CN113624731A - Super-resolution fluorescence hyperspectral microscopic imaging system - Google Patents
Super-resolution fluorescence hyperspectral microscopic imaging system Download PDFInfo
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Abstract
The invention is suitable for the technical field of microscopic imaging, and provides a super-resolution fluorescence hyperspectral microscopic imaging system, which comprises: the sample microscopic assembly comprises a sample table and an objective lens group; an excitation light source; the structure light modulation module modulates the exciting light to form a structure line with different phase translations and alternating light and shade; the structure line scanning module modulates the structure line to form a scanning structure line for scanning the sample to be detected, and the scanning structure line is focused on the sample to be detected through the objective lens group; the image detection module receives a fluorescence signal emitted by the sample to be detected after being line-scanned and excited by the scanning structure, and performs hyperspectral microimaging on the fluorescence signal. The invention scans the sample by forming the light and shade alternate structural lines which are translated in different phases, enables the sample to rotate in different directions, and realizes the isotropic frequency domain expansion and resolution improvement of the sample plane by utilizing the multidirectional multiple superposition of the sample structure and the different phase structures of the structural lines.
Description
Technical Field
The invention belongs to the technical field of microscopic imaging, and particularly relates to a super-resolution fluorescence hyperspectral microscopic imaging system.
Background
The hyperspectral imaging is taken as a branch of spectral imaging technology, is emerging since the 80 th generation of the 20 th century, and is widely applied to emerging technologies in the fields of agriculture, mineralogy, aerology, life science and the like. Compared with the traditional imaging technology, the method can obtain target image information and spectral information of specific wave bands, the spectral resolution of hyperspectral imaging can reach below 10nm, and the method can instantaneously record the spectral information of dozens or even hundreds of continuous narrow wave bands of each pixel in a field angle. In recent years, with the penetration of microscopy to various aspects of biomedical detection, many reports of combining hyperspectral technology with microscopy imaging have appeared, such as: chromosome recognition, cancer diagnosis, skin disease examination, nondestructive testing of food, agricultural products and the like, cell function research and the like.
The existing cell detection technology is difficult to obtain spatial information and composition information of a sample at the same time, and the microscopic hyperspectral technology can well present appearance information and internal chemical composition information of cells, so that the research of a microscopic hyperspectral imaging system has great significance to the cells and even the whole life science field. In the field of biomedical research, autofluorescence detection of biological tissues has been widely applied to prevention and diagnosis research of difficult and complicated diseases, while hyperspectral microimaging technology combined with microscopy and spectral imaging technology can be used for quantitative analysis of pathology, and compared with the traditional medical imaging method, the hyperspectral microimaging method can provide richer spectral component information and objective diagnosis standards, and has a wide application prospect in the field of biomedical.
At present, the existing hyperspectral microimaging system which can break through the optical diffraction limit to realize super-resolution adopts the Raman microimaging technology of structured line illumination: the method is characterized in that structured light illumination is used in a scanning Raman microscope, a sample is illuminated by a fine linear focus, and a structured light pattern is applied along a direction parallel to a line, so that the spatial resolution of the direction is improved;
however, due to the inherent characteristics of Raman microscopic imaging, only autofluorescence can be observed, and the requirements on a sample and a camera are high; and the structure line is only subjected to translation modulation, the improvement of the isotropic spatial resolution of the sample plane cannot be realized, the resolution in a certain direction can be improved, and the observation of the sample is not favorable.
Disclosure of Invention
The embodiment of the invention provides a super-resolution fluorescence hyperspectral microimaging system, and aims to solve the technical problem that the existing hyperspectral microimaging cannot improve the isotropic spatial resolution of a sample plane.
The embodiment of the invention is a super-resolution fluorescence hyperspectral microimaging system realized in such a way, and the system comprises:
the sample microscopic assembly comprises a sample table for placing a sample to be detected and an objective lens group for carrying out microscopy on the sample to be detected;
the excitation light source is used for providing excitation light for irradiating a sample to be detected;
the structure light modulation module is used for receiving the exciting light and modulating the exciting light to form a structure line with different phase translations and alternate light and shade;
the structure line scanning module receives the structure line and modulates the structure line to form a scanning structure line for scanning the sample to be detected, and the scanning structure line is focused on the sample to be detected through the objective lens group; and
the image detection module is used for receiving a fluorescence signal emitted after the to-be-detected sample is subjected to line scanning excitation by the scanning structure and performing hyperspectral microimaging on the fluorescence signal;
wherein the system further comprises:
the second dichroic mirror is arranged between the structural line scanning module and the structural light modulation module, and the structural lines penetrate through the second dichroic mirror and enter the structural line scanning module;
and the fluorescent signal is reflected by the second dichroic mirror and then is emitted into the image detection module.
Preferably, the structural line scanning module includes:
the one-dimensional scanning galvanometer receives the structure line and modulates the structure line to form a scanning structure line for scanning the sample to be detected;
the scanning structure line is focused on the same focal plane through the scanning lens, then is collimated through the telescopic lens and then enters the objective lens group. Preferably, the structured light modulation module comprises:
the spatial light modulator is used for receiving the exciting light and carrying out translation modulation on the exciting light to form light and shade alternate structured light with different phase translation;
a cylindrical lens assembly receiving the structured light and compressing the structured light into the structured line.
Preferably, the cylindrical lens assembly comprises:
the first cylindrical lens is used for carrying out primary one-dimensional focusing on the structured light;
the spatial filter is used for filtering the structured light after the primary one-dimensional focusing and selecting +/-1-order diffraction light;
the second cylindrical lens is used for defocusing the filtered structured light, aligning and adjusting the width of the structured light beam;
and the third cylindrical lens is used for carrying out secondary one-dimensional focusing on the structured light to form a required structured ray to be emitted into the structured ray scanning module.
Preferably, the number of the excitation light sources is two, and the two excitation light sources are respectively a first excitation light source and a second excitation light source;
the wavelengths of the excitation light emitted by the first excitation light source and the second excitation light source are different.
Preferably, the system further comprises a first dichroic mirror, and excitation light emitted by the first excitation light source is reflected by the first dichroic mirror and then enters the structural light modulation module; and the excitation light emitted by the second excitation light source is transmitted by the first dichroic mirror and then is emitted into the structural light modulation module.
Preferably, the fluorescent signals generated by scanning to different positions of the sample are reflected by the second dichroic mirror after being back-scanned by the structural line scanning module, and then are incident into the image detection module, wherein the image detection module comprises:
the first detection camera is used for acquiring fluorescence intensity information of the fluorescence signal;
a second detection camera for acquiring a spectral image of the fluorescence signal.
Preferably, the image detection module further comprises:
and the turnover mirror is arranged on the transmission path of the fluorescence signal and used for changing the transmission path of the fluorescence signal so as to enable the fluorescence signal to selectively emit to the first detection camera or the second detection camera.
Preferably, the image detection module further comprises:
the spectroscope is arranged on a transmission path of the fluorescence signal, part of the fluorescence signal transmitted by the spectroscope enters the second detection camera, and part of the fluorescence signal reflected by the spectroscope enters the first detection camera.
Preferably, the sample stage is arranged on a rotating stage controlled by a stepping motor, and the rotating stage drives the sample stage to rotate.
The invention achieves the following beneficial effects: the structure line is subjected to translational modulation by the structure light modulation module, the structure line is scanned by the structure line scanning module, and the isotropic frequency domain expansion and resolution improvement of the sample plane are effectively realized by performing rotation control on the sample through the high-precision rotating table. Simultaneously, the excitation light source is arranged and the structure line scanning module is matched, so that the scanning excitation of the sample to be detected is realized, the spontaneous fluorescence signal of the sample to be detected can be detected, and the fluorescence signal excited by scanning can also be detected.
Drawings
Fig. 1 is a schematic structural diagram of a super-resolution fluorescence hyperspectral microscopy imaging system in the first embodiment of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The existing high-spectrum imaging system capable of breaking through the diffraction limit and realizing super-resolution adopts a Raman microscopic imaging technology of structured line illumination, but due to the inherent characteristics of Raman microscopic imaging, only autofluorescence can be observed, and the requirements on a sample and a camera are high; the structure line is only subjected to translation modulation, the improvement of the isotropic spatial resolution of the sample plane cannot be realized, the resolution in a certain direction can be improved, and the observation of the sample is not favorable;
therefore, the invention aims to provide a super-resolution fluorescence hyperspectral microimaging system, which realizes the isotropic frequency domain expansion and resolution improvement of a sample plane by forming light and dark structure lines which are translated in different phases and enabling the sample plane to rotate in different directions and generating multi-directional multi-time superposition of a sample structure and an illumination structure line; simultaneously, the excitation light source is arranged and the structure line scanning module is matched, so that the scanning excitation of the sample to be detected is realized, the spontaneous fluorescence signal of the sample to be detected can be detected, and the fluorescence signal excited by scanning can also be detected.
Example one
Referring to fig. 1, a super-resolution fluorescence hyperspectral microscopy imaging system in accordance with a first embodiment of the present invention is shown, the system comprising:
the sample microscopic assembly 10 comprises a sample table 11 for placing a sample to be detected and an objective lens group 12 for carrying out microscopy on the sample to be detected;
an excitation light source 20 for providing excitation light for irradiating a sample to be measured;
the structure light modulation module 30 receives the exciting light and modulates the exciting light to form a structure line with different phase translations and alternate light and shade;
a structure line scanning module 40, which receives and modulates the structure lines to form scanning structure lines for scanning the sample to be tested, and the scanning structure lines are focused on the sample to be tested through the objective lens group 12; and
the image detection module 50 receives a fluorescence signal emitted by the sample to be detected after being line-scanned and excited by the scanning structure, and performs hyperspectral microimaging on the fluorescence signal.
In the present embodiment, the objective lens group 12 is disposed upside down below the sample stage 11, and the inverted microstructure is favorable for imaging semitransparent or even transparent samples, and is further favorable for realizing transmission and fluorescence imaging of cells and the like.
In addition, in the present embodiment, the number of the excitation light sources 20 is two, and the two excitation light sources are the first excitation light source 20a and the second excitation light source 20b, respectively. The super-resolution fluorescence hyperspectral microimaging system further comprises a first dichroic mirror 21, wherein excitation light emitted by the first excitation light source 20a is reflected by the first dichroic mirror 21 and then enters the structural light modulation module 30, and excitation light emitted by the second excitation light source 20b is transmitted by the first dichroic mirror 21 and then enters the structural light modulation module 30. In addition, the super-resolution fluorescence hyperspectral microimaging system further includes an Acousto-optic tunable filter 22 (AOTF for short), and excitation light emitted from the excitation light source 20 passes through the Acousto-optic tunable filter 22 and then is switched to be emitted into the structured light modulation module 30.
The wavelengths of the excitation lights emitted by the first excitation light source 20a and the second excitation light source 20b are different, and by way of example and not limitation, the wavelength of the excitation light emitted by the first excitation light source 20a may be 488nm, and the wavelength of the excitation light emitted by the second excitation light source 20b may be 561 nm.
Specifically, the structured Light modulation module 30 includes a Spatial Light Modulator (SLM) 31 and a cylindrical lens assembly 33, where the Spatial Light Modulator 31 receives the excitation Light and performs translational modulation on the excitation Light to form Light and dark structured Light with different phase translations, that is, the Spatial Light Modulator 31 generates diffracted Light through a grating pattern and modulates the Spatial Light through rotation and translation means, so that the structured lines subsequently irradiated on the surface of the sample generate translations at different positions, and the isotropic frequency domain expansion and resolution improvement of the sample plane are realized. The cylindrical lens assembly 33 is used to receive the structured light and compress the structured light into structured lines.
Specifically, in the present embodiment, the cylindrical lens assembly 33 includes a first cylindrical lens 331, a spatial filter 332, a second cylindrical lens 333 and a third cylindrical lens 334, the first cylindrical lens 331 is configured to perform one-dimensional focusing on the structured light, the spatial filter 332 is configured to filter the structured light after the one-dimensional focusing, select ± 1-order diffracted light, the second cylindrical lens 333 is configured to defocus the filtered structured light, straighten and adjust the structured light beam width, and the third cylindrical lens 334 is configured to perform one-dimensional focusing on the structured light for forming a desired structured light, and the desired structured light is incident to the structured-line scanning module 40.
The structural line scanning module 40 includes a one-dimensional scanning galvanometer 41, a scanning lens 42, and a sleeve lens 43, where the one-dimensional scanning galvanometer 41 is configured to receive the structural line output by the structural light modulation module 30 and modulate the structural line to form a scanning structural line for scanning a sample to be measured. The scan structure lines are then focused by the scan lens 42 onto the same focal plane and collimated by the telescopic lens 43 into the objective lens assembly 12.
In addition, the sample stage 11 is arranged on a rotary table (not shown) controlled by a stepping motor, which rotates the sample stage, thereby adjusting the angle of the sample. That is to say, in this embodiment, the structure line is firstly subjected to translational modulation in the first direction dimension by the spatial light modulator 31, then is subjected to scanning in the second direction dimension by the one-dimensional scanning galvanometer, and finally is emitted to the sample stage, and meanwhile, the sample on the sample stage is driven by the rotating stage to rotate in the third direction dimension, so that the structure lines in different directions are realized to scan the sample, and the isotropic frequency domain expansion and resolution improvement of the sample plane are realized.
In addition, the super-resolution fluorescence hyperspectral microscopic imaging system further comprises a second dichroic mirror 60, which is arranged between the structural line scanning module 40 and the structural light modulation module 30, and fluorescent signals generated by scanning to different positions of the sample are reflected by the structural line scanning module 40, then reflected by the second dichroic mirror 60 and then enter the image detection module. Specifically, the structural lines output by the structural light modulation module 30 will penetrate through the second dichroic mirror 60 and enter the structural line scanning module 40, so as to be emitted to the sample to be measured after being scanned by the structural line scanning module 40; because the light has reversibility, the fluorescent signal generated by the excitation of the sample by the structural line returns to the original path, that is, the fluorescent signal is subjected to inverse scanning by the structural line scanning module 40 and then reaches the second dichroic mirror 60, and at this time, the fluorescent signal is reflected by the second dichroic mirror 60 and then is emitted into the image detection module 50.
In the embodiment, the image detection module 50 includes a first detection camera sCMOS1, a second detection camera sCMOS2, and a flip mirror 51, wherein the flip mirror 51 is disposed on a transmission path of the fluorescence signal for changing the transmission path of the fluorescence signal so as to selectively emit the fluorescence signal to the first detection camera or the second detection camera, specifically, the flip mirror 51 is controlled by a motor to drive the flip mirror 51 to rotate through the motor, so as to adjust an angle of the flip mirror 51, and when the flip mirror 51 rotates to be disposed at an included angle with the transmission path of the fluorescence signal, the fluorescence signal is reflected by the flip mirror 51 to enter the first detection camera sCMOS 1; when the flip mirror 51 is turned to be parallel to the transmission path of the fluorescence signal, the fluorescence signal is reflected by the flip mirror 51 and enters the second detection camera sCMOS2 through the fixed mirror below. The first detection camera is used for acquiring fluorescence intensity information of the fluorescence signal; the second detection camera is connected with the spectrometer and is used for acquiring the spectral image of the fluorescence signal.
In other optional embodiments, the image detection module may further include a beam splitter disposed on a transmission path of the fluorescence signal, a part of the fluorescence signal transmitted by the beam splitter enters the second detection camera, and a part of the fluorescence signal reflected by the beam splitter enters the first detection camera. That is, the turning mirror 51 can be replaced by a spectroscope, and due to the semi-transmission and semi-reflection function of the spectroscope, a part of the transmitted fluorescence signal can be successfully reflected by the lower fixed mirror to enter the second detection camera, and another part of the fluorescence signal reflected by the spectroscope can be successfully reflected to enter the first detection camera.
Therefore, in the embodiment of the present invention, the fluorescence light beam is separated by the spectroscope or the turning mirror before the first detection camera, and two sCMOS detection cameras are respectively used to simultaneously obtain the original images of the fluorescence intensity information and the spectrum information; on one hand, a linear array camera or an area array camera is used for detecting a fluorescence intensity image, on the other hand, the area array camera is used for matching with a spectrometer to acquire spectral information of a structure line excitation sample, and the two cameras are used for acquiring data obtained by scanning different structure lines by using the external trigger of a scanning galvanometer to match with the scanning speed and the light splitting speed.
In addition, the super-resolution fluorescence hyperspectral microimaging system further comprises a processor (such as a computer, an upper computer and the like), wherein the processor is used for controlling the system to work cooperatively and processing the information detected by the image detection module 50. Specifically, the processor specifically includes a timing control module and an image reconstruction module. Wherein:
the time sequence control module is developed based on LABVIEW software, and is programmed to realize the time sequence synchronization of software control hardware equipment, so that the time sequence control hardware equipment and the software control hardware equipment are matched with each other to form a high-quality original image at the highest speed. The software realizes man-machine interaction by writing an LABVIEW state machine, realizes the flow processing of hardware commands by operations such as a sequence structure, a trigger event and the like, and further realizes the integrated control of hardware or a lower computer by combining the modes of serial port communication, calling a driver, a sub-VI and the like. The time sequence control module is designed to send signals to a data acquisition card by a computer, and controls the acousto-optic tunable filter, the spatial light modulator 31, the scanning galvanometer, the high-precision rotary table and the camera detector by the data acquisition card, so that the scanning of the scanning galvanometer and the exposure of the camera are synchronous, the spatial light modulator 31 is switched to modulate the phase shift of structured light or the rotary table of the sample plane is controlled to rotate the sample in one direction when the scanning of the sample plane is completed once, and simultaneously the fast light beam turning reflector 51 is controlled to enable fluorescent structure lines to sequentially enter the two camera detectors of the image detection module, and the acousto-optic tunable filter is controlled to switch laser after 9 times of structured light modulation of 3 times of phase shift in 3 directions.
The image reconstruction module includes four important parts: determining a reconstruction parameter, judging the quality of an original image, realizing a reverse convolution algorithm and mutually correcting a fluorescence image and a hyperspectral super-resolution image.
The determination part of the reconstruction parameters comprises the determination of the mode wave vector, the initial phase, the modulation depth and other parameters, wherein the accurate determination of the mode wave vector of the original image is very important, because for the original image with low signal-to-noise ratio, the slight deviation of the reconstruction parameters, especially the deviation of the mode vector, can bring about larger artifacts and destroy the authenticity of the reconstructed image. The method comprises the steps of combining images with the same wave vector and phase to reduce noise, normalizing amplitude by multi-frame averaging of an original image with low signal-to-noise ratio, performing cross-correlation by using phase information only, and separating different frequency domains by adopting the normalized cross-correlation algorithm and accurately calculating reconstruction parameters such as a mode wave vector.
And (3) using a SIMcheck plug-in of ImageJ software to judge the quality of the original image, and mainly judging the modulation signal-to-noise ratio of the original image. The selection of a later-stage image processing algorithm is determined based on the early-stage judgment of SIMcheck, the original image with a higher modulation signal-to-noise ratio value is subjected to super-resolution image reconstruction by using a wiener deconvolution reconstruction algorithm, and the original image with a lower modulation signal-to-noise ratio value is processed by a Hessian deconvolution reconstruction algorithm.
The image reconstruction part based on MATLAB comprises the steps of selecting a reconstruction algorithm according to the quality of an original image, obtaining the illumination space frequency and direction, calculating an optical transfer function, and performing deconvolution and frequency division recombination by using different deconvolution algorithms to obtain a super-resolution image.
Aiming at the original hyperspectral tiff image stack, the coordinate axis of the image stack is required to be converted, fluorescence intensity images of all wave bands are output, and then early-stage judgment based on SIMcheck and image processing based on MATLAB are carried out. And finally, comparing and correcting the super-resolution image of the fluorescence intensity and the super-resolution image of the spectral information.
In summary, the super-resolution fluorescence hyperspectral microscopy imaging system in the embodiment has at least the following technical effects compared with the structured-line illumination raman microscope:
1) the structure line is subjected to translational modulation by arranging the structure light modulation module, the structure line is scanned by matching with a one-dimensional scanning galvanometer, and the sample is rotated by arranging the rotating table, which is equivalent to that the structure line providing different rotating directions and phase shifts is used for scanning and exciting the sample, so that the multi-directional multi-time superposition of the sample structure and different phase structures of the structure line is utilized, and the isotropic frequency domain expansion and the resolution improvement of the sample plane are realized;
2) meanwhile, by arranging an excitation light source and matching with a structural line scanning module, the scanning excitation of the sample to be detected is realized, namely, the spontaneous fluorescence signal of the sample to be detected can be detected, and the fluorescence signal excited by scanning can also be detected;
3) imaging the sample by using a fluorescence inverted microscope, wherein the imaging comprises excitation and fluorescence collection, a high-resolution spectrometer and an sCMOS are matched, the sCMOS is matched with most biological samples, and the internal components of the substances are distinguished on the spectrum;
4) the spatial light modulator is adopted, so that the structure line can be controlled more flexibly and accurately, and the control of the translation of the structure line can be completed quickly and stably;
5) a spectroscope/turnover mirror and a detection camera are additionally arranged on a detection light path, so that a super-resolution image of fluorescence intensity can be acquired, and the super-resolution hyperspectral image are mutually corrected;
6) a plurality of lasers and acousto-optic tunable filters are newly added to form an excitation light control module, so that super-resolution multicolor imaging of fluorescence intensity is realized, and visual observation of a biological sample is met;
7) the super-resolution fluorescence hyperspectral microscopic imaging system can be used for observing various microscopic biological samples, the greatest advantage can be embodied in the observation of small organelles and the rapid dynamic process thereof as well as the change of the internal components of the object in the dynamic process, and the high-spatial-resolution intensity information and spectral information can be used for pathological research and drug development. Such as observation of the interaction process of lipid droplets with mitochondria.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (9)
1. A super-resolution fluorescence hyperspectral microscopy imaging system, the system comprising:
the sample microscopic assembly comprises a sample table for placing a sample to be detected and an objective lens group for carrying out microscopy on the sample to be detected;
the excitation light source is used for providing excitation light for irradiating a sample to be detected;
the structure light modulation module is used for receiving the exciting light and modulating the exciting light to form a structure line with different phase translations and alternate light and shade;
the structure line scanning module receives the structure line and modulates the structure line to form a scanning structure line for scanning the sample to be detected, and the scanning structure line is focused on the sample to be detected through the objective lens group; and
the image detection module is used for receiving a fluorescence signal emitted after the to-be-detected sample is subjected to line scanning excitation by the scanning structure and performing hyperspectral microimaging on the fluorescence signal;
the system further comprises:
the second dichroic mirror is arranged between the structural line scanning module and the structural light modulation module, and the structural lines penetrate through the second dichroic mirror and enter the structural line scanning module;
and the fluorescent signal is reflected by the second dichroic mirror and then is emitted into the image detection module.
2. The super-resolution fluorescence hyperspectral microscopic imaging system according to claim 1, wherein the structured light modulation module comprises:
the spatial light modulator is used for receiving the exciting light and carrying out translation modulation on the exciting light to form light and shade alternate structured light with different phase translation;
a cylindrical lens assembly receiving the structured light and compressing the structured light into the structured line.
3. The super-resolution fluorescence hyperspectral microscopic imaging system according to claim 2, wherein the cylindrical lens assembly comprises:
the first cylindrical lens is used for carrying out primary one-dimensional focusing on the structured light;
the spatial filter is used for filtering the structured light after the primary one-dimensional focusing and selecting +/-1-order diffraction light;
the second cylindrical lens is used for defocusing the filtered structured light, aligning and adjusting the width of the structured light beam;
and the third cylindrical lens is used for carrying out secondary one-dimensional focusing on the structured light to form a required structured ray to be emitted into the structured ray scanning module.
4. The super-resolution fluorescence hyperspectral microscopic imaging system according to claim 1, wherein the number of the excitation light sources is two, and the two excitation light sources are a first excitation light source and a second excitation light source respectively;
the wavelengths of the excitation light emitted by the first excitation light source and the second excitation light source are different.
5. The super-resolution fluorescence hyperspectral microscopic imaging system according to claim 4, wherein the system further comprises a first dichroic mirror, and excitation light emitted by the first excitation light source is reflected by the first dichroic mirror and then enters the structural light modulation module; and the excitation light emitted by the second excitation light source is transmitted by the first dichroic mirror and then is emitted into the structural light modulation module.
6. The super-resolution fluorescence hyperspectral microscopic imaging system according to claim 1, wherein the fluorescence signals generated by scanning different positions of the sample are reflected by the second dichroic mirror after being back-scanned by the structural line scanning module and then are incident into the image detection module, wherein the image detection module comprises:
the first detection camera is used for acquiring fluorescence intensity information of the fluorescence signal;
a second detection camera for acquiring a spectral image of the fluorescence signal.
7. The super-resolution fluorescence hyperspectral microscopic imaging system according to claim 6, wherein the image detection module further comprises:
and the turnover mirror is arranged on the transmission path of the fluorescence signal and used for changing the transmission path of the fluorescence signal so as to enable the fluorescence signal to selectively emit to the first detection camera or the second detection camera.
8. The super-resolution fluorescence hyperspectral microscopic imaging system according to claim 6, wherein the image detection module further comprises:
the spectroscope is arranged on a transmission path of the fluorescence signal, part of the fluorescence signal transmitted by the spectroscope enters the second detection camera, and part of the fluorescence signal reflected by the spectroscope enters the first detection camera.
9. The super-resolution fluorescence hyperspectral microscopic imaging system according to any of claims 1 to 8, wherein the sample stage is arranged on a rotating stage controlled by a stepping motor, the rotating stage driving the sample stage to rotate.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2023103960A1 (en) * | 2021-12-10 | 2023-06-15 | 中国科学院深圳先进技术研究院 | Three-dimensional imaging apparatus and imaging method |
| CN117608066A (en) * | 2024-01-19 | 2024-02-27 | 清华大学 | Microscopic imaging device and method for positive and negative integrated scanning light field |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023103960A1 (en) * | 2021-12-10 | 2023-06-15 | 中国科学院深圳先进技术研究院 | Three-dimensional imaging apparatus and imaging method |
| CN117608066A (en) * | 2024-01-19 | 2024-02-27 | 清华大学 | Microscopic imaging device and method for positive and negative integrated scanning light field |
| CN117608066B (en) * | 2024-01-19 | 2024-06-07 | 清华大学 | Microscopic imaging device and method for positive and negative integrated scanning light field |
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