CN116262034A - In-vivo two-photon microscopic imaging system - Google Patents
In-vivo two-photon microscopic imaging system Download PDFInfo
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- CN116262034A CN116262034A CN202111517904.8A CN202111517904A CN116262034A CN 116262034 A CN116262034 A CN 116262034A CN 202111517904 A CN202111517904 A CN 202111517904A CN 116262034 A CN116262034 A CN 116262034A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
- A61B5/0062—Arrangements for scanning
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
Abstract
According to the in-vivo two-photon microscopic imaging system, two different imaging dyes or probes are respectively excited and formed into images through the two excitation lights with different wavelengths by the scanning modules (5) and (9), pixel positions of cells or tissues marked by the two different dyes are determined according to the images, then the photoelectric modulators (2) and (7) are respectively controlled by using pixel clock signals of the scanning vibrating mirrors in the scanning modules, on/off of the excitation lights of the scanning modules (5) and (9) are controlled in real time, and only excitation is carried out at respective target cells or tissues, so that two-photon excitation two-color synchronous imaging is realized, and the problem of excitation/emission light crosstalk in two-color imaging is avoided.
Description
Technical Field
The application relates to the technical field of lasers, in particular to an in-vivo two-photon microscopic imaging system.
Background
The two-photon microscopic imaging technology has the advantages of non-invasiveness, high resolution, strong chromatographic capacity, small phototoxicity and strong light penetrability, is particularly suitable for observing biological tissues with strong light scattering, and becomes one of the most important research tools in the field of life science. Two beams of excitation light with different wavelengths are used to match with a double detection channel, and simultaneously, a double-color two-photon microscopic imaging technology of a double-marking technology is combined, so that synchronous imaging of two different cell types or tissue components can be realized, and the method has wide application prospects in the field of life science. Two-color simultaneous imaging often requires the selection of two dyes with very different stokes shift to avoid signal crosstalk, but imaging dyes or probes for in vivo imaging, especially for physiological function detection, have broad excitation/emission spectra, and spectral overlap is difficult to avoid.
Disclosure of Invention
In view of this, it is desirable to provide an imaging technique that can be used to reduce excitation/emission light crosstalk in two-color two-photon imaging.
In order to solve the problems, the following technical scheme is adopted in the application:
the application provides an in-vivo two-photon microscopic imaging system, which is characterized by comprising a first femtosecond pulse excitation light source (1), a first photoelectric modulator (2), a first beam expander (3), a first reflecting mirror (4), a first scanning module (5), a second femtosecond pulse excitation light source (6), a second photoelectric modulator (7), a second beam expander (8), a second scanning module (9), a first dichroic mirror (10), a scanning lens (11), a sleeve lens (12), a second dichroic mirror (13), a microscope objective (14), a third dichroic mirror (15), a first narrow-band optical filter (16), a first photomultiplier (17), a second narrow-band optical filter (18), a second photomultiplier (19), a signal control/acquisition device (20) and a computer (21), wherein:
the laser emitted by the first femtosecond pulse excitation light source (1) is modulated by the first photoelectric modulator (2) and then is subjected to beam expansion by the first beam expansion device (3), then is reflected by the first reflecting mirror (4) and vertically enters the first scanning module (5) downwards, the formed scanning beam is transmitted through the first dichroic mirror (10) and then is focused on a sample by the scanning lens (11), the sleeve lens (12) and the second dichroic mirror (13), the emitted light generated after exciting a first imaging dye is collected by the microscope objective (14) and then is reflected by the second dichroic mirror (13) to the third dichroic mirror (15), is filtered by the first narrow-band filter (16) and then is detected by the first photoelectric multiplier tube (17), and the signal control/acquisition equipment (20) and a computer (21) are used for acquiring and processing to obtain an image of the first imaging dye or tissue of the first imaging dye;
meanwhile, the laser emitted by the second femtosecond pulse excitation light source (6) is modulated by the second photoelectric modulator (7) and then is subjected to beam expansion by the second beam expansion device (8), then enters the second scanning module (9), the formed scanning beam is reflected by the first dichroic mirror (10) and vertically and downwards propagates, the scanning beam is focused on a sample by the microscope objective (14) after being reflected by the first dichroic mirror (11), the sleeve lens (12) and the second dichroic mirror (13), the emitted light generated after exciting the second dye is collected by the microscope objective (14) and reflected by the second dichroic mirror (13) to the third dichroic mirror (15), is filtered by the third dichroic mirror (15) and then is detected by the second narrow-band filter (18), and is collected and processed by the signal control/collection device (20) and the computer (21), so that an image of a cell or tissue marked by the second dye is obtained;
and (3) carrying out image segmentation on the obtained structural image, determining the pixel positions of cells or tissues marked by two different dyes, respectively controlling the photoelectric modulators (2) and (7) by using pixel clock signals of a scanning galvanometer in each scanning module, carrying out real-time control on/off of excitation light of the scanning modules (5) and (9), and only carrying out excitation at a target cell or tissue, thereby realizing double-color synchronous imaging of two-photon excitation and avoiding the problem of excitation/emission light crosstalk in double-color imaging.
In some of these embodiments, both scanning modules (5) and (9) consist of one galvanometer mirror and one high-speed resonant galvanometer mirror.
In some of these embodiments, the two scanning modules (5) and (9), one set consists of two galvanometer mirrors and the other set consists of one galvanometer mirror and one high-speed resonant mirror.
By adopting the technical scheme, the application has the following beneficial effects:
the application provides a two-photon microscopic imaging technique of two colors, including first femtosecond pulse excitation light source (1), first photoelectric modulator (2), first beam expander (3), first speculum (4), first scanning module (5), second femtosecond pulse excitation light source (6), second photoelectric modulator (7), second beam expander (8), second scanning module (9), first dichroscope (10), scanning lens (11), sleeve lens (12), second dichroscope (13), microscope objective (14), third dichroscope (15), first narrowband filter (16), first photomultiplier (17), second narrowband filter (18), second photomultiplier (19), signal control/collection device (20) and computer (21). The two different imaging dyes or probes are respectively excited by the two excitation lights with different wavelengths through the scanning modules (5) and (9) to form images, the pixel positions of cells or tissues marked by the two different dyes are determined according to the images, then the photoelectric modulators (2) and (7) are respectively controlled by using pixel clock signals of the scanning galvanometer in each scanning module, the on/off of the excitation lights of the scanning modules (5) and (9) are controlled in real time, and only the target cells or tissues are excited, so that two-photon excitation two-color synchronous imaging is realized, and the problem of excitation/emission light crosstalk in two-color imaging is avoided.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings used in the embodiments of the present application or the description of the prior art will be briefly described below. It is obvious that the drawings described below are only some embodiments of the present application, and that other drawings may be obtained from these drawings without inventive effort for a person of ordinary skill in the art.
Fig. 1 is a schematic diagram of an in-vivo two-photon microscopic imaging system according to an embodiment of the present application.
Fig. 2a is a diagram of a structure of a blood vessel and a nerve cell according to an embodiment of the present application.
Fig. 2b is a schematic diagram of a voltage signal generated by a scanning galvanometer or resonant galvanometer driving circuit during scanning line feed according to an embodiment of the present application, where the voltage signal is used to generate a pixel clock.
Detailed Description
Embodiments of the present application are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals refer to the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the drawings are exemplary and intended for the purpose of explaining the present application and are not to be construed as limiting the present application.
In the description of the present application, it should be understood that the terms "upper," "lower," "horizontal," "inner," "outer," and the like indicate an orientation or a positional relationship based on that shown in the drawings, and are merely for convenience of description and simplification of the description, and do not indicate or imply that the apparatus or element in question must have a specific orientation, be configured and operated in a specific orientation, and therefore should not be construed as limiting the present application.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. In the description of the present application, the meaning of "a plurality" is two or more, unless explicitly defined otherwise.
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be further described in detail with reference to the accompanying drawings and examples.
The working principle of the technology is described in detail by taking the functional imaging of living mouse cerebral cortex nerves and blood vessels as an example. The calcium fluorescence indicator GCaMP6f can be used for marking the activity of calcium nervonate, has a better two-photon absorption section at 920nm, and has an emission wavelength peak value of 510nm; the luminescence life of the phosphorescence oxygen probe Oxyphor2P is sensitive to oxygen concentration, can be used for measuring blood oxygen partial pressure, has a better two-photon absorption section at 950nm, and has an emission wavelength peak value of-758 nm. However, two-photon calcium-of-nerve activity imaging differs from the principle of two-photon blood oxygen partial pressure detection: neural activity causes changes in intracellular calcium ion concentration and changes in fluorescence intensity of the calcium indicator, requiring changes in fluorescence signal to be recorded at a scan rate of tens of frames per second; the two-photon blood oxygen partial pressure detection is based on triplet state molecular luminescence of ground state oxygen quenching, changes the luminescence life, and needs to repeat the excitation/collection process with single time duration of 300 mu s at discrete pixel points (such as 500-2000 times) for recording complete phosphorescence attenuation signals with high signal to noise ratio, and finally, the phosphorescence life value is converted into oxygen partial pressure according to the predetermined chemical calibration. In the synchronous measurement process of the activity of the calcium nervone and the blood oxygen partial pressure, the excitation light with the wavelength of 920nm can excite the Oxyphor2P molecule while exciting the GCaMP6f, so that phosphorescence signal interference is generated for the measurement of the oxygen partial pressure, and the accuracy of the oxygen partial pressure is affected. The two-color two-photon microscopic imaging technology provided by the application can effectively solve the problem, and the specific process is as follows:
referring to fig. 1, an in-vivo two-photon microscopic imaging system provided in the embodiments of the present application includes a first femtosecond pulse excitation light source (1), a first photoelectric modulator (2), a first beam expander (3), a first mirror (4), a first scanning module (5), a second femtosecond pulse excitation light source (6), a second photoelectric modulator (7), a second beam expander (8), a second scanning module (9), a first dichroic mirror (10), a scanning lens (11), a sleeve lens (12), a second dichroic mirror (13), a microscope objective lens (14), a third dichroic mirror (15), a first narrow-band filter (16), a first photomultiplier (17), a second narrow-band filter (18), a second photomultiplier (19), a signal control/acquisition device (20), and a computer (21). In this embodiment, the first scanning module (5) consists of two galvanometer mirrors and the second scanning module (9) consists of one galvanometer mirror and one resonant mirror. The specific operation is as follows:
structural imaging: the laser of 950nm emitted by the first femtosecond pulse excitation light source (1) is modulated by the first photoelectric modulator (2) and then is subjected to beam expansion by the first beam expander (3), then is reflected by the first reflector (4) and vertically enters the first scanning module (5) downwards, the first scanning module (5) consists of two galvanometer vibrating mirrors, the formed scanning beam is transmitted through the first dichroic mirror (10) and then is focused at a certain depth of the rat brain cortex through the microscope objective (14) through the scanning lens (11), the sleeve lens (12) and the second dichroic mirror (13), and oxygen probe (Oxyphor 2P) molecules dissolved in blood are excited and phosphorescence with 758nm as a central wavelength is emitted. The emitted phosphorescence is collected by a microscope objective lens (14), reflected by a second dichroic mirror (13) to a third dichroic mirror (15), transmitted through the third dichroic mirror (15), filtered by a first narrow-band filter (16), detected by a first photomultiplier (17), and collected and processed by a signal control/collection device (20) and a computer (21) to obtain a vascular structure image (such as a vascular image in fig. 2 a);
meanwhile, laser of 920nm emitted by a second femtosecond pulse excitation light source (6) is modulated by a second photoelectric modulator (7), then is expanded by a second beam expander (8) and enters a second scanning module (9), the second scanning module (9) consists of a galvanometer vibrating mirror and a resonance vibrating mirror, a formed scanning beam is reflected by a first dichroic mirror (10) to vertically downwards spread, the scanning beam is focused at the same depth of a rat brain cortex by a microscope objective (14) after being reflected by a scanning lens (11), a sleeve lens (12) and a second dichroic mirror (13), and a fluorescent indicator GCaMP6f for marking the activity of the nerve calcium is excited by laser of 920nm and emits fluorescence with a central wavelength of 510 nm. The emitted fluorescence is collected by a microscope objective lens (14), reflected by a second dichroic mirror (13) to a third dichroic mirror (15), reflected by the third dichroic mirror (15), filtered by a second narrow-band filter (18), detected by a second photomultiplier (19), and collected and processed by a signal control/collection device (20) and a computer (21) to obtain a nerve cell structure image (such as nerve cells in fig. 2 a);
binary segmentation is respectively carried out on the blood vessel structure image and the nerve cell structure image by utilizing an image segmentation algorithm, and the pixel position information of the blood vessel and the nerve cell to be detected is determined;
the activity of the nerve calcium is synchronously measured with the partial pressure of blood oxygen: keeping the object to be measured still, controlling the photoelectric modulator (7) by a pixel clock signal (shown in fig. 2 b) of a resonant galvanometer in the second scanning module (9), so that the excitation light (920 nm, used for exciting GCaMP6 f) of the scanning module (9) is only turned on at the pixel position of the preselected nerve cell, and turned off at other pixel positions (white lines in fig. 2a represent the scanning path of the scanning module (9), the left side shows the voltage control signal of the photoelectric modulator, and the inside of a circle shows the preselected nerve cell), thereby avoiding the excitation of the Oxyphor2P molecule to generate phosphorescence; meanwhile, the scanning module (5) is controlled according to the pixel position of the blood vessel to be detected, and the blood oxygen partial pressure measurement is carried out at the pixel position of the blood vessel to be detected without being interfered by calcium imaging excitation light, so that synchronous imaging of the activity of the calcium nervone and the blood oxygen partial pressure is realized.
The foregoing description of the preferred embodiments of the present application is provided merely for the purpose of illustrating the general principles of the present application and is not meant to limit the scope of the application in any way. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application, and other embodiments of the present application, which may occur to those skilled in the art without the exercise of inventive faculty, are intended to be included within the scope of the present application, based on the teachings herein.
Claims (3)
1. An in-vivo two-photon microscopic imaging system is characterized by comprising a first femtosecond pulse excitation light source (1), a first photoelectric modulator (2), a first beam expander (3), a first reflecting mirror (4), a first scanning module (5), a second femtosecond pulse excitation light source (6), a second photoelectric modulator (7), a second beam expander (8), a second scanning module (9), a first dichroic mirror (10), a scanning lens (11), a sleeve lens (12), a second dichroic mirror (13), a microscope objective (14), a third dichroic mirror (15), a first narrow-band filter (16), a first photomultiplier (17), a second narrow-band filter (18), a second photomultiplier (19), a signal control/acquisition device (20) and a computer (21), wherein:
the laser emitted by the first femtosecond pulse excitation light source (1) is modulated by the first photoelectric modulator (2) and then is subjected to beam expansion by the first beam expansion device (3), then is reflected by the first reflecting mirror (4) and vertically enters the first scanning module (5) downwards, the formed scanning beam is transmitted through the first dichroic mirror (10) and then is focused on a sample by the scanning lens (11), the sleeve lens (12) and the second dichroic mirror (13), the emitted light generated after exciting a first imaging dye is collected by the microscope objective (14) and then is reflected by the second dichroic mirror (13) to the third dichroic mirror (15), is filtered by the first narrow-band filter (16) and then is detected by the first photoelectric multiplier tube (17), and the signal control/acquisition equipment (20) and a computer (21) are used for acquiring and processing to obtain an image of the first imaging dye or tissue of the first imaging dye;
meanwhile, the laser emitted by the second femtosecond pulse excitation light source (6) is modulated by the second photoelectric modulator (7) and then is subjected to beam expansion by the second beam expansion device (8), then enters the second scanning module (9), the formed scanning beam is reflected by the first dichroic mirror (10) and vertically and downwards propagates, the scanning beam is focused on a sample by the microscope objective (14) after being reflected by the first dichroic mirror (11), the sleeve lens (12) and the second dichroic mirror (13), the emitted light generated after exciting the second imaging dye is collected by the microscope objective (14) and reflected by the second dichroic mirror (13) to the third dichroic mirror (15), is filtered by the third dichroic mirror (15) and then is detected by the second photomultiplier (19) after being filtered by the second narrow-band filter (18), and the image of the cell or tissue of the imaging dye is obtained by collecting and processing the signal control/collection device (20) and the computer (21);
and (3) carrying out image segmentation on the obtained structural image, determining the pixel positions of cells or tissues marked by two different dyes, respectively controlling the photoelectric modulators (2) and (7) by using pixel clock signals of a scanning galvanometer in each scanning module, carrying out real-time control on/off of excitation light of the scanning modules (5) and (9), and only carrying out excitation at a target cell or tissue, thereby realizing double-color synchronous imaging of two-photon excitation and avoiding the problem of excitation/emission light crosstalk in double-color imaging.
2. The in-vivo two-photon microscopy imaging system as in claim 1 wherein said two scanning modules (5) and (9) are each comprised of a galvanometer and a high-speed resonance galvanometer.
3. The in-vivo two-photon microscopy imaging system as in claim 1 wherein said two scanning modules (5) and (9) are comprised of two galvanometer mirrors in one set and one galvanometer mirror and one high-speed resonant mirror in the other set.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111517904.8A CN116262034A (en) | 2021-12-13 | 2021-12-13 | In-vivo two-photon microscopic imaging system |
| PCT/CN2022/137419 WO2023109625A1 (en) | 2021-12-13 | 2022-12-08 | In-vivo double-color two-photon microscopic imaging system |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111517904.8A CN116262034A (en) | 2021-12-13 | 2021-12-13 | In-vivo two-photon microscopic imaging system |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2010101894A2 (en) * | 2009-03-02 | 2010-09-10 | President & Fellows Of Harvard College | High resolution laser scanning microscopy imaging system and method using spatially patterned cumulative illumination of detection fields |
| US20100252750A1 (en) * | 2009-04-03 | 2010-10-07 | Xiaoliang Sunney Xie | Systems and methods for stimulated emission imaging |
| CN106290284B (en) * | 2016-09-19 | 2023-03-10 | 浙江大学 | Two-photon fluorescence microscope system and method with structured light illumination |
| CN108051414B (en) * | 2017-11-30 | 2019-01-08 | 哈尔滨工业大学 | Zoom optical tweezer two-photon microscopic imaging device and method |
| CN112816449B (en) * | 2020-12-31 | 2022-08-02 | 南京航空航天大学 | Dual-wavelength dual-scale nano-drug living body imaging system and time sequence control method |
| CN113390844A (en) * | 2021-06-17 | 2021-09-14 | 中国药科大学 | Multi-scale optical fiber fluorescence microscopic imaging system |
| CN113740307B (en) * | 2021-09-02 | 2022-05-20 | 深圳大学 | Multi-mode nonlinear microscopic imaging system |
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