CN115702777A - Multifunctional two-photon microscopic imaging system - Google Patents
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- 238000003384 imaging method Methods 0.000 title claims abstract description 56
- 230000005284 excitation Effects 0.000 claims abstract description 40
- 230000000638 stimulation Effects 0.000 claims abstract description 13
- 230000001766 physiological effect Effects 0.000 claims abstract description 8
- 238000001514 detection method Methods 0.000 claims description 14
- 230000003287 optical effect Effects 0.000 claims description 7
- 238000000386 microscopy Methods 0.000 claims description 5
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical group [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 abstract description 9
- 229910052791 calcium Inorganic materials 0.000 abstract description 9
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- 238000005516 engineering process Methods 0.000 abstract description 5
- 210000005036 nerve Anatomy 0.000 abstract description 4
- 230000033228 biological regulation Effects 0.000 abstract description 3
- 230000004927 fusion Effects 0.000 abstract description 3
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 5
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- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 230000009134 cell regulation Effects 0.000 description 3
- 230000001360 synchronised effect Effects 0.000 description 3
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 229910001424 calcium ion Inorganic materials 0.000 description 2
- 230000002490 cerebral effect Effects 0.000 description 2
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- 238000000149 argon plasma sintering Methods 0.000 description 1
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
Abstract
According to the multifunctional two-photon microscopic imaging system, the first excitation light source (1), the first photoelectric modulator (2), the first beam expanding device (3), the reflecting mirror (4) and the first scanning device (5) form a light stimulation module; the second excitation light source (19), the second photoelectric modulator (20), the second beam expanding device (21) and the second scanning device (22) form a point scanning imaging module; the third excitation light source (23), the third photoelectric modulator (24), the third beam expanding device (25) and the third scanning device (26) form a line scanning imaging module. The system effectively integrates the point scanning technology and the line scanning technology, and can realize the fusion and synchronization of three functions of structural imaging of living biological tissues, optogenetic regulation and control of cells and imaging of physiological activities (such as nerve calcium signals).
Description
Technical Field
The invention belongs to the technical field of optical microscopic imaging, and particularly relates to a multifunctional 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 penetration, 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. The existing scanning mode of two-photon microscopic imaging can be roughly divided into point scanning based on a galvanometer and line scanning based on a resonant galvanometer, and different application scenes can be respectively dealt with: point scanning is suitable for structural imaging of biological tissues and optogenetics-based cell regulation; line scanning is useful for observing rapidly changing physiological activity (e.g., neural calcium signals) in biological tissues. The synchronization of structural imaging, cell regulation and calcium imaging is of great significance to the research of life science, especially neuroscience.
However, the existing two-photon imaging technology cannot meet the requirement of synchronous operation of the above three functions. For example, the Ultima2P Plus system from Bruker (Bruker) can only select one of the imaging functions, point scan or line scan, when running; A1R-MP + system of Nikon (Nikon) and an FVMPE-RS system of Olympus (Olympus) have the function of synchronously operating point scanning and line scanning, but the optical stimulation and the nerve calcium imaging cannot be synchronously carried out at the same time of structural imaging. The invention provides a design scheme of a multifunctional and modularized two-photon microscopic imaging system, which has the functions of point scanning and line scanning imaging and can realize the synchronization of three functions of structural imaging of biological tissues, cell regulation and control based on optogenetics and cell calcium imaging.
Disclosure of Invention
In order to solve the problems, the invention adopts the following technical scheme:
a multi-functional two-photon microscopy imaging system, comprising: the device comprises a first excitation light source (1), a first photoelectric modulator (2), a first beam expanding device (3), a reflecting mirror (4), a first scanning device (5), a first dichroic mirror (6), a first scanning lens (7), a second dichroic mirror (8), a sleeve lens (9), a third dichroic mirror (10), a microscope objective lens (11), a fourth dichroic mirror (12), a first narrow-band filter (13), a first photoelectric detection module (14), a second narrow-band filter (15), a second photoelectric detection module (16), a signal control/acquisition device (17), a computer (18), a second excitation light source (19), a second photoelectric modulator (20), a second beam expanding device (21), a second scanning device (22), a third excitation light source (23), a third photoelectric modulator (24), a third beam expanding device (25), a third scanning device (26) and a second scanning lens (27).
Laser emitted by the first excitation light source (1) enters the first beam expanding device (3) after being modulated by the first photoelectric modulator (2), a light beam after being expanded enters the first scanning device (5) for scanning after being reflected by the reflecting mirror (4), and the scanning light beam enters the microscope objective lens (11) after passing through the first dichroic mirror (6), the first scanning lens (7), the second dichroic mirror (8), the sleeve lens (9) and the third dichroic mirror (10) in a transmission manner and is focused on biological tissues for light stimulation.
The laser emitted by the second excitation light source (19) enters the second beam expanding device (21) after being modulated by the second photoelectric modulator (20), the expanded light beam enters the second scanning device (22) for scanning, the scanning light beam is reflected by the first dichroic mirror (6), then transmits through the first scanning lens (7), the second dichroic mirror (8), the sleeve lens (9) and the third dichroic mirror (10), enters the microscope objective lens (11), is focused on the living body tissue of a living organism, the fluorescence generated by excitation is collected by the microscope objective lens (11), then is reflected by the third dichroic mirror (10), then transmits through the fourth dichroic mirror (12), is filtered by the first narrow-band optical filter (13), and then is detected by the first photoelectric detection module (14), and the signals are collected and processed by the signal control/collection device (17) and the computer (18), so that the structure or slowly-changing physiological activities of the living organism can be observed through imaging.
Laser light emitted by the third excitation light source (23) enters the third beam expanding device (25) after being modulated by the third photoelectric modulator (24), light beams after beam expansion enter the third scanning device (26) for scanning, the scanning light beams are reflected by the second dichroic mirror (8) after passing through the second scanning lens (27), then sequentially transmit through the sleeve lens (9) and the third dichroic mirror (10), enter the microscope objective lens (11), and are focused on biological living body tissues. Fluorescence generated by excitation is collected by the microscope objective lens (11), then is reflected by the third dichroic mirror and the fourth dichroic mirror (12) in sequence, is filtered by the second narrow-band filter (15), and then is detected by the second photoelectric detection module (16), and then is acquired and processed by the signal control/acquisition equipment (17) and the computer (18), so that imaging observation of rapidly changing physiological activities in biological tissues is realized.
In some of these embodiments, the first scanning device (5) consists of two galvanometer mirrors.
In some of these embodiments, the second scanning device (22) consists of two galvanometer mirrors.
In some of these embodiments, the third scanning device (26) consists of a galvanometer mirror and a high speed resonant mirror.
The technical scheme adopted by the application has the following effects:
according to the multifunctional two-photon microscopic imaging system, the first excitation light source (1), the first photoelectric modulator (2), the first beam expanding device (3), the reflecting mirror (4) and the first scanning device (5) form a light stimulation module; the second excitation light source (19), the second photoelectric modulator (20), the second beam expanding device (21) and the second scanning device (22) form a point scanning imaging module; the third excitation light source (23), the third photoelectric modulator (24), the third beam expanding device (25) and the third scanning device (26) form a line scanning imaging module. The system controls the three imaging modules through the signal control/acquisition equipment (17), and realizes the independent operation or synchronous operation of different modules. The system can realize the fusion and synchronization of optical stimulation, point scanning imaging and line scanning imaging, can perform optogenetic regulation and control on cells at mesoscale and subcellular resolution, and can quantitatively observe the structure and functional information of biological tissues.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments of the present invention 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 invention, and that for a person skilled in the art, other drawings can be derived from them without inventive effort.
Fig. 1 is a schematic structural diagram of a multifunctional two-photon microscopic imaging system according to an embodiment of the present invention.
The specific implementation mode is as follows:
reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
In the description of the present invention, it is to be understood that the terms "upper", "lower", "horizontal", "inner", "outer", and the like, indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, are merely for convenience in describing the present invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a particular orientation or be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or to implicitly indicate the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below with reference to the accompanying drawings and embodiments.
Referring to fig. 1, a schematic structural diagram of a multifunctional two-photon microscopy imaging system provided in an embodiment of the present application includes: the device comprises a first excitation light source (1), a first photoelectric modulator (2), a first beam expanding device (3), a reflecting mirror (4), a first scanning device (5), a first dichroic mirror (6), a first scanning lens (7), a second dichroic mirror (8), a sleeve lens (9), a third dichroic mirror (10), a microscope objective (11), a fourth dichroic mirror (12), a first narrow-band filter (13), a first photoelectric detection module (14), a second narrow-band filter (15), a second photoelectric detection module (16), signal control/acquisition equipment (17), a computer (18), a second excitation light source (19), a second photoelectric modulator (20), a second beam expanding device (21), a second scanning device (22), a third excitation light source (23), a third photoelectric modulator (24), a third beam expanding device (25), a third excitation light source (26) and a second scanning lens (27).
The first excitation light source (1), the first photoelectric modulator (2), the first beam expander (3), the reflector (4) and the first scanning device (5) form a light stimulation module.
The second excitation light source (19), the second photoelectric modulator (20), the second beam expanding device (21) and the second scanning device (22) form a point scanning imaging module.
The third excitation light source (23), the third photoelectric modulator (24), the third beam expanding device (25) and the third scanning device (26) form a line scanning imaging module.
The working principle of the multifunctional two-photon microscopic imaging system provided by the embodiment is as follows:
laser emitted by the first excitation light source (1) enters the first beam expanding device (3) after being modulated by the first photoelectric modulator (2), a light beam after being expanded enters the first scanning device (5) for scanning after being reflected by the reflecting mirror (4), and the scanning light beam enters the microscope objective lens (11) after passing through the first dichroic mirror (6), the first scanning lens (7), the second dichroic mirror (8), the sleeve lens (9) and the third dichroic mirror (10) in a transmission manner and is focused on biological tissues for light stimulation.
The laser emitted by the second excitation light source (19) enters the second beam expanding device (21) after being modulated by the second photoelectric modulator (20), the expanded light beam enters the second scanning device (22) for scanning, the scanning light beam is reflected by the first dichroic mirror (6), then transmits through the first scanning lens (7), the second dichroic mirror (8), the sleeve lens (9) and the third dichroic mirror (10), enters the microscope objective lens (11), is focused on the living body tissue of a living organism, the fluorescence generated by excitation is collected by the microscope objective lens (11), then is reflected by the third dichroic mirror (10), then transmits through the fourth dichroic mirror (12), is filtered by the first narrow-band optical filter (13), and then is detected by the first photoelectric detection module (14), and the signals are collected and processed by the signal control/collection device (17) and the computer (18), so that the structure or slowly-changing physiological activities of the living organism can be observed through imaging.
Laser light emitted by the third excitation light source (23) enters the third beam expanding device (25) after being modulated by the third photoelectric modulator (24), light beams after beam expansion enter the third scanning device (26) for scanning, the scanning light beams are reflected by the second dichroic mirror (8) after passing through the second scanning lens (27), then sequentially transmit through the sleeve lens (9) and the third dichroic mirror (10), enter the microscope objective lens (11), and are focused on biological living body tissues. Fluorescence generated by excitation is collected by the microscope objective lens (11), then is reflected by the third dichroic mirror and the fourth dichroic mirror (12) in sequence, is filtered by the second narrow-band filter (15), then is detected by the second photoelectric detection module (16), and then is collected and processed by the signal control/collection equipment (17) and the computer (18), so that imaging observation of rapidly changing physiological activities in biological tissues is realized.
In some of these embodiments, the first scanning means (5) consists of two galvanometer mirrors.
In some of these embodiments, the second scanning device (22) consists of two galvanometer mirrors.
Further, the first scanning means (5) and the second scanning means (22) may be replaced by a multi-beam parallel scanning technique based on structured light modulation or a random scanning technique based on acousto-optic deflectors.
In some of these embodiments, the third scanning device (26) consists of a galvanometer mirror and a high speed resonant mirror.
Further, the third scanning means (26) may be replaced by a fast zoom based solution.
According to the multifunctional two-photon microscopic imaging system, the first excitation light source (1), the first photoelectric modulator (2), the first beam expanding device (3), the reflecting mirror (4) and the first scanning device (5) form a light stimulation module; the second excitation light source (19), the second photoelectric modulator (20), the second beam expanding device (21) and the second scanning device (22) form a point scanning imaging module; the third excitation light source (23), the third photoelectric modulator (24), the third beam expanding device (25) and the third scanning device (26) form a line scanning imaging module. The system controls the three imaging modules through the signal control/acquisition equipment (17), and realizes the independent operation or synchronous operation of different modules. The system can realize the fusion and synchronization of optical stimulation, point scanning imaging and line scanning imaging, can perform optogenetic regulation and control on cells at mesoscale and subcellular resolution, and can quantitatively observe the structure and functional information of biological tissues.
The working principle of the system is described below by taking the study of the coupling mechanism of cerebral neurovascular as an example. The system can be used for photostimulating neurons in the brains of the living rats and imaging and observing the partial pressure of blood oxygen and the activity of nerve calcium:
light stimulation: 1040nm femtosecond laser emitted by the first excitation light source (1) enters the first beam expanding device (3) after being modulated by the first photoelectric modulator (2), a beam after being expanded enters the first scanning device (5) for scanning after being reflected by the reflecting mirror (4), and the scanning beam enters the microscope objective lens (11) after being transmitted through the first dichroic mirror (6), the first scanning lens (7), the second dichroic mirror (8), the sleeve lens (9) and the third dichroic mirror (10) and is focused on a target neuron for light stimulation.
Oxygen partial pressure imaging: the femtosecond laser with the wavelength of 950nm emitted by the second excitation light source (19) enters the second beam expanding device (21) after being modulated by the second photoelectric modulator (20), the expanded light beam enters the second scanning device (22) for scanning, and the scanning light beam is reflected by the first dichroic mirror (6), then is transmitted through the first scanning lens (7), the second dichroic mirror (8), the sleeve lens (9) and the third dichroic mirror (10), then enters the microscope objective lens (11), and is focused in a target blood vessel in the mouse brain. The oxygen probe is used for marking cerebral vessels of mice, and the Oxyphor2P is taken as an example, after being excited by two photons, phosphorescence with 758nm as the central wavelength is emitted. Phosphorescence is collected by the microscope objective lens (11), reflected by the third dichroic mirror (10), transmitted through the fourth dichroic mirror (12), filtered by the first narrow-band filter (13), detected by the first photoelectric detection module (14), and collected and processed by the signal control/collection device (17) and the computer (18), so that imaging measurement of blood oxygen partial pressure is realized.
Imaging of neural calcium: the 920nm femtosecond laser emitted by the third excitation light source (23) enters the third beam expanding device (25) after being modulated by the third photoelectric modulator (24), the expanded light beam enters the third scanning device (26) for scanning, the scanning light beam is reflected by the second dichroic mirror (8) after passing through the second scanning lens (27), then sequentially transmits through the sleeve lens (9) and the third dichroic mirror (10), enters the microscope objective lens (11), and is focused on a target neuron in the mouse brain. The calcium ion indicator is used for marking neurons in the brain, for example GCaMP6f, and the calcium ion indicator generates fluorescence with the central wavelength of 510nm after being excited by two photons. The fluorescence is collected by the microscope objective lens (11), then reflected by the third dichroic mirror and the fourth dichroic mirror (12) in sequence, filtered by the second narrow-band filter (15), detected by the second photoelectric detection module (16), and then collected and processed by the signal control/collection device (17) and the computer (18), so that the imaging observation of the nerve calcium activity is realized. That is, a light stimulation module is used to activate a specific neuron, and then a line scanning module and a point scanning module are used to perform imaging measurement on the calcium activity of the activated neuron and the blood oxygen partial pressure around the activated neuron. The technology has important significance for researching the functions of neurovascular units.
The above are merely examples of the present application and are not intended to limit the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.
Claims (4)
1. A multi-functional two-photon microscopy imaging system, comprising: the device comprises a first excitation light source (1), a first photoelectric modulator (2), a first beam expanding device (3), a reflecting mirror (4), a first scanning device (5), a first dichroic mirror (6), a first scanning lens (7), a second dichroic mirror (8), a sleeve lens (9), a third dichroic mirror (10), a microscope objective lens (11), a fourth dichroic mirror (12), a first narrow-band filter (13), a first photoelectric detection module (14), a second narrow-band filter (15), a second photoelectric detection module (16), a signal control/acquisition device (17), a computer (18), a second excitation light source (19), a second photoelectric modulator (20), a second beam expanding device (21), a second scanning device (22), a third excitation light source (23), a third photoelectric modulator (24), a third beam expanding device (25), a third scanning device (26) and a second scanning lens (27);
laser emitted by the first excitation light source (1) enters the first beam expanding device (3) after being modulated by the first photoelectric modulator (2), a beam after being expanded enters the first scanning device (5) for scanning after being reflected by the reflecting mirror (4), and the scanning beam enters the microscope objective lens (11) after being transmitted through the first dichroic mirror (6), the first scanning lens (7), the second dichroic mirror (8), the sleeve lens (9) and the third dichroic mirror (10) and is focused on biological tissues for light stimulation;
laser emitted by the second excitation light source (19) enters the second beam expanding device (21) after being modulated by the second photoelectric modulator (20), a beam after being expanded enters the second scanning device (22) for scanning, the scanning beam is reflected by the first dichroic mirror (6), then is transmitted through the first scanning lens (7), the second dichroic mirror (8), the sleeve lens (9) and the third dichroic mirror (10), then enters a microscope objective lens (11) and is focused on living body tissues of a living organism, fluorescence generated by excitation is collected by the microscope objective lens (11), then is reflected by the third dichroic mirror (10), then is transmitted through the fourth dichroic mirror (12), then is filtered by the first narrow-band optical filter (13), and is detected by the first photoelectric detection module (14), and is collected and processed by the signal control/collection device (17) and the computer (18), so that the structure or slowly-changing physiological activities of the living body tissues can be observed through imaging;
laser emitted by the third excitation light source (23) enters the third beam expanding device (25) after being modulated by the third photoelectric modulator (24), light beams after beam expansion enter the third scanning device (26) for scanning, the scanning light beams pass through the second scanning lens (27), are reflected by the second dichroic mirror (8), then sequentially transmit through the sleeve lens (9) and the third dichroic mirror (10), enter the microscope objective lens (11), are focused on biological living body tissues, fluorescence generated by excitation is sequentially reflected by the third dichroic mirror and the fourth dichroic mirror (12) after being collected by the microscope objective lens (11), is filtered by the second narrow-band filter (15), and is detected by the second photoelectric detection module (16), and then is collected and processed by the signal control/collection device (17) and the computer (18), so that the imaging observation of rapidly changing physiological activities in the biological tissues is realized.
2. A multifunctional two-photon microscopic imaging system according to claim 1, characterized in that said first scanning means (5) consists of two galvanometer mirrors.
3. A multifunctional two-photon microscopy imaging system as claimed in claim 1 wherein the second scanning means (22) consists of two galvanometer mirrors.
4. The multi-functional two-photon microscopy imaging system according to claim 1, wherein the third scanning means (26) is comprised of a galvanometer mirror and a high speed resonant mirror.
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| CN202110929297.XA CN115702777A (en) | 2021-08-13 | 2021-08-13 | Multifunctional two-photon microscopic imaging system |
| PCT/CN2021/138046 WO2023015798A1 (en) | 2021-08-13 | 2021-12-14 | Multi-functional two-photon microscopic imaging system |
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| JP2012008260A (en) * | 2010-06-23 | 2012-01-12 | Hamamatsu Photonics Kk | Image generation apparatus |
| CN102551661B (en) * | 2010-12-09 | 2014-11-05 | 深圳大学 | Fluorescence spectrum endoscopic imaging method and system |
| US10830701B2 (en) * | 2017-05-06 | 2020-11-10 | Howard Hughes Medical Institute | Scanned line angular projection microscopy |
| WO2019217846A1 (en) * | 2018-05-10 | 2019-11-14 | Board Of Regents, The University Of Texas System | Line excitation array detection microscopy |
| WO2021097482A1 (en) * | 2019-11-11 | 2021-05-20 | Howard Hughes Medical Institute | Random access projection microscopy |
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- 2021-12-14 WO PCT/CN2021/138046 patent/WO2023015798A1/en unknown
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| CN101915752A (en) * | 2010-07-05 | 2010-12-15 | 中国科学院深圳先进技术研究院 | Laser scanning imaging device |
| US20180328849A1 (en) * | 2017-05-15 | 2018-11-15 | Tsinghua University | Structured illumination microscopy imaging system based on line-scanning spatiotemporal focusing |
| CN110146473A (en) * | 2019-04-16 | 2019-08-20 | 浙江大学 | A kind of the two-photon fluorescence microscope equipment and method of axial super resolution |
| CN110599399A (en) * | 2019-07-26 | 2019-12-20 | 清华大学 | Fast two-photon imaging method and device based on convolutional neural network |
| CN110954523A (en) * | 2019-12-18 | 2020-04-03 | 深圳大学 | Two-photon scanning structure light microscopic imaging method and device |
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