A near-infrared structured light illumination light sheet imaging method and device based on fluorescence loss super-resolution method

Abstract

The present invention discloses a near-infrared structured light illumination light sheet imaging method and device based on the fluorescence loss super-resolution method, wherein the near-infrared excitation light and the near-infrared vortex hollow loss light generated by the beam modulation module form a coaxial coupled light beam. The near-infrared excitation light can excite the sample fluorescence, and the near-infrared vortex hollow loss light can deplete the sample fluorescence in the action area, so as to realize axial super-resolution (resolution of about 100nm) imaging. The coaxial coupled light beam leads out two coherent lights through a semi-reflective and semi-mirror lens, and each of them passes through a cylindrical lens and is conjugated to the coherent light sheet formed by the rear pupils of two objective lenses to propagate in the opposite direction in the imaging plane and interfere to form a structured illumination pattern to illuminate the sample. In the imaging module, the computer performs spectrum separation and reconstruction on the collected fluorescence images, and then takes out all the images and performs three-dimensional reconstruction, finally realizing large-field three-dimensional super-resolution (resolution of about 100nm) imaging with a large imaging depth and anti-sample scattering capability.

Description

Technical Field

[0001] The present invention relates to the technical field of optical microscopy, and in particular to a near-infrared structured light illumination light sheet imaging method and device based on a fluorescence loss super-resolution method. Background Art

[0002] Optical microscopy is an important means of studying the life activities of living cells in modern life sciences. Light sheet microscopy is a fast, highly penetrating, low-phototoxic three-dimensional microscopy technology. Structurally, it uses two or more objective lenses to illuminate the sample and detect the fluorescence signal, turning the light beam into a thin sheet to excite the sample from the side and receive the fluorescence signal in the vertical direction of the light sheet. After the advent of light sheet microscopy, how to achieve three-dimensional super-resolution imaging under a large field of view has always been the focus of research in this field. The existing laser light source of light sheet imaging technology is mainly visible light, but visible light is greatly affected by scattering in sample tissues, and the tissue penetration ability is poor, so its propagation field of view is relatively small. In order to improve the imaging field of view, some researchers have proposed a simple solution of increasing the laser wavelength, such as using near-infrared light excitation, but it often leads to a worse axial resolution of the light sheet, and the axial resolution can only achieve 1-2μm, and it is impossible to achieve axial super-resolution imaging below 100nm. In order to improve the axial resolution, some researchers have proposed a solution using a shorter excitation wavelength, but this solution will sacrifice the imaging field of view. At the same time, some researchers have proposed using structured light technology to improve the lateral or axial resolution of light sheets, but the improved resolution is mainly concentrated in a certain direction, making it difficult to achieve sub-100nm 3D super-resolution imaging. Therefore, based on the problems of existing light sheet imaging technology, a 3D super-resolution (sub-100nm) imaging technology and imaging device with a large field of view is urgently needed. Summary of the invention

[0003] In view of the shortcomings of the prior art, the present invention aims to provide a near-infrared structured light illumination light sheet imaging method and device based on the fluorescence loss super-resolution method, which specifically utilizes a beam modulation module, an optical path module, and an imaging module to structurally realize stimulated emission loss and structured light illumination at the same time, thereby realizing three-dimensional super-resolution light sheet imaging.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0005] A near-infrared structured light illumination light sheet imaging method based on fluorescence loss super-resolution method, characterized in that the method comprises the following steps:

[0006] The near-infrared excitation light and near-infrared vortex hollow loss light generated by the S1 beam modulation module form a coaxial coupled beam. The near-infrared excitation light can excite sample fluorescence, and the near-infrared vortex hollow loss light can deplete sample fluorescence in the action area. Therefore, the sandwich light sheet formed by the coaxial coupled beam through the cylindrical lens has an ultra-thin effective excitation thickness and the near-infrared has a large imaging depth and anti-sample scattering ability, which can achieve large-field axial super-resolution imaging (resolution of about ~100nm).

[0007] The S2 coaxial coupling beam leads to two coherent lights through a half-reflecting half-mirror lens, which are conjugated to the coherent light sheets formed by the rear pupils of the two objective lenses after passing through the cylindrical lenses, propagate in the opposite direction in the imaging plane and interfere to form a structured illumination pattern to illuminate the sample.

[0008] In the S3 imaging module, the computer performs spectrum separation and reconstruction on the collected fluorescence images in different phases and directions to obtain a lateral super-resolution image (resolution of about 100 nm) of a layer, and moves the stage axially to achieve a three-dimensional scan of the sample. Finally, all images are taken out and reconstructed in three dimensions to obtain a large-field three-dimensional super-resolution (resolution of about 100 nm) sample image.

[0009] It should be noted that the outermost layer of the sandwich light sheet is loss light, and the middle layer is excitation light; when irradiating the sample, the fluorescence irradiated by the overlapping part of the loss light beam and the excitation light beam is lost and no fluorescence is generated; only the fluorescent molecules in the part illuminated by the middle excitation light beam are excited to emit fluorescence; the effective excitation thickness of the excitation light beam becomes thinner, thereby improving the axial resolution of the system; the two beams of the sandwich light sheet are distributed in stripes on the xy plane due to interference, and the loss light has a different wavelength from the excitation light, so the structural stripe distribution may have a slight offset and cannot be completely overlapped. The displacement deviation can be corrected by a spatial light modulator to make them correspond one to one.

[0010] It should be noted that the phase and direction changes of the relative stripes of the illuminated sample are controlled by the stage, so as to achieve spectrum translation and expansion in all directions, thereby realizing lateral super-resolution imaging in the xy plane.

[0011] It should be noted that the biological sample is labeled with a fluorescent probe, wherein the fluorescent probe includes any one of a near-infrared excited up-conversion fluorescent probe, an organic dye, and a quantum dot.

[0012] On the other hand, the present invention also provides a device for near-infrared structured light illumination light sheet imaging based on the fluorescence loss super-resolution method, the device includes a beam modulation module, an optical path module and an imaging module, wherein the beam modulation module is composed of 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 pulsed light laser used to generate near-infrared excitation light, and the second laser is a low-power continuous light or pulsed light laser used to generate near-infrared loss light; the telescope system is respectively placed behind the first laser and the second laser; the spatial light modulator is respectively deployed behind the telescope system, for shaping the excitation light and the loss light and generating the special light beam required for the experiment;

[0013] The optical path module consists of a dichroic mirror, a half-reflecting half-mirror, a reflector group, a cylindrical lens, and an illumination objective lens; the modulated near-infrared excitation light and the near-infrared vortex hollow loss light generated by the beam modulation module are coaxially coupled through the dichroic mirror, and the half-reflecting half-mirror splits the composite light beam into two paths, which are respectively adjusted to the same optical axis through the reflector group for reverse transmission; then, each passes through the same cylindrical lens and the illumination objective lens to form two reversely transmitted light sheets to interfere with each other to generate a structured illumination pattern, and excite the fluorescent molecules in the sample to emit fluorescence;

[0014] The imaging module consists of a stage, a detection lens, a color filter, a tube lens, an imaging camera, and a computer. The fluorescence emitted by the sample on the stage is collected by the detection lens, which is located in the vertical direction of the light sheet. The collected fluorescence is filtered out by the color filter to remove other stray light except the signal light, which is beneficial to improve the signal-to-noise ratio. The filtered fluorescence is imaged on the photosensitive surface of the imaging camera through the tube lens, and the imaging camera imports the captured image into the computer. The computer separates and reconstructs the spectrum of the collected image, and finally obtains a lateral super-resolution image.

[0015] It should be noted that the stage consists of a three-dimensional electric platform and a sample chamber; the three-dimensional electric platform can move along the x, y, and z directions according to computer control and can rotate along the support axis to achieve uniform illumination of the sample at all positions and in all directions.

[0016] It should be noted that the telescope system consists of two lenses, which are used for expanding and collimating the laser beam.

[0017] It should be noted that in the beam modulation module, the spiral phase plate is located after the spatial light modulator, and is used to reshape the generated special light beam to form a near-infrared vortex hollow light beam, which enters the optical path module through the reflector.

[0018] It should be noted that the illumination objective lens is mirror-distributed relative to the optical axis of the detection objective lens and is coaxially used for sandwich light sheet interference to generate a structured illumination pattern, such as Figure 5shown.

[0019] The beneficial effects of the present invention are:

[0020] 1. Both lateral and axial resolutions are improved, achieving three-dimensional isotropic sub-100nm super-resolution imaging

[0021] 2. Since the sample is excited by near-infrared excitation light, it provides a large field of view during light sheet microscopy imaging.

[0022] 3. This super-resolution imaging technology has the advantages of no photobleaching and ultra-long time-course imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic diagram of a near-infrared structured light illumination light sheet imaging device based on the fluorescence loss super-resolution method of the present invention;

[0024] Figure 2 The present invention relates to an up-conversion fluorescent probe (NaYF4: 18% Yb 3+ ,10% Tm 3+ )'s spectral response curve and a schematic diagram of the experimental image;

[0025] Figure 3 A schematic diagram of the sandwich structured light sheet morphology and how to improve the axial resolution of the present invention;

[0026] Figure 4 A schematic diagram of how the structured light sheet of the present invention improves resolution;

[0027] Figure 5 A schematic diagram of a stage structure according to the present invention;

[0028] Figure 6 The present invention is a flow chart of a near-infrared structured light illumination light sheet imaging method based on fluorescence loss super-resolution method. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with the accompanying drawings. It should be noted that this embodiment is based on the technical solution and provides a detailed implementation method and a specific operation process, but the protection scope of the present invention is not limited to this embodiment.

[0030] Example 1

[0031] In this example, a near-infrared structured light illumination light sheet imaging method based on fluorescence loss super-resolution method is implemented by the following steps:

[0032] 1. Sample preparation: label the sample with a near-infrared excited up-conversion fluorescent probe and place it in the sample chamber of the stage 22.

[0033] 2. The coaxial coupling beam formed by the near-infrared excitation light and the near-infrared vortex hollow loss light generated by the beam modulation module leads out two coherent lights through the half-reflective half-mirror 12. After each light sheet is formed through the cylindrical lenses 16 and 20, it interferes in the imaging plane through the two illumination objective lenses 17 and 21 to form a structured illumination pattern to illuminate the sample.

[0034] 3. The near-infrared excitation light generated by the beam modulation module can stimulate fluorescence radiation, and the near-infrared vortex hollow light can consume fluorescence, making the sample non-luminescent, thereby reducing the effective excitation thickness of the sandwich light sheet formed by the two light beams on the same optical axis and having a larger imaging depth and anti-sample scattering ability, realizing large-field axial super-resolution imaging of the sample.

[0035] 4. The imaging module receives the fluorescence signal of the sample through the color filter 24, and the stage obtains the fluorescence signal of the sample illuminated by structured light of different phases and directions by translation and rotation.

[0036] 5. The imaging module performs spectrum separation and reconstruction on the collected fluorescence images to obtain a horizontal super-resolution image of a layer.

[0037] 6. The computer 27 in the imaging module controls the stage 22 to move to the next imaging area and repeat the operations of steps (4) and (5) until all areas of the sample are imaged.

[0038] 7. The imaging module takes out all the obtained images and reconstructs them in three dimensions to obtain a three-dimensional large-field-of-view super-resolution sample image.

[0039] This example is a near-infrared structured light illumination light sheet imaging device based on fluorescence loss super-resolution method. Figure 1 As shown, 1 is a semiconductor laser with a wavelength of 975nm, 2 is a semiconductor laser with a wavelength of 810nm, 3, 4, 5, 6 are lenses, 7, 8 are spatial light modulators, 9 is a spiral phase plate, 10, 13, 14, 15, 18, 19 are reflectors, 11 is a short-pass dichroic mirror, 12 is a semi-reflective and semi-transparent mirror, 16, 20 are cylindrical lenses, 17, 21 are illumination objectives, 22 is a stage, 23 is a detection objective, 24 is a color filter, 25 is a tube lens, 26 is an imaging camera, and 27 is a computer.

[0040] Furthermore, a device for near-infrared structured light illumination light sheet imaging based on fluorescence loss super-resolution method is implemented by three modules: a beam modulation module, an optical path module, and an imaging module. The beam modulation module includes semiconductor continuous lasers 1, 2, lenses 3, 4, 5, 6, spatial light modulators 7, 8, and a spiral phase plate 9. The optical path module includes a short-pass dichroic mirror 11, a half-reflecting half-mirror 12, reflectors 10, 13, 14, 15, 18, 19, cylindrical lenses 16, 20, and illumination objective lenses 17, 21. The imaging module includes a stage 22, a detection objective lens 23, a color filter 24, a tube lens 25, an imaging camera 26, and a computer 27.

[0041] In specific implementation, the semiconductor continuous laser 1 generates near-infrared excitation light with a wavelength of 975nm, which is expanded and collimated through lenses 3 and 5. The collimated laser beam is irradiated on the spatial light modulator 7, which modulates the near-infrared excitation light into a Bessel beam and reflects it onto the short-pass dichroic mirror 11. The semiconductor continuous laser 2 generates near-infrared loss light with a wavelength of 810nm, which is expanded and collimated through lenses 4 and 6. The collimated laser beam is irradiated on the spatial light modulator 8, which modulates the loss light into a Bessel beam. Then, the light beam passes through a spiral phase plate 9 to modulate the loss light into a hollow beam, and the position is adjusted through the reflector 10 to reflect the loss light onto the short-pass dichroic mirror 11. The short-pass dichroic mirror 11 has a cutoff frequency of 900nm, reflects the 975nm excitation light and conducts the 810nm hollow loss light, and couples the two beams of light to coaxial. The composite light beam is divided into two paths through the half-reflecting half-mirror 12, one of which passes through the reflector group 13, 18, and 19 to irradiate the cylindrical lens 20 and conjugate to the rear pupil of the illumination objective lens 21. The emitted composite light beam will form a sandwich light sheet, the outermost layer is the loss light, and the middle layer is the excitation light. Figure 3 (a). The other path passes through the reflector group 14, 15 to illuminate the cylindrical lens 16 and conjugate to the back pupil of the illumination lens 21. The composite light beam also forms a sandwich light sheet. The two sandwich light sheets interfere in the sample chamber in the stage 22 to produce a structured illumination pattern as shown in Figure 3 (b) (only the excitation part is drawn), Figure 3 (c) As shown. The stripe light sheet excites the upconversion fluorescent probe in the sample to generate fluorescence, which is collected by the detection objective lens 23. The collected fluorescence signal is filtered out by the color filter 24 to remove all stray light. The filtered fluorescence is imaged on the photosensitive surface of the imaging camera 26 through the tube lens 25. The imaging camera 26 transmits the captured image sequence to the computer 27 for analysis.

[0042] Specifically, the spectral response curve of the upconversion fluorescence probe is given by Figure 2As shown in the figure, when the 975 nm excitation light acts alone, it can excite the probe to emit 455 nm fluorescence, while when the 975 nm and 810 nm loss light act together, it can quench the excited fluorescence. The reason is that the 810 nm loss light causes the upconversion fluorescent probe to emit stimulated radiation, thereby losing fluorescence. Using upconversion fluorescent probes as fluorescent probes for stimulated emission depletion super-resolution can significantly reduce the saturation power of stimulated radiation, thereby reducing phototoxicity, and has the advantage of long-term imaging.

[0043] Specifically, the purpose of adjusting the directions of the two separated composite light beams through the reflector groups 13 , 14 , 15 , 18 , and 19 is to allow the light sheets finally emitted through the two illumination objective lenses 17 , 21 to interfere in the sample chamber to generate a structured illumination pattern.

[0044] Further, after the imaging camera 26 takes an image, the computer controls the stage 22 to move twice, each time moving 1 / 3 of the stripe period, and each time the computer 27 controls the imaging camera 26 to take an image. After moving twice, the computer 27 controls the stage 22 to rotate 120 degrees, and repeats the above displacement and rotation operations until the stage 22 rotates 360 degrees. Figure 4 The computer 27 calculates the collected image to obtain a super-resolution image on the xy plane, thus completing the imaging task of one position of the sample.

[0045] Furthermore, after completing the imaging task at each position, the computer 27 controls the stage 22 to move. The principle of movement is: after the imaging task of the sample in the plane where the light sheet is located is completed, the sample is controlled to move in the axial direction and the imaging task of the sample of the next layer is completed until all the imaging tasks of the sample are completed. Finally, the computer 27 performs three-dimensional reconstruction on all images to obtain a three-dimensional super-resolution image of the sample.

[0046] For those skilled in the art, various other corresponding changes and modifications can be made according to the technical solutions and concepts described above, and all of these changes and modifications should fall within the protection scope of the claims of the present invention.

Claims

1. A near-infrared structured light illumination light sheet imaging method based on fluorescence loss super-resolution method, characterized in that: The method comprises the following steps: The near-infrared excitation light and near-infrared vortex hollow loss light generated by the S1 beam modulation module form a coaxial coupled beam; the near-infrared excitation light can excite sample fluorescence, and the near-infrared vortex hollow loss light can deplete sample fluorescence in the action area. Therefore, the sandwich light sheet formed by the coaxial coupled beam through the cylindrical lens has an ultra-thin effective excitation thickness, and the near-infrared has a large imaging depth and anti-sample scattering ability, which can achieve large-field axial super-resolution imaging; The S2 coaxial coupling beam leads out two coherent lights through a half-reflecting half-mirror lens, each of which passes through a cylindrical lens and is conjugated to the coherent light sheet formed by the rear pupils of two objective lenses. The coherent light sheet propagates in the opposite direction on the imaging plane and interferes to form a structured illumination pattern to illuminate the sample; the fluorescence emitted by the sample on the stage is collected by the detection objective lens, which is located in the vertical direction of the light sheet; In the S3 imaging module, the computer performs spectrum separation and reconstruction on the collected fluorescence images of different phases and directions to obtain a horizontal super-resolution image of one layer, and moves the stage axially to realize three-dimensional scanning of the sample. Finally, all the images are taken out and reconstructed in three dimensions, and finally a large-viewing-field three-dimensional super-resolution sample image is obtained, realizing three-dimensional isotropic sub-100nm super-resolution imaging; the outermost layer of the sandwich light sheet is loss light, and the middle layer is excitation light; when irradiating the sample, the fluorescence irradiated by the overlapping part of the loss light beam and the excitation light beam is lost, and no fluorescence is generated; only the fluorescent molecules in the part illuminated by the middle excitation light beam are excited to emit fluorescence; the effective excitation thickness of the excitation light beam becomes thinner, thereby improving the axial resolution of the system; due to interference, the two sandwich light sheets are distributed in stripes on the xy plane. Due to the different wavelengths of the loss light and the excitation light, the structural stripe distribution may have a slight offset and cannot be completely overlapped. The displacement deviation can be corrected by a spatial light modulator to make them correspond one to one; Among them, the phase and direction change of the relative stripes of the illuminated sample are controlled by the stage, so as to realize the spectrum translation expansion in all directions, thereby realizing lateral super-resolution imaging in the xy plane; the biological sample is labeled with a fluorescent probe, wherein the fluorescent probe includes any one of a near-infrared excited up-conversion fluorescent probe, an organic dye, and a quantum dot.

2. A device for near-infrared structured light illumination light sheet imaging based on fluorescence loss super-resolution method is used to implement the near-infrared structured light illumination light sheet imaging method based on fluorescence loss super-resolution method as claimed in claim 1, characterized in that: The device comprises a beam modulation module, an optical path module and an imaging module, wherein the beam modulation module is composed of 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 light laser for generating near-infrared excitation light, and the second laser is a low-power continuous light or pulse light laser for generating near-infrared loss light; the telescope system is respectively arranged behind the first laser and the second laser; the spatial light modulator is respectively arranged behind the telescope system, and is used for shaping the excitation light and the loss light and generating the special beam required for the experiment; The optical path module consists of a dichroic mirror, a half-reflecting half-mirror, a reflector group, a cylindrical lens, and an illumination objective lens; the modulated near-infrared excitation light and the near-infrared vortex hollow loss light generated by the beam modulation module are coaxially coupled through the dichroic mirror, and the half-reflecting half-mirror splits the composite light beam into two paths, which are respectively adjusted to the same optical axis through the reflector group for reverse transmission; then, each passes through the same cylindrical lens and the illumination objective lens to form two reversely transmitted light sheets to interfere with each other to generate a structured illumination pattern, and excite the fluorescent molecules in the sample to emit fluorescence; The imaging module consists of a stage, a detection objective lens, a color filter, a tube lens, an imaging camera and a computer; the collected fluorescence is filtered out by the color filter to remove other stray light except the signal light, which is beneficial to improve the signal-to-noise ratio; the filtered fluorescence is imaged on the photosensitive surface of the imaging camera through the tube lens, and the imaging camera imports the captured image into the computer; the computer separates and reconstructs the spectrum of the collected image, and finally obtains a lateral super-resolution image.

3. The near-infrared structured light illumination light sheet imaging device based on fluorescence loss super-resolution method according to claim 2, characterized in that: The stage consists of a three-dimensional electric platform and a sample chamber; the three-dimensional electric platform can move along the x, y, and z directions according to computer control and can rotate along the support axis to achieve uniform illumination of the sample at all positions and in all directions.

4. The near-infrared structured light illumination light sheet imaging device based on fluorescence loss super-resolution method according to claim 2, characterized in that: The telescope system consists of two lenses and is used for expanding and collimating the laser beam.

5. The near-infrared structured light illumination light sheet imaging device based on fluorescence loss super-resolution method according to claim 2, characterized in that: In the beam modulation module, the spiral phase plate is located after the spatial light modulator and is used to reshape the generated special light beam to form a near-infrared vortex hollow beam, which enters the optical path module through the reflector.

6. The near-infrared structured light illumination light sheet imaging device based on fluorescence loss super-resolution method according to claim 2, characterized in that: The illumination objective lens is mirror-distributed relative to the optical axis of the detection objective lens and is coaxial to achieve sandwich light sheet interference to generate a structured illumination pattern.

Images