex hollow light formed by the loss light shown in fig. 3 is nested on the excitation light beam, the excitation light beam is responsible for exciting the fluorescent molecules in the focal range, and the vortex hollow loss light is responsible for causing the fluorescent molecules in the edge circle to generate excited radiation, so that the fluorescence is quenched, and the fluorescent molecules in the central range are left to continuously emit fluorescence. Wherein, the loss scope of loss light, along with the increase of loss light intensity and to fluorescence central diffusion, cause the fluorescence molecule of exciting light beam excitation to reduce from the outer lane gradually, the fluorescence facula diminishes to make resolution ratio promote, the relation of resolution ratio and loss light intensity is represented by the following formula:
where d is the limiting resolution, λ is the wavelength of the lost light, NA is the numerical aperture of the illumination objective, I is the intensity of the lost light, I is sat Is the saturation intensity, i.e., the loss intensity required to quench 90% of the luminescence of the fluorescent molecule within the illumination range. When the composite light beam passes through the cylindrical lens, a sandwich sugar-shaped light sheet is formed, and the outermost layer of light is the loss light and the exciting light and is responsible for quenching fluorescent molecules excited by the exciting light sheet. When the loss light intensity is greater than the saturation light intensity and is enhanced, the sandwich part of the composite nested light is reduced, and the equivalent thickness of the optical sheet is thinned, so that the axial resolution of the optical sheet is improved.
It should be noted that the principle of the fluorescence radial fluctuation algorithm for improving the resolution is shown in fig. 5: the fluorescence emitted by the fluorescent molecules in the image shot by the imaging camera is analyzed to have the fluorescence fluctuation property, and simultaneously, the point spread function of one fluorescent molecule is in three-dimensional Gaussian distribution. According to the algorithm, the gradient field of the point spread function is calculated according to the fluorescence fluctuation property of the point spread function, and finally, according to the convergence degree of the obtained point spread function gradient field, the point spread function after calculation is found to be converged to the center and is obviously reduced, so that plane super-resolution is realized. In addition, cross-correlation analysis of multiple pictures is also beneficial to improving the signal-to-noise ratio of the images.
The invention also provides a three-dimensional optical sheet super-resolution imaging device based on the fluorescence radial fluctuation algorithm, which comprises a light beam modulation module, a light path module and an imaging module, wherein:
the light beam modulation module comprises a first laser, a second laser, a telescope system, a spiral phase plate, a spatial light modulator and a reflector; the first laser is a low-power continuous light or pulse laser for generating exciting light with a first wavelength, and the second laser is a continuous light or pulse laser for generating loss light with a second wavelength; the telescope system is respectively arranged behind the first laser and the second laser; the spatial light modulators are respectively arranged behind the telescope modules and used for shaping the exciting light and the loss light so as to generate special light beams required by experiments, such as Gaussian light beams, bessel light beams, airy light beams and the like;
the light path module consists of a dichroic mirror, a cylindrical lens, a reflecting mirror and an illumination objective lens, wherein the modulated exciting light and the loss light are coaxially coupled through the dichroic mirror; the composite light beam is irradiated to the cylindrical lens and the illumination objective lens through the adjustment of the reflector to form a light sheet, the exciting light can excite fluorescence, and the lost light can lose the fluorescence, namely inhibit the fluorescence, so that the light sheet formed by the coaxial coupling light beam through the cylindrical lens has ultrathin effective excitation thickness, and the axial super-resolution imaging is finally realized;
the imaging module consists of an objective table, a detection objective lens, a fluorescence filter, a tube lens, an imaging camera and a computer; the objective table is used for moving the sample to enable the illuminating light sheet to excite each position of fluorescence of the sample, and three-dimensional imaging is achieved; the sample radiation fluorescence can generate the phenomenon that the fluorescence signal fluctuates along with time, and after the fluorescence signal enters a detection objective lens, the fluorescence filter filters the unwanted scattered light and other interference light signals; the lens cone lens focuses and images the preprocessed fluorescence fluctuation signals on a photosensitive surface of an imaging camera; the imaging camera records fluorescence fluctuation information to obtain an image sequence, and the computer calculates and processes the image sequence through a fluorescence radial fluctuation algorithm and reconstructs the image sequence to obtain a transverse resolution super-resolution image; after the transverse super-resolution images of all the layers are collected and calculated, the computer carries out three-dimensional reconstruction on all the images to form the required three-dimensional super-resolution images.
It should be noted that, after the sample on the plane is covered by the illuminating light sheet and imaged, the stage continues to move one layer along the axial direction, and the stage repeatedly moves in a circulating manner in sequence until all the fluorescence of the sample is excited, and then the imaging operation is completed.
In the loss light modulation optical path of the light beam adjustment module, the spiral phase plate is located behind the spatial light modulator, and is used for reshaping the loss light and modulating the loss light into a vortex hollow light beam, and the vortex hollow light beam enters the subsequent optical path module through the reflector.
The object stage consists of a three-dimensional electric support frame and a sample chamber; the three-dimensional electric support frame freely moves along the three directions of x, y and z according to the control of a computer and can rotate along the support shaft so as to realize the uniform illumination of each position and each direction of the sample.
It should be noted that the telescope system is composed of two lenses and used for expanding and collimating the laser beam.
The invention has the beneficial effects that:
1. meanwhile, the application of the super-resolution technology in the optical sheet microscopic imaging technology in the axial direction and the transverse direction is realized, and the assistance of researching the organelle of the living cell is realized.
2. The disadvantage of STED low signal-to-noise ratio is complemented with the advantage that SRRF can work under low signal-to-noise ratio and improve the signal-to-noise ratio, and the image signal-to-noise ratio is improved.
3. The invention realizes the light sheet microscopic isotropy three-dimensional imaging by utilizing the advantage of uniform imaging of the STED super-resolution technology.
Drawings
FIG. 1 is a schematic diagram of the general concept of the present invention
FIG. 2 is a schematic diagram of a three-dimensional optical sheet super-resolution imaging device based on a fluorescence radial fluctuation algorithm according to the present invention;
FIG. 3 is a schematic diagram of how stimulated radiation loss improves axial resolution according to the present invention (only the stimulated portion is shown);
FIG. 4 is a schematic view of the stage structure of the present invention;
FIG. 5 is a diagram illustrating the improvement of lateral resolution by the fluorescence radial fluctuation algorithm;
FIG. 6 is a flow chart of a three-dimensional optical sheet super-resolution imaging method based on a fluorescence radial fluctuation algorithm.
Detailed Description
The present invention will be further described with reference to the accompanying drawings, and it should be noted that the present embodiment is based on the technical solution, and the detailed implementation and the specific operation process are provided, but the protection scope of the present invention is not limited to the present embodiment.
Example 1
The method for the three-dimensional optical sheet super-resolution imaging based on the fluorescence radial fluctuation algorithm is completed by the following steps:
(1) Samples to be tested were prepared and labeled with fluorescent protein and placed in collagen in the sample chamber.
(2) The two lasers 1 and 2 respectively generate exciting light and loss light, the exciting light and the loss light are coupled and coaxially transmitted after passing through the light beam modulation module, an illuminating light sheet is formed by sequentially passing through the cylindrical lens 13 and the illuminating objective lens 14, only the sample fluorescence at the hollow position of the loss light is excited, and axial super-resolution imaging is realized.
(3) The imaging module collects and records a series of sample fluorescence images on a sample xy plane, the computer 20 analyzes fluorescence fluctuation signals of the series of fluorescence images, and calculates and reconstructs an image sequence of a fluorescence radial fluctuation algorithm to obtain a super-resolution image with transverse resolution (100 nm).
(4) After the calculation is completed, the computer 20 controls the stage 15 to move, so as to collect a fluorescence image of another position of the sample, and repeats the step (3) of completing the calculation of the position, and collecting an image signal of the next position after the reconstruction.
(5) And after collecting the super-resolution images at all positions, performing three-dimensional reconstruction on all the collected transverse super-resolution images to obtain the final three-dimensional super-resolution (-100 nm) imaging.
Fig. 2 is a schematic diagram of a three-dimensional optical sheet super-resolution imaging device based on a fluorescence radial fluctuation algorithm, in which 1 is a low-power continuous laser with a wavelength of 640nm, 2 is a pulse laser with a wavelength of 750nm, 3, 4, 5, and 6 are lenses, 7 and 8 are spatial light modulators, 9 is a spiral phase plate, 10 and 12 are reflectors, 11 is a long-pass dichroic mirror with a cutoff frequency of 700nm, 13 is a cylindrical lens, 14 is an illumination objective lens, 15 is a stage, 16 is a detection objective lens, 17 is a color filter, 18 is a tube lens, 19 is an imaging camera, and 20 is a computer.
In specific implementation, the method is implemented by the following modules including a beam modulation module, a light path module and an imaging module. The light beam modulation module is divided into two light paths, namely a light path 1 and a light path 2, and the light path 1 and the light path 2 respectively generate and modulate excitation light and vortex hollow loss light. In the light path 1, excitation light is emitted by a low-power continuous laser 1 with the wavelength of 640nm, is collimated and expanded through lenses 3 and 5 and irradiates on a spatial light modulator 7, and the spatial light modulator modulates the excitation light into a Bessel light beam and reflects the excitation light onto a long-pass dichroic mirror 11. In the optical path 2, the loss light is emitted from a pulse laser 2 having a wavelength of 750nm, collimated and expanded by lenses 4 and 6, and irradiated onto a spatial light modulator 8 at an appropriate angle to modulate the loss light into a bessel beam. The lost light then passes through the spiral phase plate 9, is modulated into a swirling hollow beam, and is reflected via a mirror 10 onto a long-pass dichroic mirror 11. In the optical path module, a long-pass dichroic mirror 11 couples the modulated excitation light and the vortex hollow loss light coaxially, and the modulated composite light beam passes through a reflecting mirror 12 and a cylindrical lens 13 and a lighting objective lens 14 in sequence to form a light sheet to excite a sample to emit fluorescence as shown in fig. 3. The fluorescent protein at the overlapped part of the loss light and the exciting light does not emit light, and only the central exciting light excites the fluorescent protein to emit fluorescence, thereby realizing axial super-resolution imaging. Fluorescence fluctuation signals emitted by the sample are collected by a detection objective lens 16, unwanted stray light is filtered by a color filter 17, and finally the stray light is imaged on a light-sensitive surface of an imaging camera 19 through a tube lens 18, and the camera shoots an image sequence and transmits the image sequence to a computer 20 to calculate the image sequence by using a fluorescence radial fluctuation algorithm and reconstruct the image sequence, so that a transverse super-resolution image is formed finally.
Further, after acquiring the transverse super-resolution image of one position, the computer 20 controls the stage 15 to move, and further collects the fluorescence image of another position of the sample, until all positions of the sample are scanned, the computer 20 performs three-dimensional reconstruction on all calculated transverse super-resolution images to form a three-dimensional super-resolution image of the sample.
Specifically, after the sample on the plane where the illuminating light sheet is located is completely covered, the object stage continues to move one layer along the axial direction, and the object stage repeatedly moves in a circulating mode in sequence until all parts of fluorescence of the sample are excited, and then imaging work is completed.
Specifically, the object stage 15 is composed of a three-dimensional electric support frame and a sample chamber, and the object stage 15 can move along the x, y and z directions under the control of the computer 20 and can rotate along a support shaft, so as to realize uniform illumination of samples at all positions and in all directions.
Various other changes and modifications to the above-described embodiments and concepts will become apparent to those skilled in the art from the above description, and all such changes and modifications are intended to be included within the scope of the present invention as defined in the appended claims.
Claims (9)
1. A method for realizing imaging of a three-dimensional optical sheet super-resolution imaging device based on a fluorescence radial fluctuation algorithm is characterized by comprising the following steps:
the method comprises the following steps that S1, a light beam modulation module is utilized to respectively generate exciting light and vortex hollow loss light, the exciting light and the vortex hollow loss light are coupled and coaxial, a light sheet is formed through a cylindrical lens and an illumination objective lens, only fluorescence at the hollow position of the loss light is excited, and axial super-resolution imaging (about 100 nm) is achieved;
s2, the imaging module collects and records a series of sample fluorescence images on a sample xy plane, the computer analyzes fluorescence fluctuation signals of the series of fluorescence images, and an image sequence is calculated and reconstructed by utilizing a fluorescence radial fluctuation algorithm to obtain a super-resolution image with transverse resolution (100 nm);
and S3, moving the objective table to realize the step-by-step scanning of the sample in the z direction, completing the step S2 at each layer, and after collecting the super-resolution images at all positions, performing three-dimensional reconstruction on all transverse super-resolution images to obtain the final three-dimensional super-resolution (-100 nm) imaging.
2. The fluorescence radial fluctuation algorithm-based three-dimensional optical sheet super-resolution imaging method according to claim 1, wherein in the step S1, a fluorescent protein, a commercial organic dye probe, a fluorescent quantum dot, an up-conversion nanoprobe, etc. are used, and the fluorescent probe has the characteristic of fluorescence fluctuation and marks the sample to be detected.
3. The method for super-resolution imaging of the three-dimensional optical sheet based on the fluorescence radial fluctuation algorithm according to claim 1, wherein the method utilizes the basic principle that the stimulated radiation loss generates super-resolution in the axial direction: vortex hollow light formed by the loss light is nested on the excitation light beam, the excitation light beam is responsible for exciting fluorescent molecules, the vortex hollow loss light is responsible for enabling the excited fluorescent molecules around to generate excited radiation to quench fluorescence, the equivalent thickness of the optical sheet is thinned, and therefore the axial resolution of the optical sheet is improved.
4. The three-dimensional optical sheet super-resolution imaging method based on the fluorescence radial fluctuation algorithm of claim 1, wherein the fluorescence radial fluctuation algorithm improves the transverse resolution principle, the algorithm calculates the gradient field of the point spread function according to the fluorescence fluctuation property of the fluorescence probe along with time, and finally finds that the calculated point spread function converges to the center and becomes significantly smaller according to the convergence degree of the obtained point spread function gradient field, thereby realizing transverse super-resolution. In addition, cross-correlation analysis of multiple pictures is also beneficial to improving the signal-to-noise ratio of the images.
5. The three-dimensional optical sheet super-resolution imaging device based on the fluorescence radial fluctuation algorithm of any one of claims 1 to 4, wherein the device comprises a beam modulation module, an optical path module and an imaging module, wherein:
the light beam modulation module comprises a first laser, a second laser, a telescope system, a spiral phase plate, a spatial light modulator and a reflector; the first laser is a low-power continuous light or pulse laser for generating exciting light with a first wavelength, and the second laser is a continuous light or pulse laser for generating loss light with a second wavelength; the telescope system is respectively arranged behind the first laser and the second laser; the spatial light modulators are respectively arranged behind the telescope modules and used for shaping the exciting light and the loss light so as to generate special light beams required by experiments, such as Gaussian light beams, bessel light beams, airy light beams and the like;
the light path module consists of a dichroic mirror, a cylindrical lens, a reflecting mirror and an illumination objective lens, wherein the modulated exciting light and the loss light are coaxially coupled through the dichroic mirror; the composite light beam is irradiated to the cylindrical lens and the illumination objective lens through the adjustment of the reflector to form a light sheet, the exciting light can excite fluorescence, and the lost light can lose the fluorescence, namely inhibit the fluorescence, so that the light sheet formed by the coaxial coupling light beam through the cylindrical lens has ultrathin effective excitation thickness, and the axial super-resolution imaging is finally realized;
the imaging module consists of an objective table, a detection objective, a fluorescence filter, a tube lens, an imaging camera and a computer; the objective table is used for moving the sample to enable the illuminating light sheet to excite each position of fluorescence of the sample, and three-dimensional imaging is achieved; the sample radiation fluorescence can generate the phenomenon that the fluorescence signal fluctuates along with time, and after the fluorescence signal enters a detection objective lens, the fluorescence filter filters the unwanted scattered light and other interference light signals; the lens cone lens focuses and images the preprocessed fluorescence fluctuation signals on a photosensitive surface of an imaging camera; the imaging camera records fluorescence fluctuation information to obtain an image sequence, and the computer calculates and processes the image sequence through a fluorescence radial fluctuation algorithm and reconstructs the image sequence to obtain a transverse resolution super-resolution image; after the transverse super-resolution images of all the layers are collected and calculated, the computer carries out three-dimensional reconstruction on all the images to form the required three-dimensional super-resolution images.
6. The fluorescence radial fluctuation algorithm-based three-dimensional optical sheet super-resolution imaging device according to claim 5, wherein after the illumination optical sheet covers the sample on the plane and images, the stage moves one layer along the axial direction again, and the stage moves in a cycle in sequence until all the fluorescence of the sample is excited, thereby completing the imaging.
7. The fluorescence radial fluctuation algorithm-based three-dimensional optical sheet super-resolution imaging device according to claim 5, wherein in the loss light modulation optical path of the light beam adjusting module, a spiral phase plate is located behind the spatial light modulator, and is used for reshaping and modulating the loss light into a vortex hollow light beam, and the vortex hollow light beam enters the subsequent optical path module through the reflector.
8. The fluorescence radial fluctuation algorithm-based three-dimensional optical sheet super-resolution imaging device according to claim 5, wherein the object stage is composed of a three-dimensional electric support frame and a sample chamber; the three-dimensional electric support frame freely moves along the three directions of x, y and z according to the control of a computer and can rotate along the support shaft so as to realize the uniform illumination of each position and each direction of the sample.
9. The fluorescence radial fluctuation algorithm-based three-dimensional optical sheet super-resolution imaging device according to claim 5, wherein the telescope system comprises two lenses for expanding and collimating the laser beam.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104764729A (en) * | 2015-04-22 | 2015-07-08 | 华南师范大学 | Up-conversion-nanocrystal-based stimulated depletion super-resolution optical microscopic method and up-conversion-nanocrystal-based stimulated depletion super-resolution optical microscopic system |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104764729A (en) * | 2015-04-22 | 2015-07-08 | 华南师范大学 | Up-conversion-nanocrystal-based stimulated depletion super-resolution optical microscopic method and up-conversion-nanocrystal-based stimulated depletion super-resolution optical microscopic system |
Non-Patent Citations (4)
| Title |
|---|
| NILS GUSTAFSSON 等: "Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations", 《NATURE COMMUNICATIONS》, 12 August 2016 (2016-08-12), pages 1 - 9 * |
| 曾志平: "基于荧光随机涨落的超分辨率显微成像", 《中国激光》, vol. 45, no. 03, 31 March 2018 (2018-03-31), pages 0307009 - 1 * |
| 李雨竹 等: "基于单分子定位成像技术的研究及展望", 《激光与光电子学进展》, vol. 57, no. 24, 31 December 2020 (2020-12-31), pages 240002 - 1 * |
| 邱雪 等: "基于微球调制光场的超分辨成像及荧光增强", 《激光与光电子学进展》, 8 August 2022 (2022-08-08), pages 11 - 28 * |
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