WO2024227348A1 - 融合光遗传刺激与光信号探测的光学系统及成像装置 - Google Patents

融合光遗传刺激与光信号探测的光学系统及成像装置 Download PDF

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WO2024227348A1
WO2024227348A1 PCT/CN2023/137699 CN2023137699W WO2024227348A1 WO 2024227348 A1 WO2024227348 A1 WO 2024227348A1 CN 2023137699 W CN2023137699 W CN 2023137699W WO 2024227348 A1 WO2024227348 A1 WO 2024227348A1
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lens
light
dichroic mirror
stimulation
signal detection
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李宝强
许亚南
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Shenzhen Institute of Advanced Technology of CAS
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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
    • A61B5/0071—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by measuring fluorescence emission
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • 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
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • 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
    • 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
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • 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
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N2021/0106—General arrangement of respective parts
    • G01N2021/0112—Apparatus in one mechanical, optical or electronic block
    • 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
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N2021/6463—Optics

Definitions

  • the present application relates to the field of fluorescence microscopy technology, and in particular to an optical system and a fluorescence microscopy device that integrate precise optogenetic stimulation and multi-channel optical signal detection.
  • Fluorescence microscopy (such as confocal or two-photon microscopy) can observe the structure and function of cells and biological tissues in detail with the help of specific labeling of fluorescent probes, and has become an indispensable research tool in the field of life sciences.
  • optogenetics can specifically activate specific types of cells, and combined with fluorescence microscopy technology, it plays a positive role in promoting the development of the field of life sciences.
  • Existing methods of combining fluorescence microscopy with optogenetic stimulation include: (1) combining the light stimulation beam and the imaging illumination beam, and controlling them through different scanning modules to realize the functions of light stimulation and imaging illumination; (2) placing the light stimulation light source outside the imaging light path to perform wide-field light stimulation on biological tissues.
  • the first method will increase the complexity of the imaging light path design; the second method cannot achieve high-resolution and precise light stimulation, and the intensity and irradiation angle of the light stimulation are easily affected by human settings, which reduces the stability of the experiment.
  • the present invention provides an optical system and a fluorescence microscopy imaging device that integrate precise optogenetic stimulation and multi-channel optical signal detection to simplify the optical path and improve experimental efficiency.
  • the specific technical solution proposed by the present invention is: including a light stimulation light source, a first lens, a scanning galvanometer, a second lens, a first dichroic mirror, a third lens, a fourth lens, a second dichroic mirror, a first emission filter, a fifth lens, a first photomultiplier tube, a second emission filter, a sixth lens, a second photomultiplier tube, a third dichroic mirror, an objective lens and an imaging scanning module, wherein:
  • the light stimulation beam is collimated by the first lens, scanned by the scanning galvanometer, and then sequentially passes through the second lens and the first dichroic mirror, and then expanded by the third lens; the expanded light stimulation beam is reflected by the third dichroic mirror, and then focused on the biological tissue by the objective lens, thereby completing the light stimulation regulation of the biological tissue;
  • the illumination light beam is scanned by the imaging scanning module, and then is focused on the biological tissue by the objective lens after passing through the third dichroic mirror; the emission light generated after the illumination light beam excites the biological tissue is reflected by the objective lens and the third dichroic mirror in sequence, and then is separated by the second dichroic mirror after passing through the third lens, the first dichroic mirror, and the fourth lens; the emission light reflected by the second dichroic mirror passes through the first emission filter, and is then focused on the photosensitive surface of the first photomultiplier tube by the fifth lens; the emission light transmitted through the second dichroic mirror passes through the second emission filter, and is focused on the photosensitive surface of the second photomultiplier tube by the sixth lens.
  • the light stimulation light source is used to stimulate the activity of neurons marked by commonly used light-sensitive proteins.
  • the first dichroic mirror is used to separate the light stimulation beam and the emission light.
  • the light stimulation beam is reflected by the first dichroic mirror; the emission light is transmitted by the first dichroic mirror.
  • the second dichroic mirror is used to separate emission light of different wavelength ranges; the emission light reflected by the second dichroic mirror is detected by the first photomultiplier tube, and the emission light transmitted by the second dichroic mirror is detected by the second photomultiplier tube.
  • the first emission filter is used for receiving filtering before the first photomultiplier tube; the second emission filter is used for receiving filtering before the second photomultiplier tube.
  • the first photomultiplier tube and the second photomultiplier tube are used for detecting emitted light.
  • the optical system provided in the present application integrates precise optogenetic stimulation and multi-channel optical signal detection, shares part of the detection optical path during the optogenetic stimulation process, simplifies the optical path design, and realizes a compact layout of the system; compared with optical stimulation as an independent module, it reduces the use of materials and reduces the manufacturing cost; and can achieve precise focusing of optical stimulation, improve its working stability, help simplify the operation process in biological experiments, and improve experimental efficiency.
  • FIG1 is a schematic diagram of the structure of an optical system integrating precise optogenetic stimulation and multi-channel optical signal detection provided in Example 1 of the present application.
  • first and second are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features.
  • a feature defined as “first” or “second” may explicitly or implicitly include one or more of the features.
  • the meaning of “plurality” is two or more, unless otherwise clearly and specifically defined.
  • FIG1 is a schematic diagram of a structure of a module design that integrates precise optogenetic stimulation and multi-channel optical signal detection provided in an embodiment of the present application, including: an optical stimulation light source 10, a first lens 11, a scanning galvanometer 12, a second lens 13, a first dichroic mirror 14, a third lens 15, a fourth lens 16, a second dichroic mirror 17, a first emission filter 18, a fifth lens 19, a first photomultiplier tube 20, a second emission filter 21, a sixth lens 22, a second photomultiplier tube 23, a third dichroic mirror 24, an objective lens 25, and an imaging scanning module 26.
  • an optical stimulation light source 10 a first lens 11, a scanning galvanometer 12, a second lens 13, a first dichroic mirror 14, a third lens 15, a fourth lens 16, a second dichroic mirror 17, a first emission filter 18, a fifth lens 19, a first photomultiplier tube 20, a second emission filter 21, a sixth lens 22, a second photomultiplier tube 23, a third dichroic mirror 24, an objective lens 25, and
  • the two-photon microscopic imaging of the living mouse brain is used as an example for illustration.
  • the wavelength of the light source for light stimulation is 470 ⁇ 14 nanometers; a genetically encoded calcium fluorescent indicator (GCaMP) is used to mark neuronal calcium activity; and a near-infrared fluorescent dye (Alexa Fluor 680) is used to mark blood vessels.
  • GCaMP genetically encoded calcium fluorescent indicator
  • Alexa Fluor 680 a near-infrared fluorescent dye
  • the peak two-photon excitation wavelength of GCaMP is about 920 nanometers, and the peak emission wavelength is about 515 nanometers; the peak two-photon excitation wavelength of Alexa Fluor 680 is about 1280 nanometers, and the peak emission wavelength is about 710 nanometers.
  • the first dichroic mirror 14 is used to reflect light less than 490 nanometers and transmit light greater than 500 nanometers.
  • the second dichroic mirror 17 is used to reflect light of 500-630 nanometers and transmit light of 640-840 nanometers.
  • the third dichroic mirror 24 is used to reflect light of 450-840 nanometers and transmit light of more than 850 nanometers.
  • the first emission filter 18 is used for receiving and filtering calcium fluorescence (eg, 500-600 nanometers).
  • the second emission filter 21 is used for receiving and filtering the Alexa Fluor 680 fluorescence (eg, 670-840 nanometers).
  • the light stimulation light beam is collimated by the first lens 11 and scanned by the scanning galvanometer 12, and then sequentially passes through the second lens 13 and the first dichroic mirror 14, and is then expanded by the third lens 15; the expanded light stimulation light beam is reflected by the third dichroic mirror 24, and then focused on the biological tissue by the objective lens 25 to activate neurons expressing light-sensitive proteins, thereby completing light stimulation regulation of the biological tissue.
  • the illumination beam in the fluorescence imaging process is scanned by the imaging scanning module (picture omitted) and focused on the mouse cerebral cortex through the objective lens; the illumination beam (920 nanometers) excites the calcium fluorescence produced by GCaMP (the peak emission wavelength is about 515 nanometers; marking neural calcium activity) or excites the fluorescence produced by Alexa Fluor 680 (1280 nanometers) (the peak emission wavelength is about
  • the light beam is scanned by the imaging scanning module 26, and then is focused on the biological tissue by the objective lens 25 after passing through the third dichroic mirror 24.
  • the emission light generated after the biological tissue is excited by the illumination light beam is reflected by the objective lens 25 and the third dichroic mirror 24 in sequence, and then enters the second dichroic mirror 17 through the third lens 15, the first dichroic mirror 14, and the fourth lens 16. Subsequently, the fluorescence generated by exciting GCaMP or Alexa Fluor 680 is separated by the second dichroic mirror 17.
  • the fluorescence reflected by the second dichroic mirror 17 (such as the calcium fluorescence generated by exciting GCaMP) passes through the first emission filter 18 and is focused on the photosensitive surface of the first photomultiplier tube 20 by the fifth lens 19.
  • the fluorescence transmitted through the second dichroic mirror 17 (such as the fluorescence generated by exciting Alexa Fluor 680) passes through the second emission filter 21 and is focused on the photosensitive surface of the second photomultiplier tube 23 by the sixth lens 22.
  • the optical system provided in the above-mentioned embodiments of the present application that integrates precise optogenetic stimulation and multi-channel optical signal detection can realize the separation and detection of fluorescence (GCaMP or Alexa Fluor 680) in different wavelength ranges, and integrates the optogenetic stimulation function.
  • GCaMP or Alexa Fluor 680 fluorescence
  • part of the detection optical path is shared, which simplifies the optical path design and realizes a compact layout of the system.
  • the use of materials is reduced and the manufacturing cost is reduced. It can also achieve precise focusing of optical stimulation and improve its working stability, which helps to simplify the operation process in biological experiments and improve experimental efficiency.

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Abstract

在荧光显微成像设备中使用的融合精准光遗传刺激与多通道光信号探测的光学系统，包括光刺激光源（10）、第一透镜（11）、扫描振镜（12）、第二透镜（13）、第一二向色镜（14）、第三透镜（15）、第四透镜（16）、第二二向色镜（17）、第一发射滤光片（18）、第五透镜（19）、第一光电倍增管（20）、第二发射滤光片（21）、第六透镜（22）、第二光电倍增管（23）。光学系统在光信号探测中融入了光刺激光源（10），其优点是：光刺激光路共享部分探测光路，简化了光路设计，实现了系统的紧凑布局；与光刺激作为独立模块相比，减少了材料的使用，降低了制造成本；且可以实现光刺激的精准聚焦，提高了工作稳定性，有助于简化在生物实验中的操作过程，提高实验效率。

Description

融合光遗传刺激与光信号探测的光学系统及成像装置 技术领域
本申请涉及荧光显微成像技术领域，特别涉及一种融合精准光遗传刺激与多通道光信号探测的光学系统及荧光显微成像装置。
 背景技术
荧光显微成像（如共聚焦或双光子显微成像），借助荧光探针的特异性标记，能够精细地观测细胞与生物组织的结构及功能，已成为生命科学领域中不可缺少的研究工具。与此同时，光遗传技术可以特异性地激活特定类型的细胞，与荧光显微成像技术相结合，对生命科学领域的发展起着积极的推动作用。
现有的荧光显微成像与光遗传刺激相结合的方式包括：（1）将光刺激光束与成像照明光束合束，分别通过不同的扫描模块控制实现光刺激与成像照明的功能；（2）将光刺激光源置于成像光路之外，对生物组织进行宽场光刺激。第1种方式将增加成像光路设计的复杂性；第2种方式无法实现高分辨率的精准光刺激，并且光刺激的强度和照射角度易受人为设置的影响，降低了实验的稳定性。
 技术问题
鉴于此，本发明提供一种融合精准光遗传刺激与多通道光信号探测的光学系统及荧光显微成像装置以简化光路，提高实验效率。
 技术解决方案
本发明提出的具体技术方案为：包括光刺激光源、第一透镜、扫描振镜、第二透镜、第一二向色镜、第三透镜、第四透镜、第二二向色镜、第一发射滤光片、第五透镜、第一光电倍增管、第二发射滤光片、第六透镜、第二光电倍增管、第三二向色镜、物镜及成像扫描模块，其中：
在光遗传刺激过程中：所述光刺激光束经所述第一透镜准直后经所述扫描振镜扫描，然后依次经所述第二透镜、所述第一二向色镜，再由所述第三透镜进行扩束；扩束后的光刺激光束经所述第三二向色镜反射，再由所述物镜聚焦于生物组织，完成对生物组织的光刺激调控；
在成像过程中：照明光束经所述成像扫描模块进行扫描，再通过所述第三二向色镜后由所述物镜聚焦于生物组织处，经所述照明光束激发生物组织后所产生的发射光依次经所述物镜、所述第三二向色镜反射，再通过所述第三透镜、所述第一二向色镜、所述第四透镜后由所述第二二向色镜分离；经所述第二二向色镜反射的发射光通过所述第一发射滤光片，再由所述第五透镜聚焦于所述第一光电倍增管的光敏面；透射通过所述第二二向色镜的发射光通过所述第二发射滤光片，由所述第六透镜聚焦于第二光电倍增管的光敏面。
具体地，所述光刺激光源用于激发由常用光敏感蛋白标记的神经元的活动。
具体地，所述第一二向色镜用于分离光刺激光束和发射光。光刺激光束被第一二向色镜反射；发射光被第一二向色镜透射。
具体地，所述第二二向色镜用于分离不同波长范围的发射光；经第二二向色镜反射的发射光由第一光电倍增管探测，经第二二向色镜透射的发射光由第二光电倍增管探测。
具体地，所述第一发射滤光片用于第一光电倍增管前的接收滤波；所述第二发射滤光片用于第二光电倍增管前的接收滤波。
具体地，所述第一光电倍增管和第二光电倍增管用于发射光的探测。
 有益效果
本申请采用上述技术方案，其有益效果如下：
本申请提供的融合精准光遗传刺激与多通道光信号探测的光学系统，在光遗传刺激过程中共享部分探测光路，简化了光路设计，实现了系统的紧凑布局；与光刺激作为独立模块相比，减少了材料的使用，降低了制造成本；且可以实现光刺激的精准聚焦，提高了其工作稳定性，有助于简化在生物实验中的操作过程，提高实验效率。
 附图说明
为了更清楚地说明本申请实施例的技术方案，下面将对本申请实施例或现有技术描述中所需要使用的附图作简单地介绍。显而易见地，下面所描述的附图仅仅是本申请的一些实施例，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他的附图。
图1为本申请实施例1提供的融合精准光遗传刺激与多通道光信号探测的光学系统的结构示意图。
 本发明的实施方式
下面详细描述本申请的实施例，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，旨在用于解释本申请，而不能理解为对本申请的限制。
在本申请的描述中，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申请和简化描述，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、以特定的方位构造和操作，因此不能理解为对本申请的限制。
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。在本申请的描述中，“多个”的含义是两个或两个以上，除非另有明确具体的限定。
为了使本申请的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本申请进行进一步地详细说明。
实施例1
请参阅图1，本申请实施例提供的一种融合精准光遗传刺激与多通道光信号探测模块设计的结构示意图。包括：光刺激光源10、第一透镜11、扫描振镜12、第二透镜13、第一二向色镜14、第三透镜15、第四透镜16、第二二向色镜17、第一发射滤光片18、第五透镜19、第一光电倍增管20、第二发射滤光片21、第六透镜22、第二光电倍增管23、第三二向色镜24、物镜25及成像扫描模块26。
以活体鼠脑双光子显微成像为实施例进行说明。在实施例中，光刺激光源的波长为470±14纳米；采用基因编码钙荧光指示剂（GCaMP）标记神经元钙活动；采用近红外荧光染料（Alexa Fluor 680）标记血管。GCaMP的双光子激发波长峰值约为920纳米，发射光波长峰值约为515纳米；Alexa Fluor 680的双光子激发波长峰值约为1280纳米，发射光波长峰值约为710纳米。
在本实施例中，第一二向色镜14用于反射小于490纳米的光，透射大于500纳米的光。
在实施例中，第二二向色镜17用于反射500~630纳米的光，透射640~840纳米的光。
在实施例中，第三二向色镜24用于反射450~840纳米的光，透射850纳米以上的光。
在实施例中，第一发射滤光片18用于钙荧光的接收滤光（如500~600纳米）。
在实施例中，第二发射滤光片21用于Alexa Fluor 680荧光的接收滤光（如670~840纳米）。
本申请上述实施例提供的融合精准光遗传刺激与多通道光信号探测模块设计，其工作方式如下：
光刺激光束经所述第一透镜11准直后经所述扫描振镜12扫描，然后依次经所述第二透镜13、所述第一二向色镜14，再由所述第三透镜15进行扩束；扩束后的光刺激光束经所述第三二向色镜24反射，再由所述物镜25聚焦于生物组织，以激活表达光敏感蛋白的神经元，完成对生物组织的光刺激调控。
与此同时，在荧光成像过程中的照明光束通过成像扫描模块（图略）进行扫描，经物镜聚焦于鼠脑皮层中；经照明光束（920纳米）激发GCaMP产生的钙荧光（发射光波长峰值约为515纳米；标记神经钙活动）或激发Alexa Fluor 680（1280纳米）产生的荧光（发射光波长峰值约为 纳米；标记血管）经所述成像扫描模块26进行扫描，再通过所述第三二向色镜24后由所述物镜25聚焦于生物组织处，经所述照明光束激发生物组织后所产生的发射光依次经所述物镜25、所述第三二向色镜24反射，再通过所述第三透镜15、所述第一二向色镜14、所述第四透镜16进入所述第二二向色镜17；随后，激发GCaMP或Alexa Fluor 680产生的荧光由所述第二二向色镜17分离；经第二二向色镜17反射的荧光（如激发GCaMP产生的钙荧光）通过第一发射滤光片18，由第五透镜19聚焦于第一光电倍增管20的光敏面；透射通过第二二向色镜17的荧光（如激发Alexa Fluor 680产生的荧光）通过第二发射滤光片21，由第六透镜22聚焦于第二光电倍增管23的光敏面。
本申请上述实施例提供的融合精准光遗传刺激与多通道光信号探测的光学系统，可实现对不同波长范围的荧光（GCaMP或Alexa Fluor 680）的分离与探测，并融合了光遗传刺激功能，在光遗传刺激过程中共享部分探测光路，简化了光路设计，实现了系统的紧凑布局；与光刺激作为独立模块相比，减少了材料的使用，降低了制造成本；且可以实现光刺激的精准聚焦，提高了其工作稳定性，有助于简化在生物实验中的操作过程，提高实验效率。
可以理解，以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明记载的范围。
以上仅为本申请的较佳实施例，仅具体描述了本申请的技术原理，这些描述只是为了解释本申请的原理，不能以任何方式解释为对本申请保护范围的限制。基于此处解释，凡在本申请的精神和原则之内所作的任何修改、等同替换和改进，及本领域的技术人员不需要付出创造性的劳动即可联想到本申请的其他具体实施方式，均应包含在本申请的保护范围之内。

Claims (8)

  1. 一种融合精准光遗传刺激与多通道光信号探测的光学系统，其特征在于，包括光刺激光源、第一透镜、扫描振镜、第二透镜、第一二向色镜、第三透镜、第四透镜、第二二向色镜、第一发射滤光片、第五透镜、第一光电倍增管、第二发射滤光片、第六透镜、第二光电倍增管、第三二向色镜、物镜及成像扫描模块，其中：
    在光遗传刺激过程中：所述光刺激光束经所述第一透镜准直后经所述扫描振镜扫描，然后依次经所述第二透镜、所述第一二向色镜，再由所述第三透镜进行扩束；扩束后的光刺激光束经所述第三二向色镜反射，再由所述物镜聚焦于生物组织，完成对生物组织的光刺激调控；
    在成像过程中：照明光束经所述成像扫描模块进行扫描，再通过所述第三二向色镜后由所述物镜聚焦于生物组织处，经所述照明光束激发生物组织后所产生的发射光依次经所述物镜、所述第三二向色镜反射，再通过所述第三透镜、所述第一二向色镜、所述第四透镜后由所述第二二向色镜分离；经所述第二二向色镜反射的发射光通过所述第一发射滤光片，再由所述第五透镜聚焦于所述第一光电倍增管的光敏面；透射通过所述第二二向色镜的发射光通过所述第二发射滤光片，由所述第六透镜聚焦于第二光电倍增管的光敏面。
  2. 如权利要求1所述的融合精准光遗传刺激与多通道光信号探测的光学系统，其特征在于，所述第一透镜设置于光刺激光源和扫描振镜之间，用于光刺激光束的准直。
  3. 如权利要求1所述的融合精准光遗传刺激与多通道光信号探测的光学系统，其特征在于，所述第一二向色镜用于反射光刺激光束，并透射通过成像过程中产生的发射光。
  4. 如权利要求1所述的融合精准光遗传刺激与多通道光信号探测的光学系统，其特征在于，在光刺激光路中，所述第二透镜和第三透镜对光刺激光束进行扩束；在发射光接收光路中，所述第三透镜和第四透镜用来对发射光进行光学接力。
  5. 如权利要求1所述的融合精准光遗传刺激与多通道光信号探测的光学系统，其特征在于，所述第二二向色镜用于分离不同波长范围的发射光。
  6. 如权利要求1所述的融合精准光遗传刺激与多通道光信号探测的光学系统，其特征在于，所述第一发射滤光片和第二发射滤光片用于选择性地通过发射光的特征波段光谱。
  7. 如权利要求1所述的融合精准光遗传刺激与多通道光信号探测的光学系统，其特征在于，所述第一光电倍增管和第二光电倍增管用于发射光的探测。
  8. 一种荧光显微成像装置，其特征在于，包括如权利要求1至7任一项所述的融合精准光遗传刺激与多通道光信号探测的光学系统。
PCT/CN2023/137699 2023-05-04 2023-12-09 融合光遗传刺激与光信号探测的光学系统及成像装置 Ceased WO2024227348A1 (zh)

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