A fluorescence emission ratio super-resolution imaging method

Abstract

The present invention provides a fluorescence emission ratio super-resolution imaging method, including a pulsed laser, wherein the laser emitted by the pulsed laser passes through a half-wave plate, and then passes through a Glan laser prism, wherein one beam of laser light passes through the Glan laser prism to a high-speed photodiode detector, and the other beam of laser light passes through a first beam splitter, and respectively illuminates a first reflector and a spiral phase plate, wherein the laser light passing through the spiral phase plate passes through a second reflector, and then passes through the second reflector to a corner reflector, wherein the laser light passing through the first reflector passes through a second beam splitter, and then passes through a corner reflector and a third reflector, and then passes through a dichroic mirror, a galvanometer, a scanning lens, a tube lens, and a quarter glass slide in sequence, and then is focused by an objective lens and irradiated onto a sample on a stage. The present invention reduces the requirements of super-resolution technology for fluorescent dyes and sample preparation, broadens the selection range of dyes, and provides technical support for low-cost in vivo super-resolution imaging research.

Description

Technical Field

[0001] The invention belongs to the technical field of optical microscope imaging methods, and in particular relates to a fluorescence emission ratio super-resolution imaging method. Background Art

[0002] Humans mainly absorb information through their eyes, but the sensitivity of human eyes is closely related to the wavelength of light. Since they can only recognize light waves with a wavelength range of 400 to 760 nm, normal human eyes can only distinguish submillimeter objects at a distance of 25 cm. If you want to observe smaller objects, you need to use a microscope. The invention of the optical microscope allowed humans to observe living organisms with cells as basic structural and functional units for the first time, and it has now been widely used in various fields of life and scientific research. Optical microscopy imaging technology for living cell research has the advantages of non-contact, non-destructive and specific. It can conduct in-situ, real-time and dynamic research on cells, and deeply understand the interactions and physiological processes between organelles, proteins and molecules, which has greatly promoted the progress and development of life sciences. However, the resolution of optical microscopes is limited by optical diffraction, and it is impossible to clearly distinguish biological structures below 200 nm in size. In order to study and reveal the interactions and action laws of subcellular structures and related molecules, imaging technology that breaks through the optical diffraction limit is urgently needed.

[0003] In order to meet the imaging needs of nanoscale resolution, super-resolution microscopy technology came into being. In the past three decades, super-resolution imaging methods based on different principles have been proposed one after another, making optical microscopes gradually enter the era of nano-imaging. In recent years, the continuous development of super-resolution optical imaging technology has made optical microscopes more closely connected with fields such as biomedicine. From the perspective of optical engineering, the performance of microscopy technology is determined by some hard indicators, such as imaging resolution, imaging depth, positioning accuracy and imaging rate. In addition, the effective number of photons, labeling specificity and sample state are all limiting factors for obtaining the best imaging effect in actual biological applications. Therefore, an advanced optical imaging system not only needs to maintain the biological characteristics of the observed sample itself, but also should maximize the authenticity and effectiveness of the information obtained.

[0004] However, almost all super-resolution imaging modes will generate new problems when pursuing the super-resolution limit, such as high laser power, low imaging speed, complex imaging system and expensive experimental cost. The stimulated emission depletion technology consumes extremely high laser power, resulting in serious sample damage and dye photobleaching, so it is not suitable for long-term super-resolution imaging of living samples such as cells; single-molecule localization microscopy technology requires image reconstruction, so the temporal resolution is low and the imaging depth is limited, which limits its application in dynamic imaging of biological samples. In short, the current super-resolution imaging technology still faces a series of problems such as complex imaging system, high cost, low image quality and complex sample preparation process, as well as the need for special fluorescent probes when realizing three-dimensional, multi-color and deep super-resolution imaging.

[0005] Based on this, a fluorescence emission ratio super-resolution imaging method was proposed. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a fluorescence emission ratio super-resolution imaging method in view of the shortcomings of the above-mentioned prior art, reduce the requirements of super-resolution technology for fluorescent dyes and sample preparation, broaden the selection range of dyes, provide technical support for low-cost in vivo super-resolution imaging research, and solve the problems raised in the above-mentioned background technology.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is: a fluorescence emission ratio super-resolution imaging method, comprising a pulsed laser, wherein the laser light emitted by the pulsed laser passes through a half-wave plate, and then passes through a Glan laser prism, and is divided into two laser beams by the Glan laser prism, wherein one laser beam is transmitted to a high-speed photodiode detector, and the other laser beam is transmitted to a first beam splitter, and is again divided into two laser beams by the beam splitter, and respectively illuminates a first reflector and a spiral phase plate;

[0008] The laser light passing through the spiral phase plate is transmitted to the second reflector, and then to the corner reflector after passing through the second reflector;

[0009] The laser beam passing through the first reflector is transmitted to the second beam splitter, and is split into two beams again by the second beam splitter, and is transmitted to the corner reflector and the third reflector respectively;

[0010] The laser light passing through the third reflector passes through the dichroic mirror, galvanometer, scanning lens, tube lens and quarter glass slide in sequence, and is focused by the objective lens and irradiated onto the sample on the stage.

[0011] Furthermore, the high-speed photodiode detector is connected to a time-correlated single-photon counter, and the time-correlated single-photon counter is connected to a computer.

[0012] Furthermore, the sample on the stage generates fluorescence after being excited. The fluorescence is collected by the objective lens and then passes through a quarter glass slide, a tube lens, a scanning lens, a galvanometer and a dichroic mirror. After being reflected by the dichroic mirror, it reaches the photomultiplier tube through a filter. The filter is used to remove stray light other than fluorescence and improve the image signal-to-noise ratio. The time-correlated single-photon counter simultaneously receives the reference signal and the fluorescence signal collected by the high-speed photodiode detector and the photomultiplier tube, and transmits the data to a computer for storage and processing.

[0013] Furthermore, the pulse laser generates 40 MHz pulsed excitation light by a picosecond laser, the half-wave plate is used to adjust the polarization direction of the laser, and the Glan laser prism is used to separate lasers with different polarization directions.

[0014] Furthermore, the spiral phase plate is used to convert the wavefront of the laser from Gaussian to annular, and the corner reflector controls the pulse interval between the Gaussian laser and the annular laser in time by extending or shortening the optical path of the annular excitation spot.

[0015] Further, the dichroic mirror is used to transmit the excitation light and reflect the fluorescence signal;

[0016] The galvanometer is used to synchronously scan the excitation light to achieve area array imaging of the sample;

[0017] The scanning lens is used to collect the laser beam for area array scanning.

[0018] Furthermore, the objective lens is used to focus the laser onto a focal plane and collect the fluorescent signal reflected from the sample at the same time. The tube lens and the objective lens are combined to form a microscope system.

[0019] Furthermore, the quarter glass is used to convert linearly polarized laser light into right-handed circularly polarized light.

[0020] Furthermore, the stage is used to place and fix the sample and to move the sample in three dimensions.

[0021] Furthermore, the first beam splitter and the second beam splitter are respectively used for laser beam splitting or beam combining, and the first reflector, the second reflector and the third reflector are respectively used for changing the transmission direction of the laser.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] The fluorescence emission ratio nano-microscopy imaging technology of the present invention breaks through the optical diffraction limit at low laser power, reduces the requirements of super-resolution technology on fluorescent dyes and sample preparation, broadens the selection range of dyes, and provides technical support for low-cost in vivo super-resolution imaging research. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a system optical path diagram of fluorescence emission ratio nanomicroscopy imaging provided by an embodiment of the present invention;

[0025] Figure 2 It is a principle diagram of fluorescence emission ratio nano-microscopy imaging implemented in the present invention;

[0026] Figure 3 It is a method for determining the gray value of each pixel in the fluorescence emission ratio nano-microscope image implemented by the present invention;

[0027] Figure 4 It is the theoretical simulation result of the fluorescence emission ratio nano-microscopy imaging implemented by the present invention.

[0028] Description of reference numerals:

[0029] Pulse laser 1, half-wave plate 2, Glan laser prism 3, first beam splitter 4, first reflector 5, spiral phase plate 6, second beam splitter 7, third reflector 8, second reflector 9, corner reflector 10, high-speed photodiode detector 11, time-correlated single photon counter 12, computer 13, photomultiplier tube 14, dichroic mirror 15, filter 16, galvanometer 17, scanning lens 18, tube lens 19, quarter slide 20, objective lens 21 and stage 22. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] like Figure 1-4 As shown, the present invention provides a technical solution: a fluorescence emission ratio super-resolution imaging method, comprising a pulse laser 1, the laser emitted by the pulse laser 1 passes through a half-wave plate 2, and then passes through a Glan laser prism 3, the pulse laser 1 generates 40MHz pulsed excitation light by a picosecond laser, the half-wave plate 2 is used to adjust the polarization direction of the laser, and the Glan laser prism 3 is used to separate lasers with different polarization directions;

[0032] One of the laser beams is transmitted to a high-speed photodiode detector 11, which is connected to a time-correlated single-photon counter 12, which is connected to a computer 13. The time-correlated single-photon counter 12 simultaneously receives the reference signal and the fluorescence signal collected by the high-speed photodiode detector 11 and the photomultiplier tube 14, and transmits the data to the computer 13 for storage and processing.

[0033] Another laser beam is transmitted to the first beam splitter 4, and is split into two laser beams again by the beam splitter 4, and respectively illuminates the first reflector 5 and the spiral phase plate 6, wherein the spiral phase plate 6 is used to convert the wavefront of the laser from a Gaussian type to a ring type.

[0034] The laser light passing through the spiral phase plate 6 is transmitted to the second reflector 9, and then to the corner reflector 10. The corner reflector 10 controls the pulse interval between the Gaussian laser light and the ring laser light in time by extending or shortening the optical path of the ring-shaped excitation light spot.

[0035] The laser beam passing through the first reflector 5 is transmitted to the second beam splitter 7, and is split into two beams again by the second beam splitter 7, and is transmitted to the corner reflector 10 and the third reflector 8 respectively;

[0036] The laser light passing through the third reflector 8 passes through the dichroic mirror 15 , the galvanometer mirror 17 , the scanning lens 18 , the tube lens 19 and the quarter glass slide 20 in sequence, and is focused by the objective lens 21 and irradiated onto the sample on the stage 22 .

[0037] The dichroic mirror 15 is used to transmit the excitation light and reflect the fluorescence signal;

[0038] The galvanometer 17 is used to synchronously scan the excitation light to achieve area array imaging of the sample;

[0039] The scanning lens 18 is used to collect the laser beam for area array scanning.

[0040] The quarter glass 20 is used to convert the linearly polarized laser light into right-handed circularly polarized light.

[0041] The objective lens 21 is used to focus the laser onto a focal plane and collect the fluorescent signal reflected by the sample. The tube lens 19 and the objective lens 21 form a microscope system.

[0042] The stage 22 is used to place and fix the sample and to move the sample in three dimensions.

[0043] The sample on the stage 22 generates fluorescence after being excited. The fluorescence is collected by the objective lens 21 and then passes through the quarter glass slide 20, the tube lens 19, the scanning lens 18, the galvanometer 17 and the dichroic mirror 15. After being reflected by the dichroic mirror 15, it reaches the photomultiplier tube 14 through the filter 16. The filter 16 is used to remove stray light other than fluorescence and improve the image signal-to-noise ratio. The time-correlated single photon counter 12 simultaneously receives the reference signal and the fluorescence signal collected by the high-speed photodiode detector 11 and the photomultiplier tube 14, and transmits the data to the computer 13 for storage and processing.

[0044] The first beam splitter 4 and the second beam splitter 7 are used for laser beam splitting or beam combining, respectively. The first reflector 5, the second reflector 9 and the third reflector 8 are used for changing the transmission direction of the laser, respectively.

[0045] Pulse laser 1, half-wave plate 2, Glan laser prism 3, first beam splitter 4, first reflector 5, spiral phase plate 6, second beam splitter 7, third reflector 8, second reflector 9, corner reflector 10, high-speed photodiode detector 11, time-correlated single photon counter 12, computer 13, photomultiplier tube 14, dichroic mirror 15, filter 16, galvanometer 17, scanning lens 18, tube lens 19, quarter slide 20, objective lens 21 and stage 22

[0046] Specifically, the laser of the pulse laser 1 is emitted and split by the Glan laser prism 2 and the first beam splitter 4 to form three light paths, one of which is collected by the high-speed photodiode detector 11 and used as a reference signal for fluorescence lifetime imaging;

[0047] The other two light beams are used to excite the sample, and their wavefronts are Gaussian and annular respectively, and the pulse interval is half of the laser pulse period; the two light beams are combined after passing through the second beam splitter 7, and pass through the dichroic mirror 15, the galvanometer 17, the scanning lens 18, the tube lens 19 and the quarter glass slide 20 in sequence, and then are focused by the objective lens 21 to irradiate the sample.

[0048] The gold nanoparticle sample was imaged and the spot was adjusted through real-time imaging so that the focal planes of the Gaussian laser and the ring laser overlapped precisely in space.

[0049] After the sample is excited, it generates fluorescence, which is collected by the same objective lens 21 and then returns to the original path, passing through the quarter glass slide 20, tube lens 19, scanning lens 18, galvanometer 17 and dichroic mirror 15, and then reflected by the dichroic mirror 15 and reaches the photomultiplier tube 14 through the filter 16. The time-correlated single-photon counter 12 simultaneously receives the reference signal and the fluorescence signal collected by the high-speed photodiode detector 11 and the photomultiplier tube 14, and transmits the data to the computer 13 for storage and processing.

[0050] Figure 2 The schematic diagram of the present invention for realizing fluorescence emission ratio nano-microscopic imaging. Fluorescence lifetime imaging is performed on the dye-labeled sample, and the fluorescence photons are collected and their spatiotemporal information is obtained by the time-correlated single photon counter 12, and then the fluorescence lifetime data is post-processed. First, the fluorescence signal is divided into two parts based on the time channel where the ring laser pulse is located. The fluorescence photons in the first half are excited by Gaussian laser to form an image composed of Gaussian light spot expansion function, and the fluorescence photons in the remaining part are excited by ring laser to form an image composed of ring light spot expansion function.

[0051] When the laser pulse frequency is 40MHz, the time difference between the Gaussian image and the annular image is in the nanosecond range, which is equivalent to recording the spatial position information of the Gaussian spot and the annular spot in real time. Then, the grayscale values ​​of the corresponding coordinates of the two images are determined, and the grayscale values ​​of each coordinate of the fluorescence emission ratio nanomicroscope image are obtained according to the determination scheme to achieve super-resolution imaging.

[0052] Figure 3 This is a solution for determining the grayscale value of a pixel in a fluorescence emission ratio nano-microscope image. After signal processing of the collected fluorescence lifetime data, two images are obtained, and the grayscale value of each pixel in the image is represented by IGaussian(x,y) and IDount(x,y), respectively. First, determine whether the grayscale value of the pixel at each coordinate position of IGaussian(x,y) is zero. If IGaussian(x,y)=0, the grayscale value of the corresponding coordinate pixel in the IFERN(x,y) image is 0; if IGaussian(x,y)≠0, determine whether the grayscale value of the pixel at the corresponding coordinate position of IDount(x,y) is zero. At this time, if IDount(x,y)=0, the grayscale value of the corresponding coordinate pixel in the IFERN(x,y) image is the same as IGaussian(x,y); if IDount(x,y)≠0, the grayscale value of the corresponding coordinate pixel in the IFERN(x,y) image is IGaussian(x,y) / IDount(x,y). According to the above judgment, the fluorescence emission ratio nanomicroscope image is finally obtained.

[0053] Figure 4 This is the theoretical simulation result of the fluorescence emission ratio nano-microscopy imaging achieved by the present invention. Figure (a) is the fluorescence decay curve collected after two laser beams are irradiated. The pulse interval between the Gaussian light and the annular light is half of the laser pulse period (T / 2). The power of the two laser beams is in the microwatt level and can be adjusted by rotating the angle of the half-wave plate in the system.

[0054] Taking 9 point objects uniformly distributed in a 3×3 shape as the imaging objects, Figures (b) and (c) are the Gaussian image (IGaussian) and annular image (IDount) obtained after data processing, respectively.

[0055] use Figure 3 The scheme shown in the figure is used for image processing to obtain the fluorescence emission ratio image (IFERN) shown in Figure (d). Comparing Figure (b) and Figure (d), it can be seen that the maximum half-height full width of the spot in Figure (d) is smaller, so the resolution is higher. Therefore, the fluorescence emission ratio nanomicroscopy imaging method proposed in the present invention can achieve super-resolution imaging at microwatt-level laser power, broaden the selection range of fluorescent dyes, and provide technical support for the study of dynamic processes and interactions of subcellular structures in living cells.

[0056] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0057] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A fluorescence emission ratio super-resolution imaging method, characterized in that: The invention comprises a pulse laser (1), wherein the laser light emitted by the pulse laser (1) passes through a half-wave plate (2), and then passes through a Glan laser prism (3), and is split into two laser beams by the Glan laser prism (3), wherein one laser beam is transmitted to a high-speed photodiode detector (11), wherein the high-speed photodiode detector (11) is connected to a time-correlated single photon counter (12), and wherein the time-correlated single photon counter (12) is connected to a computer (13), and the other laser beam is transmitted to a first beam splitter (4), and is split into two laser beams again by the first beam splitter (4), and is irradiated to a first reflector (5) and a spiral phase plate (6) respectively; The laser light passing through the spiral phase plate (6) is transmitted to the second reflector (9), and then to the corner reflector (10) after passing through the second reflector (9); the spiral phase plate (6) is used to convert the wavefront of the laser light from a Gaussian type to a ring type, and the corner reflector (10) controls the pulse interval between the Gaussian laser light and the ring laser light in terms of time by extending or shortening the optical path of the ring-shaped excitation light spot; The laser light passing through the first reflector (5) is transmitted to the second beam splitter (7), and is again split into two beams by the second beam splitter (7), and is transmitted to the corner reflector (10) and the third reflector (8) respectively; The laser light passing through the third reflector (8) passes through the dichroic mirror (15), the galvanometer mirror (17), the scanning lens (18), the tube lens (19) and the quarter glass slide (20) in sequence, and is focused by the objective lens (21) and irradiated onto the sample on the stage (22); The sample on the stage (22) generates fluorescence after being excited. The fluorescence is collected by the objective lens (21) and then passes through a quarter glass slide (20), a tube lens (19), a scanning lens (18), a galvanometer (17) and a dichroic mirror (15). After being reflected by the dichroic mirror (15), the fluorescence reaches the photomultiplier tube (14) through a filter (16). The filter (16) is used to remove stray light other than fluorescence and improve the image signal-to-noise ratio. The time-correlated single photon counter (12) simultaneously receives the reference signal and the fluorescence signal collected by the high-speed photodiode detector (11) and the photomultiplier tube (14), and transmits the data to a computer (13) for storage and processing; Firstly, the fluorescence signal is divided into two parts based on the time channel where the ring laser pulse is located. The fluorescence photons of the first half are excited by Gaussian laser to form a Gaussian image composed of Gaussian light spot expansion function, and the fluorescence photons of the remaining part are excited by ring laser to form a ring image composed of ring light spot expansion function. Then, the grayscale values ​​of the corresponding coordinates of the Gaussian image and the ring image are judged, and the grayscale values ​​of each coordinate of the fluorescence emission ratio nanomicroscope image are obtained according to the judgment scheme. The grayscale values ​​of each pixel in the Gaussian image and the ring image are respectively IGaussian(x, y) and IDount(x, y) express; judge first IGaussian(x, y) Whether the gray value of the pixel at each coordinate position is zero, if IGaussian(x, y) = 0 ,but IFERN(x, y) The gray value of the corresponding coordinate pixel in the image is 0; if IGaussian(x, y) ≠ 0 , then judge IDount(x, y) Whether the gray value of the pixel at the corresponding coordinate position is zero, if IDount(x, y) = 0 ,but IFERN (x, y) The grayscale value of the corresponding coordinate pixel in the image is IGaussian(x, y) Same; if IDount(x, y) ≠ 0 ,but IFERN(x, y) The gray value of the corresponding coordinate pixel in the image is IGaussian(x, y) / IDount(x, y), According to the above determination, a fluorescence emission ratio nano-microscope image is finally obtained.

2. A fluorescence emission ratio super-resolution imaging method according to claim 1, characterized in that: The pulse laser (1) generates 40 MHz pulsed excitation light from a picosecond laser, the half-wave plate (2) is used to adjust the polarization direction of the laser, and the Glan laser prism (3) is used to separate lasers with different polarization directions.

3. The fluorescence emission ratio super-resolution imaging method according to claim 1, characterized in that: The dichroic mirror (15) is used to transmit the excitation light and reflect the fluorescence signal; The galvanometer (17) is used to synchronously scan the excitation light to achieve area array imaging of the sample; The scanning lens (18) is used to collect the laser beam for area array scanning.

4. The fluorescence emission ratio super-resolution imaging method according to claim 1, characterized in that: The objective lens (21) is used to focus the laser onto a focal plane and simultaneously collect the fluorescent signal reflected back by the sample. The tube lens (19) and the objective lens (21) are combined to form a microscope system.

5. The fluorescence emission ratio super-resolution imaging method according to claim 1, characterized in that: The quarter glass (20) is used to convert linearly polarized laser light into right-handed circularly polarized light.

6. The fluorescence emission ratio super-resolution imaging method according to claim 1, characterized in that: The stage (22) is used to place and fix the sample, and to move the sample in three dimensions.

7. The fluorescence emission ratio super-resolution imaging method according to claim 1, characterized in that: The first beam splitter (4) and the second beam splitter (7) are respectively used for laser beam splitting or beam combining, and the first reflector (5), the second reflector (9) and the third reflector (8) are respectively used for changing the transmission direction of the laser.

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