A high-precision fluorescence modulation super-resolution microscopy imaging method

Abstract

The invention discloses a high-precision fluorescence modulation super-resolution microscopic imaging method, which is applied in the field of optical microscopic imaging technology. The invention uses a first-order Bessel ring light with a smaller point spread function to replace the ordinary ring light generated by modulating Gaussian light through a vortex phase plate in the traditional technology. The high-precision fluorescence modulation super-resolution imaging new technology can solve the problem of insufficient resolution in the existing fluorescence subtraction microscopic technology and the image distortion problem caused by negative side lobes, enhance the visibility and authenticity of fine structures in the image, and when facing any type of laser scanning confocal microscopic system and fluorescence subtraction super-resolution imaging technology, only a wavefront control optical path including a spatial light modulator and a Fourier lens needs to be added to the optical system. Compared with the traditional super-resolution technology, the technology reduces the complexity and cost of the system and reduces the harm of photobleaching. It can be used in combination with other imaging technologies to improve imaging accuracy and imaging quality.

Description

Technical Field

[0001] The invention belongs to the technical field of optical microscopic imaging, and in particular relates to a high-precision fluorescence modulation super-resolution microscopic imaging method. Background Art

[0002] In biological research, optical microscopes are essential tools for observing cells, tissues and tiny structures, and are widely used in basic and applied research. For biomedical imaging, optical microscopes can capture the internal details of biological samples with high spatial resolution, providing key image support for disease diagnosis, drug development and other fields. However, traditional microscopy technology is limited by the optical diffraction limit and cannot distinguish structures with a size smaller than half the wavelength, which in turn hinders the precise imaging of microscopic structures. The emergence of super-resolution optical imaging technology has broken this bottleneck. They break through the diffraction limit through different principles, significantly improve the spatial resolution, and promote the further development of fluorescence microscopy.

[0003] In recent years, a microscopy technique based on fluorescence intensity subtraction has been proposed, including fluorescence emission difference microscopy (FED), intensity weighted difference microscopy (IWSM), and fluorescence spatiotemporal modulation (FSTM) microscopy. Among them, FED is the first fluorescence subtraction microscopy technique proposed, which uses the intensity difference between two different acquired images to improve the imaging resolution. The deformation-free FED (dfFED) and common-path parallel FED (cpFED) schemes proposed on this basis further improve the image quality. IWSM is also a method that has attracted much attention. It improves the resolution while reducing the image distortion of this type of technology. FSTM combines time-resolved detection technology to eliminate the pixel misalignment problem caused by sample drift and provide high-quality super-resolution imaging. In short, fluorescence subtraction microscopy achieves super-resolution imaging with a simple optical path and low cost. Its low-light-intensity imaging reduces photobleaching and is suitable for in vivo imaging. It has been applied to single-photon, two-photon microscopy and label-free microscopy.

[0004] However, fluorescence subtraction microscopy also has some shortcomings. For example, the differential image is distorted, which is mainly due to the mismatch between the point spread function (PSF) of Gaussian light and annular light, and obvious negative pixels will be generated after the difference. If they are simply taken as zero, information loss will occur, that is, when a strong signal in the negative confocal image corresponds to a weak signal in the negative confocal image, the intensity of the weak signal becomes negative after the image difference. In addition, a too large differential coefficient will cause image deformation, which is manifested as the disappearance of object points, discontinuous lines, or shrinkage and deformation of the center of the image. In short, although the spatial resolution of fluorescence subtraction microscopy can break through the diffraction limit, compared with traditional super-resolution technologies such as stimulated emission depletion (STED) and structured light microscopy (SIM), the resolution improvement is limited, and the image quality needs to be further improved and enhanced. Summary of the invention

[0005] The purpose of the present invention is to propose a high-precision fluorescence modulation super-resolution microscopy imaging method, which has the advantage of using a first-order Bessel ring light with a smaller point spread function to replace the ordinary ring light generated by Gaussian light modulated by a vortex phase plate in the traditional technology. The high-precision fluorescence modulation super-resolution imaging new technology can solve the problem of insufficient resolution in the existing fluorescence subtraction microscopy technology and the image distortion problem caused by negative side lobes, enhance the visibility and authenticity of fine structures in the image, and when facing any type of laser scanning confocal microscopy system and fluorescence subtraction super-resolution imaging technology, it is only necessary to add a wavefront control optical path including a spatial light modulator and a Fourier lens to the optical system. Compared with traditional super-resolution technology, this technology reduces the complexity and cost of the system, reduces the harm of photobleaching, and has higher flexibility. It can be used in combination with other imaging technologies to improve imaging accuracy and imaging quality.

[0006] The above technical objectives of the present invention are achieved through the following technical solutions: A high-precision fluorescence modulation super-resolution microscopy imaging method, comprising the following steps:

[0007] S1. Select a laser light source, the type of laser light source is a continuous laser or a pulsed laser;

[0008] S2. After the laser is emitted, it is divided into two beams through a half-wave plate and a polarizer. One of the reflected beams is modulated by a spatial light modulator that simultaneously loads the axicon phase hologram and the vortex phase image, and outputs a beam carrying a focused conical phase. The other transmitted light is Gaussian light, which serves as the excitation light for the confocal image.

[0009] S3. The light beam modulated by the spatial light modulator is focused by the lens to generate a first-order Bessel beam with non-diffraction characteristics, and then focused by the objective lens to obtain negative confocal light;

[0010] S4. The two beams are combined at the beam splitter, modulated into left-handed circular polarization state by a quarter glass slide, and sequentially pass through a dichroic mirror, a galvanometer, and a tube lens, and then focused by an objective lens to illuminate the sample;

[0011] S5. Using gold nanoparticles as samples, the spot is adjusted in real time through imaging, so that the focal planes of the Gaussian beam and the annular beam overlap accurately in space. After the sample is excited, fluorescence is generated, which is collected by the same objective lens and returned along the original path. After being reflected by the dichroic mirror, it passes through the lens and filter to reach the detector. The fluorescence data is transmitted to the computer for storage and processing to obtain two images.

[0012] S6. Apply the two images to phase subtraction microscopy to obtain high-precision fluorescence modulation super-resolution images with higher resolution and quality.

[0013] The present invention is further configured as follows: the phase mask of the spatial light modulator is expressed as: exp(iar+ilθ), wherein a is the axicon parameter, l is the topological charge, and r and θ are polar coordinates.

[0014] The present invention is further configured as follows: during the high-precision fluorescence modulation process, two different images are collected, one is a traditional confocal image, and the other is a negative confocal image excited by first-order Bessel vortex light, wherein the Bessel ring light is generated by focusing an ideal vortex using an objective lens.

[0015] The present invention is further configured as follows: the process of generating an ideal vortex by focusing the Bessel annular light with an objective lens is divided into two stages:

[0016] Firstly, a spatial light modulator is used to generate a Bessel beam with controllable radial wave vector and topological charge l.

[0017] Then, the light beam is converted into Bessel vortex light with a smaller center point spread function than ordinary ring light by using the Fourier transform property of a simple lens and the focusing of an objective lens;

[0018] The final high-precision fluorescence modulation image is obtained by intensity difference between a confocal image and a negative confocal image.

[0019] The present invention is further configured as follows: the image intensity is expressed as I=Ic-αIn, where I c represents the light intensity distribution of the confocal image, I n is the intensity distribution of the negative confocal image generated by the first-order Bessel vortex light, α is the subtraction coefficient, the optimal value of the subtraction coefficient depends on the sample type and imaging mode, and is adjusted according to the imaging parameters. The resolution of the system is mainly determined by the distribution of the point spread function.

[0020] The present invention is further configured such that when Gaussian light is used to scan a sample, the point spread function is expressed by the formula: where PSF e (x, y) is the PSF of the excitation light after it is focused by the microscope objective lens. f (x,y) PSF of the fluorescence collected by the objective evaluated at the fluorescence emission wavelength.

[0021] The present invention is further configured as follows: the confocal light obtains information of the sample point itself and some surrounding areas, while the negative confocal light obtains information of the area outside the center of the sample point, the PSF of the conventional annular light is much larger than the PSF of the Gaussian light beam, and the Bessel Gaussian light is used to replace the conventional annular light used in the fluorescence subtraction microscopy technology. Since the central dark core of the Bessel annular light beam is smaller than that of the ordinary annular light beam, adjacent and closer object points can be separated more accurately.

[0022] The present invention is further configured as follows: taking a difference between a Gaussian beam and a first-order Bessel annular beam, not only can the low-frequency information of the edge of the structure in the image be subtracted and the high-frequency information of the center of the sample be retained, but also the interference to the normal fluorescence signal of the adjacent object points can be reduced, and the accuracy of the compression of the ordinary Gaussian point spread function can be improved. By adjusting the axis pyramid parameter a and the subtraction coefficient α of the SLM phase, the resolution can be further improved while ensuring the image quality.

[0023] The present invention is further configured as follows: compared with conventional ring light, the Bessel ring light has a narrower central PSF, and the super-resolution spot obtained in the phase subtraction microscope has a smaller PSF and a weaker negative value distribution.

[0024] The present invention is further configured as follows: a first-order Bessel light beam with non-diffraction characteristics is generated through the focusing action of a lens.

[0025] In summary, the present invention has the following beneficial effects:

[0026] The new high-precision fluorescence modulation super-resolution imaging technology uses first-order Bessel ring light with a smaller point spread function to replace the ordinary ring light generated by modulating Gaussian light through a vortex phase plate in the traditional technology. It can solve the problem of insufficient resolution in the existing fluorescence subtraction microscopy technology and the image distortion problem caused by negative side lobes, enhance the visibility and authenticity of fine structures in the image, and for any type of laser scanning confocal microscopy system and fluorescence subtraction super-resolution imaging technology, it only needs to add a wavefront control optical path including a spatial light modulator and a Fourier lens to the optical system. Compared with traditional super-resolution technology, this technology reduces the complexity and cost of the system, reduces the harm of photobleaching, and has higher flexibility. It can be used in combination with other imaging technologies to improve imaging accuracy and quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a schematic diagram of an improved laser scanning confocal system based on the present invention;

[0028] Figure 2 Schematic diagram of the high-precision fluorescence modulation super-resolution microscopy imaging implemented in the present invention;

[0029] Figure 3 This is a diagram showing the effect of high-precision fluorescence modulation super-resolution microscopic imaging achieved by the present invention. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with the accompanying drawings.

[0031] Embodiment 1:

[0032] A high-precision fluorescence modulation super-resolution microscopy imaging method comprises the following steps:

[0033] S1. Select a laser light source, the type of laser light source is a continuous laser or a pulsed laser;

[0034] S2. After the laser is emitted, it is divided into two beams through a half-wave plate and a polarizer. One of the reflected beams is modulated by a spatial light modulator that simultaneously loads the axicon phase hologram and the vortex phase image, and outputs a beam carrying a focused conical phase. The other transmitted light is Gaussian light, which serves as the excitation light for the confocal image.

[0035] S3. The light beam modulated by the spatial light modulator is focused by the lens to generate a first-order Bessel beam with non-diffraction characteristics, and then focused by the objective lens to obtain negative confocal light;

[0036] S4. The two beams are combined at the beam splitter, modulated into left-handed circular polarization state by a quarter glass slide, and sequentially pass through a dichroic mirror, a galvanometer, and a tube lens, and then focused by an objective lens to illuminate the sample;

[0037] S5. Using gold nanoparticles as samples, the spot is adjusted in real time through imaging, so that the focal planes of the Gaussian beam and the annular beam overlap accurately in space. After the sample is excited, fluorescence is generated, which is collected by the same objective lens and returned along the original path. After being reflected by the dichroic mirror, it passes through the lens and filter to reach the detector. The fluorescence data is transmitted to the computer for storage and processing to obtain two images.

[0038] S6. Apply the two images to phase subtraction microscopy to obtain high-precision fluorescence modulation super-resolution images with higher resolution and quality.

[0039] Embodiment 2:

[0040] like Figure 1As shown, this system has no restrictions on the type of laser light source, which can be a continuous laser or a pulsed laser. After the laser is emitted, it passes through a half-wave plate and a polarizer and is divided into two beams; among them, the reflected light beam is modulated by a spatial light modulator that simultaneously loads the axicon phase hologram and the vortex phase image, and then outputs a light beam carrying a focused conical phase. The phase mask of the spatial light modulator is expressed as: exp(iar+ilθ), where a is the axicon parameter, l is the topological charge number, and r and θ are polar coordinates; then, through the focusing effect of the lens, a first-order Bessel light beam with non-diffraction characteristics is generated, and finally the negative confocal light is obtained by focusing through the objective lens; the other transmitted light is Gaussian light, which serves as the excitation light of the confocal image, and the two beams are split in the light beam. The laser beams are combined at the mirror and modulated into a left-handed circular polarization state by a 1 / 4 glass slide to further improve the quality of the annular light spot. The combined laser beams pass through a dichroic mirror, a galvanometer, and a tube lens in sequence, and are focused by an objective lens before irradiating the sample. Gold nanoparticles are used as samples, and the light spot is adjusted in real time through imaging, so that the focal planes of the Gaussian beam and the annular beam coincide precisely in space. After the sample is excited, fluorescence is generated, which is collected by the same objective lens and returned along the original path. After being reflected by the dichroic mirror, the fluorescence reaches the detector through a lens and a filter, and the fluorescence data is transmitted to a computer for storage and processing to obtain two images. Finally, the two images are applied to a phase subtraction microscope, such as a fluorescence differential microscope, to obtain a high-precision fluorescence modulation super-resolution image with higher resolution and higher quality.

[0041] like Figure 2 As shown in the figure, in the process of high-precision fluorescence modulation, two different images are collected, one is a traditional confocal image, and the other is a negative confocal image excited by a first-order Bessel vortex light, wherein the Bessel ring light is generated by focusing an ideal vortex with an objective lens. The process is divided into two stages; first, a spatial light modulator is used to generate a Bessel beam with a controllable radial wave vector and a topological charge of l, and then the Fourier transform characteristics of a simple lens and the focusing of the objective lens are used to convert the beam into a Bessel vortex light with a smaller center point spread function than ordinary ring light; the final high-precision fluorescence modulation image is obtained by intensity difference between a confocal image and a negative confocal image, and the image intensity is expressed as I=Ic-αIn, where I c represents the light intensity distribution of the confocal image, I n is the intensity distribution of the negative confocal image generated by the first-order Bessel vortex light, α is the subtraction coefficient, the optimal value of the subtraction coefficient depends on the sample type and imaging mode, so it needs to be adjusted according to the imaging parameters. The resolution of the system is mainly determined by the distribution of the point spread function. When Gaussian light is used to scan the sample, the point spread function is expressed by this formula: where PSF e (x, y) is the PSF of the excitation light after it is focused by the microscope objective lens. f(x, y) is the PSF of the fluorescence collected by the objective lens evaluated using the fluorescence emission wavelength; for convenience, the wavelength difference between the excitation light and the fluorescence is generally ignored, that is, the PSF e (x,y)=PSF f (x, y), p(x, y) refers to the small hole, which is generally represented by an infinitely small hole, that is, let p(x, y) = δ(x, y), so the above formula can be simplified to Similarly, the point spread function of negative confocal light is: The electric field distribution on the sample can be obtained by the Richard Wolf diffraction integral; from the simulation results of Figure a, it can be seen that the Bessel ring light has a smaller central ring width than the traditional ring light, which has a certain effect on improving resolution and reducing image distortion. In the method proposed in the present invention, the confocal light can obtain information about the sample point itself and some surrounding areas, while the negative confocal light obtains information about the area outside the center of the sample point. The PSF of conventional ring light is much larger than the PSF of Gaussian beam. Bessel Gaussian light is used instead of conventional ring light used in fluorescence subtraction microscopy technology. Since the central dark core of the Bessel ring light is smaller than that of the ordinary ring light, it can be more accurately Separate adjacent object points that are close to each other; make a difference between the Gaussian beam and the first-order Bessel ring beam, which can not only subtract the low-frequency information of the edge of the structure in the image and retain the high-frequency information of the center of the sample, but also reduce the interference to the normal fluorescence signal of the adjacent object points and improve the accuracy of the compression of the ordinary Gaussian point spread function. By adjusting the axis pyramid parameter a and the subtraction coefficient α of the SLM phase, the resolution can be further improved while ensuring the image quality; Figure b is the intensity distribution curve of the simulated point spread function of Figure a; compared with conventional ring light, Bessel ring light has a narrower central PSF, so the super-resolution spot obtained in the phase subtraction microscope has a smaller PSF and a weaker negative value distribution.

[0042] like Figure 3 As shown, it is a scattering image of gold nanoparticles collected by the optical system of the present invention. It can be seen from the figure that the ring radius of the Bessel ring light is smaller than that of the conventional ring light. Therefore, the light spot in the super-resolution image after intensity difference has a smaller PSF, and the resolution is improved by about 33%, which proves the effect of the method proposed in the present invention in improving the resolution; in the super-resolution image, the blue part represents the negative value generated after the fluorescence difference, and the greater the blue depth, the higher the negative value. Since the Bessel ring light has a smaller central ring width, there are fewer negative values ​​in the high-precision fluorescence modulation super-resolution microscopy imaging image, which greatly improves the distortion problem existing in the microscope technology of fluorescence intensity subtraction; in short, the present invention can improve the imaging accuracy and imaging resolution of subtraction microscopes such as fluorescence emission difference microscopy (FED) and intensity weighted difference microscopy (IWSM), and further promote the application of super-resolution imaging technology in biomedical imaging.

[0043] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A high-precision fluorescence modulation super-resolution microscopy imaging method, characterized in that: The following steps are involved: S1. Select a laser light source, the type of laser light source is a continuous laser or a pulsed laser; S2. After the laser is emitted, it is divided into two beams through a half-wave plate and a polarizer. One of the reflected beams is modulated by a spatial light modulator that simultaneously loads the axicon phase hologram and the vortex phase image, and outputs a beam carrying a focused conical phase. The other transmitted light is Gaussian light, which serves as the excitation light for the confocal image. S3. The light beam modulated by the spatial light modulator is focused by the lens to generate a first-order Bessel beam with non-diffraction characteristics, and then focused by the objective lens to obtain negative confocal light; S4. The two beams are combined at the beam splitter, modulated into left-handed circular polarization state by a quarter glass slide, and sequentially pass through a dichroic mirror, a galvanometer, and a tube lens, and then focused by an objective lens to illuminate the sample; S5. Using gold nanoparticles as samples, the spot is adjusted in real time through imaging, so that the focal planes of the Gaussian beam and the annular beam overlap accurately in space. After the sample is excited, fluorescence is generated, which is collected by the same objective lens and returned along the original path. After being reflected by the dichroic mirror, it passes through the lens and filter to reach the detector. The fluorescence data is transmitted to the computer for storage and processing to obtain two images. S6. Apply the two images to phase subtraction microscopy to obtain high-precision fluorescence modulation super-resolution images with higher resolution and quality.

2. A high-precision fluorescence modulation super-resolution microscopy imaging method according to claim 1, characterized in that: The phase mask of the spatial light modulator is expressed as: exp(iar+ilθ), where a is the axicon parameter, l is the topological charge, and r and θ are polar coordinates.

3. A high-precision fluorescence modulation super-resolution microscopy imaging method according to claim 2, characterized in that: During the high-precision fluorescence modulation process, two different images are collected, one is a traditional confocal image, and the other is a negative confocal image excited by first-order Bessel vortex light, where the Bessel ring light is generated by focusing the ideal vortex using an objective lens.

4. A high-precision fluorescence modulation super-resolution microscopy imaging method according to claim 3, characterized in that: The process of generating an ideal vortex by focusing Bessel ring light with an objective lens is divided into two stages: Firstly, a spatial light modulator is used to generate a Bessel beam with controllable radial wave vector and topological charge l. Then, the Fourier transform property of a simple lens and the focusing of an objective lens are used to transform the light beam into a Bessel vortex light having a smaller central point spread function than that of ordinary annular light. The final high-precision fluorescence modulation image is obtained by intensity difference between a confocal image and a negative confocal image.

5. A high-precision fluorescence modulation super-resolution microscopy imaging method according to claim 4, characterized in that: Image intensity is expressed as I = Ic-αIn, where I c represents the light intensity distribution of the confocal image, I n is the intensity distribution of the negative confocal image generated by the first-order Bessel vortex light, α is the subtraction coefficient, the optimal value of the subtraction coefficient depends on the sample type and imaging mode, and is adjusted according to the imaging parameters. The resolution of the system is mainly determined by the distribution of the point spread function.

6. A high-precision fluorescence modulation super-resolution microscopy imaging method according to claim 5, characterized in that: When Gaussian light is used to scan a sample, the point spread function is expressed as: where PSF e (x, y) is the PSF of the excitation light after it is focused by the microscope objective. f (x,y) PSF of the fluorescence collected by the objective evaluated at the fluorescence emission wavelength.

7. A high-precision fluorescence modulation super-resolution microscopy imaging method according to claim 6, characterized in that: Confocal light obtains information about the sample point itself and some surrounding areas, while negative confocal light obtains information about the area outside the center of the sample point. The PSF of conventional annular light is much larger than the PSF of Gaussian beam. Bessel Gaussian light is used instead of conventional annular light used in fluorescence subtraction microscopy. Since the central dark core of the Bessel annular beam is smaller than that of the ordinary annular beam, adjacent and closer object points can be separated more accurately.

8. A high-precision fluorescence modulation super-resolution microscopy imaging method according to claim 7, characterized in that: Taking the difference between the Gaussian beam and the first-order Bessel annular beam can not only subtract the low-frequency information of the structure edge in the image and retain the high-frequency information of the sample center, but also reduce the interference to the normal fluorescence signal of the adjacent object points and improve the accuracy of the compression of the ordinary Gaussian point spread function. By adjusting the axicon parameter a and the subtraction coefficient α of the SLM phase, the resolution can be further improved while ensuring the image quality.

9. A high-precision fluorescence modulation super-resolution microscopy imaging method according to claim 8, characterized in that: Compared with conventional ring light, Bessel ring light has a narrower central PSF. The super-resolution spot obtained in phase subtraction microscopy has a smaller PSF and a weaker negative value distribution.

10. The high-precision fluorescence modulation super-resolution microscopy imaging method according to claim 1, characterized in that: A first-order Bessel beam with non-diffraction characteristics generated by the focusing effect of the lens.

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