CN115656129A - Fluorescence emission ratio super-resolution imaging method - Google Patents
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- 229910052737 gold Inorganic materials 0.000 description 1
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Abstract
The invention provides a fluorescence emission ratio super-resolution imaging method, which comprises a pulse laser, wherein laser emitted by the pulse laser passes through a half-wave plate, then passes through a Glan laser prism, passes through one laser beam of the Glan laser prism and is transmitted to a high-speed photodiode detector, the other laser beam of the Glan laser prism is transmitted to a first beam splitter, respectively irradiates to a first reflector and a spiral phase plate, passes through the laser of the spiral phase plate and is transmitted to a second reflector, passes through the second reflector and is transmitted to an angle reflector, passes through the laser of the first reflector and is transmitted to the second beam splitter, respectively transmits to the angle reflector and a third reflector, and the laser passing through the third reflector sequentially passes through a dichroic mirror, a vibrating mirror, a scanning lens, a tube mirror and a quarter glass slide and irradiates to a sample of an objective table after being focused by an objective lens. The invention reduces the requirements of the super-resolution technology on the preparation of fluorescent dyes and samples, widens the selection range of the dyes and provides technical support for the low-cost living body super-resolution imaging research.
Description
Technical Field
The invention belongs to the technical field of optical microscope imaging methods, and particularly relates to a fluorescence emission ratio super-resolution imaging method.
Background
Human uptake of information is primarily through the eye, but the sensitivity of the human eye is closely related to the wavelength of light. Because only light waves with the wavelength ranging from 400 nm to 760nm can be recognized, the human eyes with normal eyesight can only distinguish submillimeter-level objects at a distance of 25cm from the photopic vision. If smaller objects are to be observed, the aid of a microscope is required. The invention of the optical microscope, which allows human beings to observe living organisms having cells as basic structural and functional units for the first time, is now widely used in various fields of life and scientific research. The optical microscopic imaging technology oriented to living cell research has the advantages of non-contact, no damage and specificity, can carry out in-situ, real-time and dynamic research on cells, deeply understand the interaction and physiological process among organelles, proteins and molecules, and greatly promote the progress and development of life science. However, the resolution of the optical microscope is limited by optical diffraction, and biological structures having a size of 200nm or less cannot be clearly distinguished. In order to study and reveal the subcellular structures and the interaction and law of action of related molecules, imaging techniques that break through the optical diffraction limit are urgently needed.
In order to meet the imaging requirements of nanoscale resolution, the super-resolution microscopic imaging technology is developed. In the last thirty years, super-resolution imaging methods based on different principles are proposed successively, so that the optical microscope gradually enters the nanometer imaging era. In recent years, the development of super-resolution optical imaging technology has made the connection between optical microscopes and the fields of biomedicine and the like closer. From the optical engineering point of view, the performance of microscopy techniques is determined by some rigid criteria, such as imaging resolution, imaging depth, positioning accuracy and imaging rate. In addition, the number of available photons, the specificity of the label, the state of the sample, etc. are all limiting factors in achieving optimal imaging in practical biological applications. Therefore, an advanced optical imaging system is required to maintain the biological characteristics of the observed sample, and to ensure the authenticity and validity of the acquired information to the maximum extent.
However, almost all super-resolution imaging modes create new problems when the super-resolution limit is pursued, such as high laser power, low imaging speed, complex imaging system and expensive experimental cost. The stimulated radiation loss technology has extremely high laser power loss, causes serious sample damage and dye photobleaching, and is not suitable for long-time super-resolution imaging of living samples such as cells and the like; the single molecule positioning microscope technology needs image reconstruction, so the time resolution is low, the imaging depth is limited, and the application in the dynamic imaging of the biological sample is limited. In summary, the current super-resolution imaging technology still faces a series of problems such as complex imaging system, high cost, low image quality, complex sample preparation process, and the need of special fluorescent probe when realizing three-dimensional, multi-color and deep super-resolution imaging.
Based on the method, a fluorescence emission ratio super-resolution imaging method is provided.
Disclosure of Invention
The technical problem to be solved by the present invention is to provide a fluorescence emission ratio super-resolution imaging method, which aims at overcoming the shortcomings of the prior art, reduces the requirements of the super-resolution technology on the preparation of fluorescent dyes and samples, widens the selection range of the dyes, provides technical support for low-cost living body super-resolution imaging research, and solves the problems proposed in the background technology.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a fluorescence emission ratio super-resolution imaging method comprises a pulse laser, wherein laser emitted by the pulse laser passes through a half-wave plate, passes through a Glan laser prism, and is divided into two beams of laser by the Glan laser prism, wherein one beam of laser is transmitted to a high-speed photodiode detector, the other beam of laser is transmitted to a first beam splitter, and is divided into two beams of laser by the beam splitter again, and the two beams of laser respectively irradiate a first reflector and a spiral phase plate;
the laser passing through the spiral phase plate is transmitted to the second reflector and then transmitted to the corner reflector after passing through the second reflector;
the laser passing through the first reflector is transmitted to the second beam splitter, and is divided into two beams again by the second beam splitter, and the two beams are transmitted to the corner reflector and the third reflector respectively;
and the laser passing through the third reflector sequentially passes through the dichroic mirror, the vibrating mirror, the scanning lens, the tube lens and the quarter glass slide, is focused by the objective lens and then irradiates on a sample on the objective table.
Furthermore, the high-speed photodiode detector is connected with a time-dependent single photon counter, and the time-dependent single photon counter is connected with a computer.
Furthermore, the sample of the objective table is excited to generate fluorescence, the fluorescence is collected by the objective lens, then passes through the quarter glass, the tube lens, the scanning lens, the vibrating mirror and the dichroic mirror, is reflected by the dichroic mirror, then reaches the photomultiplier through the optical filter, the optical filter is used for removing stray light except the fluorescence, the signal to noise ratio of the image is improved, the time-dependent single photon counter simultaneously receives the reference signal and the fluorescence signal collected by the high-speed photodiode detector and the photomultiplier, and transmits the data to the computer for storage and processing.
Furthermore, the pulsed laser generates 40MHz pulsed excitation light by picosecond laser, the half-wave plate is used for adjusting the polarization direction of the laser, and the glan laser prism is used for separating the laser with different polarization directions.
Further, the spiral phase plate is used for converting the wave front of the laser light from a Gaussian type to a ring shape, and the corner reflector controls the pulse interval between the Gaussian type laser light and the ring type laser light in time by prolonging or shortening the optical path of the ring type excitation light spot.
Further, the dichroic mirror is used for transmitting the exciting light and reflecting the fluorescent signal;
the galvanometer is used for synchronously scanning exciting light to realize area array imaging of a sample;
the scanning lens is used for collecting laser beams scanned by the area array.
Furthermore, the objective lens is used for focusing laser on a focal plane and collecting a fluorescence signal reflected by a sample, and the tube lens and the objective lens are matched to form a microscope system.
Further, the quarter glass is used for converting the linearly polarized laser light into right-handed circularly polarized light.
Further, the object stage is used for placing and fixing the sample and performing three-dimensional movement on the sample.
Further, the first beam splitter and the second beam splitter are respectively used for splitting or combining laser beams, and the first reflecting mirror, the second reflecting mirror and the third reflecting mirror are respectively used for changing the transmission direction of the laser beams.
Compared with the prior art, the invention has the following advantages:
the fluorescence emission ratio nanometer microscopic imaging technology breaks through the optical diffraction limit under low laser power, reduces the requirements of the super-resolution technology on the preparation of fluorescent dye and samples, widens the selection range of the dye, and provides technical support for the low-cost living body super-resolution imaging research.
Drawings
FIG. 1 is an optical path diagram of a fluorescence emission ratio nano-microscopic imaging system provided by an embodiment of the present invention;
FIG. 2 is a schematic diagram of fluorescence emission ratio nano-microscopy imaging performed in accordance with the present invention;
FIG. 3 is a graph showing the manner of determining the gray level value of each pixel in a fluorescence emission ratio nano-microscopic image according to the embodiment of the present invention;
FIG. 4 is the theoretical simulation results of fluorescence emission ratio nano-microscopy imaging performed in accordance with the present invention.
Description of the reference numerals:
the device comprises a pulse laser 1, a half-wave plate 2, a Glan laser prism 3, a first beam splitter 4, a first reflecting mirror 5, a spiral phase plate 6, a second beam splitter 7, a third reflecting mirror 8, a second reflecting mirror 9, an angle reflector 10, a high-speed photodiode detector 11, a time-dependent single photon counter 12, a computer 13, a photomultiplier 14, a dichroic mirror 15, a light filter 16, a galvanometer 17, a scanning lens 18, a tube mirror 19, a quarter glass 20, an objective lens 21 and a stage 22.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
As shown in fig. 1 to 4, the present invention provides a technical solution: a fluorescence emission ratio super-resolution imaging method comprises a pulse laser 1, wherein 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 is used for generating 40MHz pulse type excitation light by a picosecond laser, the half-wave plate 2 is used for adjusting the polarization direction of the laser, and the Glan laser prism 3 is used for separating the laser in different polarization directions;
one laser beam is transmitted to the high-speed photodiode detector 11, the high-speed photodiode detector 11 is connected with a time-dependent single photon counter 12, the time-dependent single photon counter 12 is connected with a computer 13, and the time-dependent single photon counter 12 simultaneously receives a reference signal and a fluorescence signal which are acquired by the high-speed photodiode detector 11 and a photomultiplier 14 and transmits data to the computer 13 for storage and processing.
The other laser beam is transmitted to the first beam splitter 4, and is split into two laser beams again through the beam splitter 4, and the two laser beams respectively irradiate the first reflector 5 and the spiral phase plate 6, and the spiral phase plate 6 is used for converting the wave front of the laser beams into a ring from a Gaussian shape.
The laser light passing through the spiral phase plate 6 passes to the second mirror 9, passes through the second mirror 9, and then passes to the corner reflector 10, and the corner reflector 10 controls the pulse interval between the gaussian laser and the ring laser in time by lengthening or shortening the optical path of the ring-shaped excitation spot.
The laser passing through the first reflector 5 is transmitted to a second beam splitter 7, and is split into two beams again by the second beam splitter 7, and the two beams are respectively transmitted to a corner reflector 10 and a third reflector 8;
the laser light passing through the third reflector 8 passes through the dichroic mirror 15, the galvanometer 17, the scanning lens 18, the tube lens 19 and the quarter glass 20 in sequence, is focused by the objective lens 21, and is irradiated onto a sample on the stage 22.
The dichroic mirror 15 is used for transmitting the excitation light and reflecting the fluorescence signal;
the galvanometer 17 is used for synchronously scanning exciting light to realize area array imaging of a sample;
the scanning lens 18 is used for collecting the laser beams of the area array scanning.
The quarter-glass 20 is used to convert linearly polarized laser light into right-handed circularly polarized light.
The objective lens 21 is used for focusing laser on a focal plane and collecting a fluorescence signal reflected by a sample, and the tube lens 19 and the objective lens 21 are matched to form a microscope system.
The stage 22 is used to place and hold a sample and to move the sample in three dimensions.
The sample of the objective table 22 is excited to generate fluorescence, the fluorescence is collected by the objective lens 21, then passes through the quarter glass 20, the tube lens 19, the scanning lens 18, the vibrating mirror 17 and the dichroic mirror 15, is reflected by the dichroic mirror 15, then reaches the photomultiplier 14 through the optical filter 16, the optical filter 16 is used for removing stray light except for the fluorescence, so that the image signal-to-noise ratio is improved, the time-dependent single photon counter 12 simultaneously receives the reference signal and the fluorescence signal collected by the high-speed photodiode detector 11 and the photomultiplier 14, and transmits the data to the computer 13 for storage and processing.
The first beam splitter 4 and the second beam splitter 7 are respectively used for splitting or combining laser beams, 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 beams.
The device comprises a pulse laser 1, a half-wave plate 2, a Glan laser prism 3, a first beam splitter 4, a first reflector 5, a spiral phase plate 6, a second beam splitter 7, a third reflector 8, a second reflector 9, an angle reflector 10, a high-speed photodiode detector 11, a time-dependent single photon counter 12, a computer 13, a photomultiplier 14, a dichroic mirror 15, a light filter 16, a galvanometer 17, a scanning lens 18, a tube mirror 19, a quarter-glass 20, an objective lens 21 and a stage 22
Specifically, after being emitted, the laser of the pulse laser 1 is split into three light paths by the Glan laser prism 2 and the first beam splitter 4, wherein one light path is collected by the high-speed photodiode detector 11 and then is used as a reference signal during fluorescence lifetime imaging;
the other two paths of light are used for exciting the sample, the wave fronts of the two paths of light are respectively in a Gaussian shape and a circular shape, and the pulse interval is half of the pulse period of the laser; the two paths of light are combined after passing through a second beam splitter 7, sequentially pass through a dichroic mirror 15, a vibrating mirror 17, a scanning lens 18, a tube lens 19 and a quarter glass 20, are focused through an objective lens 21, and then irradiate a sample.
Imaging is carried out on the gold nanoparticle sample, and light spot adjustment is carried out through real-time imaging, so that the focal planes of the Gaussian laser and the annular laser are accurately superposed in space.
The sample is excited to generate fluorescence, the fluorescence is collected by the same objective lens 21 and returns to the original path, and the fluorescence passes through a quarter glass 20, a tube lens 19, a scanning lens 18, a vibrating lens 17 and a dichroic mirror 15, is reflected by the dichroic mirror 15 and then reaches the photomultiplier 14 through an optical filter 16. The time-correlated single photon counter 12 receives the reference signal and the fluorescence signal collected by the high-speed photodiode detector 11 and the photomultiplier 14 at the same time, and transmits the data to the computer 13 for storage and processing.
FIG. 2 is a schematic diagram of fluorescence emission ratio nano-microscopic imaging realized by the present invention. And (3) carrying out fluorescence lifetime imaging on the sample marked by the dye, collecting fluorescence photons through a time-correlated single photon counter 12, acquiring the time-space information of the fluorescence photons, and then carrying out post-processing on fluorescence lifetime data. Firstly, a time channel where the annular laser pulse is located is used as a boundary to divide a fluorescence signal into two parts, wherein the fluorescence photons of the first half part are excited by Gaussian laser to form an image consisting of a Gaussian light spot expansion function, and the fluorescence photons of the remaining part are excited by annular laser to form an image consisting of an annular light spot expansion function.
When the laser pulse frequency is 40MHz, the time difference between the Gaussian image and the annular image is in nanosecond order, which is equivalent to real-time recording of the spatial position information of the Gaussian spot and the annular spot. And then, judging the gray values of the coordinates corresponding to the two images, and obtaining the gray value of each coordinate of the fluorescence emission ratio nanometer microscopic image according to a judging scheme to realize super-resolution imaging.
FIG. 3 is a scheme for determining pixel gray scale values of fluorescence emission ratio nanometer micrographs according to the present invention. And performing signal processing on the acquired fluorescence lifetime data to obtain two graphs, wherein the gray value of each pixel in the graph is represented by IGaussian (x, y) and IDount (x, y). Firstly, judging whether the gray value of each coordinate position pixel of IGaussian (x, y) is zero, if IGaussian (x, y) =0, the gray value of the corresponding coordinate pixel in the IFERN (x, y) image is 0; if IGaussian (x, y) ≠ 0, then it is determined whether the gray-level value of the pixel at the coordinate position corresponding to IDount (x, y) is zero. At this time, if id (x, y) =0, the grayscale value of the corresponding coordinate pixel in the IFERN (x, y) image is the same as the iassian (x, y); if IDount (x, y) ≠ 0, the gray-level value of the corresponding coordinate pixel in the IFERN (x, y) image is IGaussian (x, y)/IDount (x, y). And finally obtaining the fluorescence emission ratio nanometer microscopic image according to the judgment.
FIG. 4 shows the theoretical simulation results of the present invention for fluorescence emission ratio nano-microscopy. The graph (a) shows the fluorescence attenuation curves collected after irradiation of the two laser beams. The pulse interval between the Gaussian light and the annular light is half (T/2) of the laser pulse period, the power of the two beams of laser is in the micro watt level, and the adjustment can be carried out through the angle of a half-wave plate in the rotating system.
With 9 point objects uniformly distributed at 3 × 3 as imaging objects, the graphs (b) and (c) are a gaussian image (igausian) and an annular image (IDount) obtained after data processing, respectively.
Image processing was performed using the scheme shown in fig. 3, and a fluorescence emission ratio Image (IFERN) shown in fig. (d) was obtained. Comparing graph (b) with graph (d), it can be seen that the maximum full width at half maximum of the spot in graph (d) is smaller and therefore the resolution is higher. Therefore, the fluorescence emission ratio nano-microscopic imaging method provided by the invention can realize super-resolution imaging under microwatt level laser power, broadens the selection range of fluorescent dyes and provides technical support for research on dynamic process and interaction of subcellular structures in living cells.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A fluorescence emission ratio super-resolution imaging method is characterized in that: the laser beam splitting device comprises a pulse laser (1), wherein laser emitted by the pulse laser (1) passes through a half-wave plate (2), then passes through a Glan laser prism (3), and is split into two laser beams through the Glan laser prism (3), wherein one laser beam is transmitted to a high-speed photodiode detector (11), the other laser beam is transmitted to a first beam splitter (4), and is split into two laser beams through the beam splitter (4) again, and the two laser beams irradiate a first reflector (5) and a spiral phase plate (6) respectively;
the laser passing through the spiral phase plate (6) is transmitted to the second reflector (9), and then transmitted to the corner reflector (10) after passing through the second reflector (9);
the laser 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 the two beams are transmitted to the corner reflector (10) and the third reflector (8) respectively;
the laser passing through the third reflector (8) sequentially passes through a dichroic mirror (15), a vibrating mirror (17), a scanning lens (18), a tube mirror (19) and a quarter glass slide (20), is focused through an objective lens (21) and then is irradiated onto a sample of an objective table (22).
2. A fluorescence emission ratio super-resolution imaging method according to claim 1, characterized in that the high speed photodiode detector (11) is connected to a time dependent single photon counter (12), and the time dependent single photon counter (12) is connected to a computer (13).
3. The fluorescence emission ratio super-resolution imaging method according to claim 2, wherein the sample of the stage (22) is excited to generate fluorescence, the fluorescence is collected by the objective lens (21), passes through the quarter glass (20), the tube lens (19), the scanning lens (18), the galvanometer (17) and the dichroic mirror (15), is reflected by the dichroic mirror (15), passes through the optical filter (16) and reaches the photomultiplier (14), the optical filter (16) is used for removing stray light except for fluorescence, improving the signal-to-noise ratio of the image, and the time-dependent single photon counter (12) receives the reference signal and the fluorescence signal collected by the high-speed photodiode detector (11) and the photomultiplier (14) at the same time and transmits the data to the computer (13) for storage and processing.
4. The fluorescence emission ratio super-resolution imaging method according to claim 1, wherein the pulsed laser (1) is a picosecond laser to generate 40MHz pulsed excitation light, the half-wave plate (2) is used for adjusting the polarization direction of the laser light, and the Glan laser prism (3) is used for separating the laser light with different polarization directions.
5. The fluorescence emission ratio super-resolution imaging method according to claim 1, wherein the spiral phase plate (6) is used to convert the wave front of the laser from gaussian to ring shape, and the corner reflector (10) controls the pulse interval between the gaussian laser and the ring laser in time by lengthening or shortening the optical path of the ring-shaped excitation spot.
6. A fluorescence emission ratio super-resolution imaging method according to claim 1, wherein the dichroic mirror (15) is used to transmit excitation light and reflect fluorescence signals;
the galvanometer (17) is used for synchronously scanning exciting light to realize area array imaging of a sample;
the scanning lens (18) is used for collecting laser beams scanned by the area array.
7. The fluorescence emission ratio super-resolution imaging method according to claim 1, wherein the objective lens (21) is used for focusing laser light to a focal plane and collecting fluorescence signals reflected by the sample, and the tube lens (19) and the objective lens (21) are matched to form a microscope system.
8. A fluorescence emission ratio super-resolution imaging method according to claim 1, wherein said quarter-glass (20) is used to convert linearly polarized laser light into right-handed circularly polarized light.
9. The fluorescence emission ratio super-resolution imaging method according to claim 1, wherein the stage (22) is used for placing and fixing the sample and moving the sample in three dimensions.
10. The fluorescence emission ratio super-resolution imaging method according to claim 1, wherein the first beam splitter (4) and the second beam splitter (7) are used for splitting or combining laser beams respectively, and the first mirror (5), the second mirror (9) and the third mirror (8) are used for changing the transmission direction of laser beams respectively.
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| CN105241857B (en) * | 2015-09-30 | 2017-12-29 | 深圳大学 | A kind of super-resolution imaging system |
| CN107024457B (en) * | 2017-03-22 | 2019-05-14 | 华南理工大学 | A kind of far-field optics super-resolution microscopic method |
| CN111579486B (en) * | 2020-06-04 | 2021-02-26 | 深圳大学 | Super-resolution imaging method and imaging system based on low-power stimulated emission loss |
| CN111521596B (en) * | 2020-06-04 | 2021-02-05 | 深圳大学 | Fluorescence differential super-resolution imaging method and imaging system |
| CN111879740A (en) * | 2020-07-14 | 2020-11-03 | 哈尔滨工业大学 | All-optical super-resolution microscopic device based on photon reset technology |
| CN114895450B (en) * | 2022-05-10 | 2023-05-19 | 深圳大学 | Super-resolution microscopic imaging system and method based on second harmonic |
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| WO2024087614A1 (en) * | 2022-10-29 | 2024-05-02 | 深圳大学 | Ratiometric fluorescence emission super-resolution imaging method |
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