WO2023015798A1 - 一种多功能双光子显微成像系统 - Google Patents

一种多功能双光子显微成像系统 Download PDF

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WO2023015798A1
WO2023015798A1 PCT/CN2021/138046 CN2021138046W WO2023015798A1 WO 2023015798 A1 WO2023015798 A1 WO 2023015798A1 CN 2021138046 W CN2021138046 W CN 2021138046W WO 2023015798 A1 WO2023015798 A1 WO 2023015798A1
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scanning
dichroic mirror
lens
light source
excitation light
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PCT/CN2021/138046
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French (fr)
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李宝强
毕国强
王洋
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深圳先进技术研究院
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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
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes

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  • the invention belongs to the technical field of optical microscopic imaging, and in particular relates to a multifunctional two-photon microscopic imaging system.
  • Two-photon microscopy has the advantages of non-invasiveness, high resolution, strong tomographic ability, low phototoxicity and strong light penetration. It is especially suitable for observing biological tissues with strong light scattering.
  • the existing two-photon microscopy imaging scanning methods can be roughly divided into point scanning based on galvanometer galvanometer and line scanning based on resonant galvanometer, which respectively deal with different application scenarios: point scanning is suitable for structural imaging of biological tissues, And cell regulation based on optogenetics; line scanning is suitable for observing rapidly changing physiological activities (such as neural calcium signals) in biological tissues.
  • point scanning is suitable for structural imaging of biological tissues, And cell regulation based on optogenetics; line scanning is suitable for observing rapidly changing physiological activities (such as 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 sciences, especially neuroscience.
  • the existing two-photon imaging technology cannot meet the requirement of synchronous operation of the above three functions.
  • Bruker's Ultima2P Plus system can only select one of the imaging functions of point scan or line scan during operation; Nikon's A1R-MP+ system and Olympus' FVMPE-
  • the RS system has the function of synchronous operation of point scanning and line scanning, it cannot simultaneously perform structural imaging while performing light stimulation and neural calcium imaging.
  • the present invention proposes a multifunctional and modular two-photon microscopic imaging system design scheme, which has both point scanning and line scanning imaging functions, and can realize structural imaging of biological tissues, cell regulation based on optogenetics, and cell calcium Synchronization of the three imaging functions.
  • the present invention adopts the following technical solutions:
  • a multifunctional two-photon microscopic imaging system comprising: a first excitation light source (1), a first photoelectric modulator (2), a first beam expander (3), a mirror (4), a first scanning device ( 5), the first dichroic mirror (6), the first scanning lens (7), the second dichroic mirror (8), the sleeve lens (9), the third dichroic mirror (10), the microscope objective lens (11), the fourth dichroic mirror (12), the first narrowband filter (13), the first photodetection module (14), the second narrowband filter (15), the second photodetection module (16 ), signal control/acquisition equipment (17), computer (18), second excitation light source (19), second photoelectric modulator (20), second beam expander (21), second scanning device (22), A third excitation light source (23), a third photoelectric modulator (24), a third beam expander (25), a third scanning device (26) and a second scanning lens (27).
  • the laser light emitted by the first excitation light source (1) is modulated by the first photoelectric modulator (2) and then enters the first beam expander (3), and the beam expanded by the reflector (4) After being reflected, it enters the first scanning device (5) for scanning, and the scanning light beam is transmitted through the first dichroic mirror (6), the first scanning lens (7), the second dichroic The mirror (8), the sleeve lens (9) and the third dichroic mirror (10) enter the microscope objective lens (11) and focus on the biological tissue for photostimulation.
  • the laser light emitted by the second excitation light source (19) enters the second beam expander (21) after being modulated by the second photoelectric modulator (20), and the expanded beam enters the second scanning device ( 22) Scanning, the scanning beam is reflected by the first dichroic mirror (6) and then transmitted through the first scanning lens (7), the second dichroic mirror (8), the sleeve After the barrel lens (9) and the third dichroic mirror (10) enter the microscope objective lens (11), and focus on the living tissue, the excited fluorescence is collected by the microscope objective lens (11) Reflected by the third dichroic mirror (10), then transmitted through the fourth dichroic mirror (12), filtered by the first narrow-band filter (13) and then filtered by the first photodetection module (14) detection, and the signal control/acquisition device (17) and the computer (18) collect and process the signal, so as to realize the imaging observation of the structure of biological tissue or slowly changing physiological activities.
  • the laser light emitted by the third excitation light source (23) enters the third beam expander (25) after being modulated by the third photoelectric modulator (24), and the expanded beam enters the third scanning device ( 26) Scanning, the scanning light beam is reflected by the second dichroic mirror (8) after passing through the second scanning lens (27), and then transmitted through the sleeve lens (9) and the first The three dichroic mirrors (10) enter the microscope objective lens (11) and focus on the living tissue.
  • Fluorescence generated by excitation is collected by the microscope objective lens (11), reflected by the third dichroic mirror and the fourth dichroic mirror (12) in turn, and then passed through the second narrow-band filter (15) ) is detected by the second photoelectric detection module (16) after light filtering, and then the signal is collected and processed by the signal control/acquisition device (17) and the computer (18), realizing the detection of Imaging observations of rapidly changing physiological activities.
  • the first scanning device (5) is composed of two galvanometer vibrating mirrors.
  • the second scanning device (22) is composed of two galvanometer vibrating mirrors.
  • the third scanning device (26) is composed of a galvanometer vibrating mirror and a high-speed resonant vibrating mirror.
  • the first excitation light source (1), the first photoelectric modulator (2), the first beam expander (3), and the mirror ( 4) and the first scanning device (5) constitute a light stimulation module;
  • the second excitation light source (19), the second photoelectric modulator (20), the second beam expander (21) and the The second scanning device (22) constitutes a point scanning imaging module;
  • the third excitation light source (23), the third photoelectric modulator (24), the third beam expander (25) and the third The scanning device (26) constitutes a line scanning imaging module.
  • the system controls the three imaging modules through the signal control/acquisition device (17), so as to realize the separate operation or synchronous operation of different modules.
  • This system can realize the fusion and synchronization of photostimulation, point-scanning imaging and line-scanning imaging, and can perform optogenetic regulation on cells at mesoscopic scale with subcellular resolution, and can also quantitatively observe the structural and functional information of biological tissues .
  • FIG. 1 is a schematic structural diagram of a multifunctional two-photon microscopic imaging system provided by an embodiment of the present invention.
  • first and second are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features. Thus, a feature defined as “first” and “second” may explicitly or implicitly include one or more of these features.
  • “plurality” means two or more, unless otherwise specifically defined.
  • Figure 1 is a schematic structural diagram of the multifunctional two-photon microscopic imaging system provided by the embodiment of the present application, including: the first excitation light source (1), the first photoelectric modulator (2), and the first beam expander (3 ), mirror (4), first scanning device (5), first dichroic mirror (6), first scanning lens (7), second dichroic mirror (8), sleeve lens (9) , the third dichroic mirror (10), the microscope objective lens (11), the fourth dichroic mirror (12), the first narrow-band filter (13), the first photodetection module (14), the second narrow-band filter Light sheet (15), second photoelectric detection module (16), signal control/acquisition device (17), computer (18), second excitation light source (19), second photoelectric modulator (20), second beam expander device (21), second scanning device (22), third excitation light source (23), third photoelectric modulator (24), third beam expander (25), third scanning device (26) and second scanning Lens (27).
  • the first excitation light source (1), the first photoelectric modulator (2), the first beam expander (3), the mirror (4) and the first scanning device (5) constitute Photostimulation module.
  • the second excitation light source (19), the second photoelectric modulator (20), the second beam expander (21) and the second scanning device (22) constitute a point scanning imaging module.
  • the third excitation light source (23), the third photoelectric modulator (24), the third beam expander (25) and the third scanning device (26) constitute a line scan imaging module.
  • the laser light emitted by the first excitation light source (1) is modulated by the first photoelectric modulator (2) and then enters the first beam expander (3), and the beam expanded by the reflector (4) After being reflected, it enters the first scanning device (5) for scanning, and the scanning light beam is transmitted through the first dichroic mirror (6), the first scanning lens (7), the second dichroic The mirror (8), the sleeve lens (9) and the third dichroic mirror (10) enter the microscope objective lens (11) and focus on the biological tissue for photostimulation.
  • the laser light emitted by the second excitation light source (19) enters the second beam expander (21) after being modulated by the second photoelectric modulator (20), and the expanded beam enters the second scanning device ( 22) Scanning, the scanning beam is reflected by the first dichroic mirror (6) and then transmitted through the first scanning lens (7), the second dichroic mirror (8), the sleeve After the tube lens (9) and the third dichroic mirror (10) enter the microscope objective lens (11), and focus on the living tissue, the excited fluorescence is collected by the microscope objective lens (11) Reflected by the third dichroic mirror (10), then transmitted through the fourth dichroic mirror (12), filtered by the first narrow-band filter (13) and then filtered by the first photodetection module (14) detection, and the signal control/acquisition device (17) and the computer (18) collect and process the signal, so as to realize the imaging observation of the structure of biological tissue or slowly changing physiological activities.
  • the laser light emitted by the third excitation light source (23) enters the third beam expander (25) after being modulated by the third photoelectric modulator (24), and the expanded beam enters the third scanning device ( 26) Scanning, the scanning light beam is reflected by the second dichroic mirror (8) after passing through the second scanning lens (27), and then transmitted through the sleeve lens (9) and the first The three dichroic mirrors (10) enter the microscope objective lens (11) and focus on the living tissue.
  • Fluorescence generated by excitation is collected by the microscope objective lens (11), reflected by the third dichroic mirror and the fourth dichroic mirror (12) in turn, and then passed through the second narrow-band filter (15) ) is detected by the second photoelectric detection module (16) after light filtering, and then the signal is collected and processed by the signal control/acquisition device (17) and the computer (18), realizing the detection of Imaging observations of rapidly changing physiological activities.
  • the first scanning device (5) is composed of two galvanometer vibrating mirrors.
  • the second scanning device (22) is composed of two galvanometer vibrating mirrors.
  • first scanning device (5) and the second scanning device (22) can be replaced by a multi-beam parallel scanning technology based on structured light modulation or a random scanning technology based on an acousto-optic deflector.
  • the third scanning device (26) is composed of a galvanometer vibrating mirror and a high-speed resonant vibrating mirror.
  • the third scanning device (26) can be replaced by a technical solution based on a fast zoom device.
  • the first excitation light source (1), the first photoelectric modulator (2), the first beam expander (3), and the mirror ( 4) and the first scanning device (5) constitute a light stimulation module;
  • the second excitation light source (19), the second photoelectric modulator (20), the second beam expander (21) and the The second scanning device (22) constitutes a point scanning imaging module;
  • the third excitation light source (23), the third photoelectric modulator (24), the third beam expander (25) and the third The scanning device (26) constitutes a line scanning imaging module.
  • the system controls the three imaging modules through the signal control/acquisition device (17), so as to realize the separate operation or synchronous operation of different modules.
  • This system can realize the fusion and synchronization of photostimulation, point-scanning imaging and line-scanning imaging, and can perform optogenetic regulation on cells at mesoscopic scale with subcellular resolution, and can also quantitatively observe the structural and functional information of biological tissues .
  • the following is an example of studying the neurovascular coupling mechanism of the brain to describe the working principle of the system.
  • the system can be used to light-stimulate the neurons in the brain of living mice, and to perform imaging observations on blood oxygen partial pressure and nerve calcium activity:
  • the 1040 nm femtosecond laser emitted by the first excitation light source (1) enters the first beam expander (3) after being modulated by the first photoelectric modulator (2), and the expanded beam After being reflected by the reflecting mirror (4), it enters the first scanning device (5) for scanning, and the scanning light beam is 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) enter the microscope objective lens (11) and focus on the target neuron Perform photostimulation.
  • Oxygen partial pressure imaging the 950 nm femtosecond laser emitted by the second excitation light source (19) enters the second beam expander (21) after being modulated by the second photoelectric modulator (20), and after beam expansion
  • the light beam enters the second scanning device (22) for scanning, and the scanning light beam is reflected by the first dichroic mirror (6) and then transmitted through the first scanning lens (7), the second two
  • the dichroic mirror (8), the sleeve lens (9) and the third dichroic mirror (10) then enter the microscope objective lens (11) and focus on the target blood vessel in the rat brain.
  • Oxygen probes were used to label rat brain blood vessels.
  • Oxyphor2P emitted phosphorescence with a center wavelength of 758 nm after two-photon excitation.
  • the phosphorescence is collected by the microscope objective lens (11), reflected by the third dichroic mirror (10), then transmitted through the fourth dichroic mirror (12), and then passed through the first narrow-band filter (13) After filtering, it is detected by the first photoelectric detection module (14), and the signal is collected and processed by the signal control/acquisition device (17) and the computer (18) to realize blood oxygenation Imaging measurements of partial pressures.
  • the 920 nm femtosecond laser emitted by the third excitation light source (23) enters the third beam expander (25) after being modulated by the third photoelectric modulator (24), and the beam expanded
  • the light beam enters the third scanning device (26) for scanning, the scanning light beam passes through the second scanning lens (27), is reflected by the second dichroic mirror (8), and then transmits through the set
  • the tube lens (9) and the third dichroic mirror (10) enter the microscope objective lens (11) and focus on the target neurons in the rat brain. Neurons in the brain were labeled with calcium ion indicators.
  • the two-photon excitation produced 510 nm is the central wavelength of fluorescence.
  • Fluorescence is collected by the microscope objective lens (11), reflected by the third dichroic mirror and the fourth dichroic mirror (12) in turn, and then filtered by the second narrow-band filter (15)
  • the signal is collected and processed by the signal control/acquisition device (17) and the computer (18), so as to realize the imaging observation of nerve calcium activity . That is, the specific neuron is activated by using the light stimulation module, and then the calcium activity of the activated neuron and the blood oxygen partial pressure around it are imaged and measured by using the line scanning module and the point scanning module. This technique is of great significance for studying the function of the neurovascular unit.

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Abstract

多功能双光子显微成像系统，第一激发光源（1）、第一光电调制器（2）、第一扩束装置（3）、反射镜（4）及第一扫描装置（5）构成光刺激模块；第二激发光源（19）、第二光电调制器（20）、第二扩束装置（21）及第二扫描装置（22）构成点扫描成像模块；第三激发光源（23）、第三光电调制器（24）、第三扩束装置（25）及第三扫描装置（26）构成线扫描成像模块。系统有效集成了点扫描和线扫描技术，可实现活体生物组织的结构成像、细胞的光遗传学调控、以及生理活动（如神经钙信号）成像三种功能的融合及同步。

Description

一种多功能双光子显微成像系统 技术领域
本发明属于光学显微成像技术领域，具体涉及一种多功能双光子显微成像系统。
 背景技术
双光子显微成像技术具有非侵入性、分辨率高、层析能力强、光毒性小和光穿透力强的优点，特别适用于观测光散射较强的生物组织，已成为生命科学领域中最重要的研究工具之一。现有的双光子显微成像的扫描方式可大体分为基于检流计振镜的点扫描和基于共振振镜的线扫描，分别应对不同的应用场景：点扫描适用于生物组织的结构成像、以及基于光遗传学的细胞调控；线扫描适用于观测生物组织中快速变化的生理活动（如神经钙信号）。结构成像、细胞调控和钙成像的同步，对于生命科学，特别是神经科学的研究，具有十分重要的意义。
 技术问题
然而，现有的双光子成像技术还不能满足以上三种功能同步运行的要求。如，布鲁克（Bruker）公司的Ultima2P Plus系统在运行时仅能选用点扫描或线扫描其中一种成像功能；尼康（Nikon）公司的A1R-MP+系统和奥林巴斯（Olympus）公司的FVMPE-RS系统虽然具有点扫描和线扫描同步运行的功能，但是，在进行光刺激和神经钙成像的同时无法同步进行结构成像。本发明提出了一种多功能、模块化的双光子显微成像系统设计方案，兼具点扫描和线扫描成像功能，可实现生物组织的结构成像、基于光遗传学的细胞调控、和细胞钙成像三种功能的同步。
 技术解决方案
为解决上述问题，本发明采用下述技术方案：
一种多功能双光子显微成像系统，包括：第一激发光源（1）、第一光电调制器（2）、第一扩束装置（3）、反射镜（4）、第一扫描装置（5）、第一二向色镜（6）、第一扫描透镜（7）、第二二向色镜（8）、套筒透镜（9）、第三二向色镜（10）、显微镜物镜（11）、第四二向色镜（12）、第一窄带滤光片（13）、第一光电探测模块（14）、第二窄带滤光片（15）、第二光电探测模块（16）、信号控制/采集设备（17）、电脑（18）、第二激发光源（19）、第二光电调制器（20）、第二扩束装置（21）、第二扫描装置（22）、第三激发光源（23）、第三光电调制器（24）、第三扩束装置（25）、第三扫描装置（26）及第二扫描透镜（27）。
所述第一激发光源（1）发出的激光经所述第一光电调制器（2）调制后进入所述第一扩束装置（3），扩束后的光束由所述反射镜（4）反射后进入所述第一扫描装置（5）进行扫描，所述扫描光束透射通过所述第一二向色镜（6）、所述第一扫描透镜（7）、所述第二二向色镜（8）、所述套筒透镜（9）及所述第三二向色镜（10）后进入所述显微镜物镜（11），并聚焦在生物组织上进行光刺激。
所述第二激发光源（19）发出的激光经所述第二光电调制器（20）调制后进入所述第二扩束装置（21），扩束后的光束进入所述第二扫描装置（22）进行扫描，所述扫描光束经所述第一二向色镜（6）反射后透射通过所述第一扫描透镜（7）、所述第二二向色镜（8）、所述套筒透镜（9）及所述第三二向色镜（10）后进入显微镜物镜（11），并聚焦在生物活体组织上，激发产生的荧光经所述显微镜物镜（11）收集后由所述第三二向色镜（10）反射，然后透射通过所述第四二向色镜（12），再经所述第一窄带滤光片（13）滤光后被所述第一光电探测模块（14）探测，并由所述信号控制/采集设备（17）和所述电脑（18）对所述信号进行采集和处理，实现对生物组织的结构或慢速变化的生理活动进行成像观测。
所述第三激发光源（23）发出的激光经所述第三光电调制器（24）调制后进入所述第三扩束装置（25），扩束后的光束进入所述第三扫描装置（26）进行扫描，所述扫描光束经所述第二扫描透镜（27）后由所述第二二向色镜（8）反射，然后依次透射通过所述套筒透镜（9）及所述第三二向色镜（10）后进入显微镜物镜（11），并聚焦在生物活体组织上。激发产生的荧光经所述显微镜物镜（11）收集后依次由所述第三二向色镜和所述第四二向色镜（12）反射，再经所述第二窄带滤光片（15）滤光后被所述第二光电探测模块（16）探测，然后由所述信号控制/采集设备（17）和所述电脑（18）对所述信号进行采集和处理，实现对生物组织中快速变化的生理活动进行成像观测。
在其中一些实施例中，所述第一扫描装置（5）由两个检流计振镜组成。
在其中一些实施例中，所述第二扫描装置（22）由两个检流计振镜组成。
在其中一些实施例中，所述第三扫描装置（26）由一个检流计振镜和一个高速共振振镜组成。
 有益效果
本申请采用的上述技术方案具备下述效果：
本申请提供的多功能双光子显微成像系统，所述第一激发光源（1）、所述第一光电调制器（2）、所述第一扩束装置（3）、所述反射镜（4）及所述第一扫描装置（5）构成光刺激模块；所述第二激发光源（19）、所述第二光电调制器（20）、所述第二扩束装置（21）及所述第二扫描装置（22）构成点扫描成像模块；所述第三激发光源（23）、所述第三光电调制器（24）、所述第三扩束装置（25）及所述第三扫描装置（26）构成线扫描成像模块。此系统通过所述信号控制/采集设备（17）对三个成像模块进行控制，实现不同模块的单独运行或同步运行。此系统可实现光刺激、点扫描成像和线扫描成像的融合及同步，能够在介观尺度、以亚细胞分辨率对细胞进行光遗传调控，同时可对生物组织的结构和功能信息进行量化观测。
 附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对本发明实施例或现有技术描述中所需要使用的附图作简单的介绍。显而易见地，下面所描述的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其它的附图。
图1为本发明实施例提供的多功能双光子显微成像系统的结构示意图。
 本发明的实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
在本发明的描述中，需要理解的是，术语“上”、“下”、“水平”、“内”、“外”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本发明和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位或以特定的方位构造和操作，因此不能理解为对本发明的限制。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。在本发明的描述中，“多个”的含义是两个或两个以上，除非另有明确具体的限定。
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步地详细说明。
请参阅图1，为本申请实施例提供的多功能双光子显微成像系统的结构示意图，包括：第一激发光源（1）、第一光电调制器（2）、第一扩束装置（3）、反射镜（4）、第一扫描装置（5）、第一二向色镜（6）、第一扫描透镜（7）、第二二向色镜（8）、套筒透镜（9）、第三二向色镜（10）、显微镜物镜（11）、第四二向色镜（12）、第一窄带滤光片（13）、第一光电探测模块（14）、第二窄带滤光片（15）、第二光电探测模块（16）、信号控制/采集设备（17）、电脑（18）、第二激发光源（19）、第二光电调制器（20）、第二扩束装置（21）、第二扫描装置（22）、第三激发光源（23）、第三光电调制器（24）、第三扩束装置（25）、第三扫描装置（26）及第二扫描透镜（27）。
所述第一激发光源（1）、所述第一光电调制器（2）、所述第一扩束装置（3）、所述反射镜（4）及所述第一扫描装置（5）构成光刺激模块。
所述第二激发光源（19）、所述第二光电调制器（20）、所述第二扩束装置（21）及所述第二扫描装置（22）构成点扫描成像模块。
所述第三激发光源（23）、所述第三光电调制器（24）、所述第三扩束装置（25）及所述第三扫描装置（26）构成线扫描成像模块。
上述实施例提供的多功能双光子显微成像系统的工作原理如下：
所述第一激发光源（1）发出的激光经所述第一光电调制器（2）调制后进入所述第一扩束装置（3），扩束后的光束由所述反射镜（4）反射后进入所述第一扫描装置（5）进行扫描，所述扫描光束透射通过所述第一二向色镜（6）、所述第一扫描透镜（7）、所述第二二向色镜（8）、所述套筒透镜（9）及所述第三二向色镜（10）后进入所述显微镜物镜（11），并聚焦在生物组织上进行光刺激。
所述第二激发光源（19）发出的激光经所述第二光电调制器（20）调制后进入所述第二扩束装置（21），扩束后的光束进入所述第二扫描装置（22）进行扫描，所述扫描光束经所述第一二向色镜（6）反射后透射通过所述第一扫描透镜（7）、所述第二二向色镜（8）、所述套筒透镜（9）及所述第三二向色镜（10）后进入显微镜物镜（11），并聚焦在生物活体组织上，激发产生的荧光经所述显微镜物镜（11）收集后由所述第三二向色镜（10）反射，然后透射通过所述第四二向色镜（12），再经所述第一窄带滤光片（13）滤光后被所述第一光电探测模块（14）探测，并由所述信号控制/采集设备（17）和所述电脑（18）对所述信号进行采集和处理，实现对生物组织的结构或慢速变化的生理活动进行成像观测。
所述第三激发光源（23）发出的激光经所述第三光电调制器（24）调制后进入所述第三扩束装置（25），扩束后的光束进入所述第三扫描装置（26）进行扫描，所述扫描光束经所述第二扫描透镜（27）后由所述第二二向色镜（8）反射，然后依次透射通过所述套筒透镜（9）及所述第三二向色镜（10）后进入显微镜物镜（11），并聚焦在生物活体组织上。激发产生的荧光经所述显微镜物镜（11）收集后依次由所述第三二向色镜和所述第四二向色镜（12）反射，再经所述第二窄带滤光片（15）滤光后被所述第二光电探测模块（16）探测，然后由所述信号控制/采集设备（17）和所述电脑（18）对所述信号进行采集和处理，实现对生物组织中快速变化的生理活动进行成像观测。
在其中一些实施例中，所述第一扫描装置（5）由两个检流计振镜组成。
在其中一些实施例中，所述第二扫描装置（22）由两个检流计振镜组成。
进一步地，所述第一扫描装置（5）和第二扫描装置（22）可由基于结构光调制的多光束并行扫描技术或基于声光偏转器的随机扫描技术替代。
在其中一些实施例中，所述第三扫描装置（26）由一个检流计振镜和一个高速共振振镜组成。
进一步地，所述第三扫描装置（26）可由基于快速变焦器件的技术方案替代。
本申请提供的多功能双光子显微成像系统，所述第一激发光源（1）、所述第一光电调制器（2）、所述第一扩束装置（3）、所述反射镜（4）及所述第一扫描装置（5）构成光刺激模块；所述第二激发光源（19）、所述第二光电调制器（20）、所述第二扩束装置（21）及所述第二扫描装置（22）构成点扫描成像模块；所述第三激发光源（23）、所述第三光电调制器（24）、所述第三扩束装置（25）及所述第三扫描装置（26）构成线扫描成像模块。此系统通过所述信号控制/采集设备（17）对三个成像模块进行控制，实现不同模块的单独运行或同步运行。此系统可实现光刺激、点扫描成像和线扫描成像的融合及同步，能够在介观尺度、以亚细胞分辨率对细胞进行光遗传调控，同时可对生物组织的结构和功能信息进行量化观测。
下面以研究大脑神经血管耦合机制为例，讲述该系统的工作原理。应用该系统可以对活体鼠脑内的神经元进行光刺激，并对血氧分压和神经钙活动进行成像观测：
光刺激：所述第一激发光源（1）发出的1040 nm的飞秒激光经所述第一光电调制器（2）调制后进入所述第一扩束装置（3），扩束后的光束由所述反射镜（4）反射后进入所述第一扫描装置（5）进行扫描，所述扫描光束透射通过所述第一二向色镜（6）、所述第一扫描透镜（7）、所述第二二向色镜（8）、所述套筒透镜（9）及所述第三二向色镜（10）后进入所述显微镜物镜（11），并聚焦在目标神经元上进行光刺激。
氧分压成像：所述第二激发光源（19）发出的950 nm的飞秒激光经所述第二光电调制器（20）调制后进入所述第二扩束装置（21），扩束后的光束进入所述第二扫描装置（22）进行扫描，所述扫描光束经所述第一二向色镜（6）反射后透射通过所述第一扫描透镜（7）、所述第二二向色镜（8）、所述套筒透镜（9）及所述第三二向色镜（10）后进入显微镜物镜（11），并聚焦在鼠脑内的目标血管中。利用氧气探针对鼠脑血管进行标记，以Oxyphor2P为例，对其双光子激发后发射出以758 nm为中心波长的磷光。磷光经所述显微镜物镜（11）收集后由所述第三二向色镜（10）反射，然后透射通过所述第四二向色镜（12），再经所述第一窄带滤光片（13）滤光后被所述第一光电探测模块（14）探测，并由所述信号控制/采集设备（17）和所述电脑（18）对所述信号进行采集和处理，实现血氧分压的成像测量。
神经钙成像：所述第三激发光源（23）发出的920 nm的飞秒激光经所述第三光电调制器（24）调制后进入所述第三扩束装置（25），扩束后的光束进入所述第三扫描装置（26）进行扫描，所述扫描光束经所述第二扫描透镜（27）后由所述第二二向色镜（8）反射，然后依次透射通过所述套筒透镜（9）及所述第三二向色镜（10）后进入显微镜物镜（11），并聚焦在鼠脑内的目标神经元上。采用钙离子指示剂对脑内的神经元进行标记，以GCaMP6f为例，对其双光子激发后产生以510 nm为中心波长的荧光。荧光经所述显微镜物镜（11）收集后依次由所述第三二向色镜和所述第四二向色镜（12）反射，再经所述第二窄带滤光片（15）滤光后被所述第二光电探测模块（16）探测，然后由所述信号控制/采集设备（17）和所述电脑（18）对所述信号进行采集和处理，实现对神经钙活动的成像观测。即，应用光刺激模块激活特定的神经元，然后应用线扫描模块和点扫描模块对被激活神经元的钙活动和其周围的血氧分压进行成像测量。该技术对研究神经血管单元的功能具有重要的意义。
以上仅为本申请的实施例，并不用于限制本申请。对于本领域技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原理之内所作的任何修改、等同替换、改进等，均应包含在本申请的权利要求范围之内。

Claims (4)

  1. 一种多功能双光子显微成像系统，其特征在于，包括：第一激发光源（1）、第一光电调制器（2）、第一扩束装置（3）、反射镜（4）、第一扫描装置（5）、第一二向色镜（6）、第一扫描透镜（7）、第二二向色镜（8）、套筒透镜（9）、第三二向色镜（10）、显微镜物镜（11）、第四二向色镜（12）、第一窄带滤光片（13）、第一光电探测模块（14）、第二窄带滤光片（15）、第二光电探测模块（16）、信号控制/采集设备（17）、电脑（18）、第二激发光源（19）、第二光电调制器（20）、第二扩束装置（21）、第二扫描装置（22）、第三激发光源（23）、第三光电调制器（24）、第三扩束装置（25）、第三扫描装置（26）及第二扫描透镜（27）；
    所述第一激发光源（1）发出的激光经所述第一光电调制器（2）调制后进入所述第一扩束装置（3），扩束后的光束由所述反射镜（4）反射后进入所述第一扫描装置（5）进行扫描，所述扫描光束透射通过所述第一二向色镜（6）、所述第一扫描透镜（7）、所述第二二向色镜（8）、所述套筒透镜（9）及所述第三二向色镜（10）后进入所述显微镜物镜（11），并聚焦在生物组织上进行光刺激；
    所述第二激发光源（19）发出的激光经所述第二光电调制器（20）调制后进入所述第二扩束装置（21），扩束后的光束进入所述第二扫描装置（22）进行扫描，所述扫描光束经所述第一二向色镜（6）反射后透射通过所述第一扫描透镜（7）、所述第二二向色镜（8）、所述套筒透镜（9）及所述第三二向色镜（10）后进入显微镜物镜（11），并聚焦在生物活体组织上，激发产生的荧光经所述显微镜物镜（11）收集后由所述第三二向色镜（10）反射，然后透射通过所述第四二向色镜（12），再经所述第一窄带滤光片（13）滤光后被所述第一光电探测模块（14）探测，并由所述信号控制/采集设备（17）和所述电脑（18）对所述信号进行采集和处理，实现对生物组织的结构或慢速变化的生理活动进行成像观测；
    所述第三激发光源（23）发出的激光经所述第三光电调制器（24）调制后进入所述第三扩束装置（25），扩束后的光束进入所述第三扫描装置（26）进行扫描，所述扫描光束经所述第二扫描透镜（27）后由所述第二二向色镜（8）反射，然后依次透射通过所述套筒透镜（9）及所述第三二向色镜（10）后进入显微镜物镜（11），并聚焦在生物活体组织上，激发产生的荧光经所述显微镜物镜（11）收集后依次由所述第三二向色镜和所述第四二向色镜（12）反射，再经所述第二窄带滤光片（15）滤光后被所述第二光电探测模块（16）探测，然后由所述信号控制/采集设备（17）和所述电脑（18）对所述信号进行采集和处理，实现对生物组织中快速变化的生理活动进行成像观测。
  2. 根据权利要求1所述的多功能双光子显微成像系统，其特征在于，所述第一扫描装置（5）由两个检流计振镜组成。
  3. 根据权利要求1所述的多功能双光子显微成像系统，其特征在于，所述第二扫描装置（22）由两个检流计振镜组成。
  4. 根据权利要求1所述的多功能双光子显微成像系统，其特征在于，所述第三扫描装置（26）由一个检流计振镜和一个高速共振振镜组成。
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