WO2023109625A1 - In-vivo double-color two-photon microscopic imaging system - Google Patents

In-vivo double-color two-photon microscopic imaging system Download PDF

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WO2023109625A1
WO2023109625A1 PCT/CN2022/137419 CN2022137419W WO2023109625A1 WO 2023109625 A1 WO2023109625 A1 WO 2023109625A1 CN 2022137419 W CN2022137419 W CN 2022137419W WO 2023109625 A1 WO2023109625 A1 WO 2023109625A1
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scanning
dichroic mirror
color
mirror
lens
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PCT/CN2022/137419
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French (fr)
Chinese (zh)
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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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Publication of WO2023109625A1 publication Critical patent/WO2023109625A1/en

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • 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
    • 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

Definitions

  • the present application relates to the field of laser technology, in particular to an in vivo two-color 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.
  • One of the important research tools Using two beams of excitation light with different wavelengths, combined with dual detection channels, combined with dual-label two-color two-photon microscopic imaging technology, can realize simultaneous imaging of two different cell types or tissue components, and has a wide range of applications in the field of life sciences prospect.
  • Two-color simultaneous imaging often requires the selection of two dyes with very different Stokes shifts to avoid signal crosstalk.
  • imaging dyes or probes used for in vivo imaging, especially for physiological function detection have broad excitation/emission spectra. Spectral overlap is unavoidable.
  • Two-color simultaneous imaging often requires the selection of two dyes with very different Stokes shifts to avoid signal crosstalk.
  • imaging dyes or probes used for in vivo imaging especially for physiological function detection, have broad excitation/emission spectra. Spectral overlap is unavoidable.
  • the present application provides an in vivo two-color two-photon microscopic imaging system, which is characterized in that it includes a first femtosecond pulse excitation light source (1), a first photoelectric modulator (2), a first beam expander (3), The first mirror (4), the first scanning module (5), the second femtosecond pulse excitation light source (6), the second photoelectric modulator (7), the second beam expander (8), and the second scanning module ( 9), first dichroic mirror (10), scanning lens (11), sleeve lens (12), second dichroic mirror (13), microscope objective lens (14), third dichroic mirror (15 ), the first narrow-band filter (16), the first photomultiplier tube (17), the second narrow-band filter (18), the second photomultiplier tube (19), signal control/acquisition equipment (20) and computer (21), where:
  • the laser beam emitted by the first femtosecond pulse excitation light source (1) is modulated by the first photoelectric modulator (2), then expanded by the first beam expander (3), and then reflected by the first After being reflected by the mirror (4), it enters the first scanning module (5) vertically downwards, and the formed scanning beam transmits through the first dichroic mirror (10) and then passes through the scanning lens (11), the set After the tube lens (12) and the second dichroic mirror (13), it is focused on the sample by the microscope objective lens (14), and the emitted light generated after exciting the first imaging dye is captured by the microscope objective lens (14 ) is collected by the second dichroic mirror (13) and reflected to the third dichroic mirror (15), transmitted through the third dichroic mirror (15) and then passed through the first narrow-band filter
  • the light sheet (16) is filtered and detected by the first photomultiplier tube (17), and is collected and processed by the signal control/acquisition device (20) and computer (21) to obtain the first imaging dye Structural images of labeled
  • the laser emitted by the second femtosecond pulse excitation light source (6) is modulated by the second photoelectric modulator (7) and then expanded by the second beam expander (8), and then enters the first Two scanning modules (9), the scanning beam formed is reflected by the first dichroic mirror (10) and propagates vertically downward, and the scanning light passes through the scanning lens (11), the sleeve lens (12) and After the second dichroic mirror (13), it is focused on the sample by the microscope objective lens (14), and the emitted light generated after exciting the second dye is collected by the microscope objective lens (14) and then emitted by the microscope objective lens (14).
  • the second dichroic mirror (13) is reflected to the third dichroic mirror (15), and after being reflected by the third dichroic mirror (15), it is filtered by the second narrow-band filter (18)
  • the light is detected by the second photomultiplier tube (19), collected and processed by the signal control/acquisition device (20) and computer (21), and the structure of the cell or tissue marked by the second dye is obtained image;
  • Carry out image segmentation on the structural image obtained above determine the pixel positions of cells or tissues labeled with two different dyes, and then use the pixel clock signals of the scanning mirrors in each scanning module to control the photoelectric modulators (2) and (7), respectively,
  • the on/off of the excitation light of the scanning modules (5) and (9) is controlled in real time, and only the target cells or tissues are excited to realize two-color simultaneous imaging of two-photon excitation and avoid excitation/emission in two-color imaging Optical crosstalk problem.
  • the two scanning modules (5) and (9) are both composed of a galvanometer vibrating mirror and a high-speed resonant vibrating mirror.
  • one group of the two scanning modules (5) and (9) consists of two galvanometer vibrating mirrors, and the other group consists of one galvanometer vibrating mirror and a high-speed resonant vibrating mirror .
  • the two-color two-photon microscopic imaging technology includes a first femtosecond pulse excitation light source (1), a first photoelectric modulator (2), a first beam expander (3), a first mirror (4), The first scanning module (5), the second femtosecond pulse excitation light source (6), the second photoelectric modulator (7), the second beam expander (8), the second scanning module (9), the first dichroic mirror (10), scanning lens (11), tube lens (12), second dichroic mirror (13), microscope objective lens (14), third dichroic mirror (15), first narrow-band filter (16), a first photomultiplier tube (17), a second narrow-band filter (18), a second photomultiplier tube (19), a signal control/acquisition device (20) and a computer (21).
  • the excitation light of two different wavelengths excites two different imaging dyes or probes respectively through the scanning modules (5) and (9) and forms an image, and determines the pixel position of the cells or tissues labeled by the two different dyes according to the image, Then use the pixel clock signal of the scanning galvanometer in each scanning module to control the photoelectric modulators (2) and (7) respectively, and control the on/off of the excitation light of the scanning modules (5) and (9) in real time.
  • the cell or tissue is excited to realize two-color simultaneous imaging of two-photon excitation, and avoid the excitation/emission light crosstalk problem in two-color imaging.
  • Fig. 1 is a schematic diagram of an in vivo two-color two-photon microscopic imaging system provided in an embodiment of the present application.
  • FIG. 2 is a structural diagram of blood vessels and nerve cells provided in the embodiment of the present application.
  • FIG. 2 is a schematic diagram of the voltage signal generated by the scanning vibrating mirror (galvanometer vibrating mirror or resonant vibrating mirror) driving circuit provided by the embodiment of the present application when scanning and changing lines, and the signal is used to generate a pixel clock.
  • the scanning vibrating mirror galvanometer vibrating mirror or resonant vibrating mirror
  • 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.
  • Calcium fluorescent indicator GCaMP6f can be used to label neural calcium activity, which is at 920 nm has a good two-photon absorption cross-section, and the peak emission wavelength is at ⁇ 510 nm; the luminescence lifetime of the phosphorescent oxygen probe Oxyphor2P is sensitive to oxygen concentration, and can be used to measure blood oxygen partial pressure, which has a good two-photon absorption at 950 nm Cross-section, the emission wavelength peaks at ⁇ 758 nm.
  • neural activity causes changes in the concentration of calcium ions in cells and changes the fluorescence intensity of calcium indicators, requiring scanning at tens of frames per second speed to record changes in fluorescence signals;
  • two-photon blood oxygen partial pressure detection is based on ground state oxygen quenching triplet molecular luminescence, changing its luminescence lifetime, which needs to be repeated multiple times (eg: 500-2000 times) at discrete pixel points
  • a 300 ⁇ s excitation/acquisition sequence is used to record the complete phosphorescence decay signal with high signal-to-noise ratio, and finally, the phosphorescence lifetime value is converted to oxygen partial pressure according to a predetermined chemical calibration.
  • the in vivo two-color two-photon microscopic imaging system includes a first femtosecond pulse excitation light source (1), a first photoelectric modulator (2), and a first beam expander (3 ), the first mirror (4), the first scanning module (5), the second femtosecond pulse excitation light source (6), the second photoelectric modulator (7), the second beam expander (8), the second scanning Module (9), first dichroic mirror (10), scan lens (11), tube lens (12), second dichroic mirror (13), microscope objective (14), third dichroic mirror (15), first narrow-band filter (16), first photomultiplier tube (17), second narrow-band filter (18), second photomultiplier tube (19), signal control/acquisition device (20) and computers (21).
  • the first scanning module (5) is composed of two galvanometer vibration mirrors
  • the second scanning module (9) is composed of a galvanometer vibration mirror and a resonant vibration mirror. The specific operation is as follows:
  • the 950 nm laser emitted by the first femtosecond pulse excitation light source (1) is modulated by the first photoelectric modulator (2) and then expanded by the first beam expander (3), and then the beam is expanded by the first mirror ( 4) After reflection, it enters the first scanning module (5) vertically downwards.
  • the first scanning module (5) is composed of two galvanometer vibrating mirrors, and the formed scanning beam is transmitted through the first dichroic mirror (10) and then passes through the The scanning lens (11), the sleeve lens (12) and the second dichroic mirror (13) are focused on a certain depth of the mouse brain cortex by the microscope objective lens (14), and the oxygen probe (Oxyphor2P) dissolved in the blood is excited.
  • the filter (16) is filtered, it is detected by the first photomultiplier tube (17), and is collected and processed by the signal control/acquisition device (20) and the computer (21) to obtain an image of the blood vessel structure (as shown in Figure 2a blood vessel image);
  • the 920 nm laser emitted by the second femtosecond pulse excitation light source (6) is modulated by the second photoelectric modulator (7) and then expanded by the second beam expander (8) before entering the second scanning module (9)
  • the second scanning module (9) is composed of a galvanometer vibrating mirror and a resonant vibrating mirror
  • the formed scanning beam is reflected by the first dichroic mirror (10) and propagates vertically downward
  • the scanning beam passes through the scanning lens (11) , the sleeve lens (12) and the second dichroic mirror (13)
  • the microscope objective lens (14) is focused on the same depth of the mouse cerebral cortex
  • the fluorescent indicator GCaMP6f which marks neural calcium activity, is excited by a 920 nm laser Fluorescence with a center wavelength of 510 nm is emitted.
  • the emitted fluorescence is collected by the microscope objective lens (14), reflected by the second dichroic mirror (13) to the third dichroic mirror (15), reflected by the third dichroic mirror (15), and passed through the second narrow band After the filter (18) is filtered, it is detected by the second photomultiplier tube (19), collected and processed by the signal control/acquisition device (20) and the computer (21), and the image of the nerve cell structure is obtained (as shown in Figure 2a nerve cells);
  • Synchronous measurement of nerve calcium activity and blood oxygen partial pressure keep the measured object still, and control the photoelectric modulator (7) through the pixel clock signal (as shown in Figure 2b) of the resonant galvanometer in the second scanning module (9),
  • the excitation light (920 nm, used to excite GCaMP6f) of the scanning module (9) is only turned on at the pixel positions of the pre-selected neurons, and turned off at other pixel positions (the white line in Figure 2a represents the scanning module (9)
  • the scanning path, the left side of the figure is the voltage control signal of the photoelectric modulator, the circle in the figure is the pre-selected nerve cell), so as to avoid the excitation of Oxyphor2P molecules to generate phosphorescence;
  • the scanning module is controlled according to the pixel position of the blood vessel to be measured (5 ), the blood oxygen partial pressure is measured at the pixel position of the blood vessel to be measured, without interference from the calcium imaging excitation light, so as to realize the synchronous imaging of nerve calcium activity and blood oxygen

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Abstract

An in-vivo double-color two-photon microscopic imaging system. Two excitation lights having different wavelengths respectively excite two different imaging dyes or probes by means of a first scanning module (5) and a second scanning module (9) to form images; pixel positions of cells or tissues marked by two different dyes are determined according to the images; and then, a first photoelectric modulator (2) and a second photoelectric modulator (7) are respectively controlled by using pixel clock signals of scanning galvanometers in the first scanning module (5) and the second scanning module (9). Real-time control is performed on the turning-on/off of the excitation lights of the first scanning module (5) and the second scanning module (9), such that excitation is only performed at respective target cells or tissues, so as to realize synchronous double-color imaging via two-photon excitation, and avoid the problem of the crosstalk of excitation/emission lights during double-color imaging.

Description

一种在体双色双光子显微成像***An in vivo two-color two-photon microscopy imaging system 技术领域technical field
本申请涉及激光技术领域，特别涉及一种在体双色双光子显微成像***。The present application relates to the field of laser technology, in particular to an in vivo two-color two-photon microscopic imaging system.
背景技术Background technique
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。使用两束不同波长的激发光，配合双探测通道，同时结合双标记技术的双色双光子显微成像技术，可以实现两种不同细胞类型或组织成分的同步成像，在生命科学领域有广泛的应用前景。双色同步成像往往需要选取两种斯托克斯位移差别很大的染料以避免信号串扰，但是，用于在体成像，特别是生理功能检测的成像染料或探针的激发/发射光谱较宽，光谱重叠难以避免。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. One of the important research tools. Using two beams of excitation light with different wavelengths, combined with dual detection channels, combined with dual-label two-color two-photon microscopic imaging technology, can realize simultaneous imaging of two different cell types or tissue components, and has a wide range of applications in the field of life sciences prospect. Two-color simultaneous imaging often requires the selection of two dyes with very different Stokes shifts to avoid signal crosstalk. However, imaging dyes or probes used for in vivo imaging, especially for physiological function detection, have broad excitation/emission spectra. Spectral overlap is unavoidable.
技术问题technical problem
双色同步成像往往需要选取两种斯托克斯位移差别很大的染料以避免信号串扰，但是，用于在体成像，特别是生理功能检测的成像染料或探针的激发/发射光谱较宽，光谱重叠难以避免。Two-color simultaneous imaging often requires the selection of two dyes with very different Stokes shifts to avoid signal crosstalk. However, imaging dyes or probes used for in vivo imaging, especially for physiological function detection, have broad excitation/emission spectra. Spectral overlap is unavoidable.
技术解决方案technical solution
鉴于此，有必要提供一种可用于减少双色双光子成像中激发/发射光串扰的成像技术。In view of this, it is necessary to provide an imaging technique that can be used to reduce the excitation/emission light crosstalk in two-color two-photon imaging.
为解决上述问题，本申请采用下述技术方案：In order to solve the above problems, the application adopts the following technical solutions:
本申请提供了一种在体双色双光子显微成像***，其特征在于，包括第一飞秒脉冲激发光源（1）、第一光电调制器（2）、第一扩束装置（3）、第一反射镜（4）、第一扫描模块（5）、第二飞秒脉冲激发光源（6）、第二光电调制器（7）、第二扩束装置（8）、第二扫描模块（9）、第一二向色镜（10）、扫描透镜（11）、套筒透镜（12）、第二二向色镜（13）、显微镜物镜（14）、第三二向色镜（15）、第一窄带滤光片（16）、第一光电倍增管（17）、第二窄带滤光片（18）、第二光电倍增管（19）、信号控制/采集设备（20）及电脑（21），其中：The present application provides an in vivo two-color two-photon microscopic imaging system, which is characterized in that it includes a first femtosecond pulse excitation light source (1), a first photoelectric modulator (2), a first beam expander (3), The first mirror (4), the first scanning module (5), the second femtosecond pulse excitation light source (6), the second photoelectric modulator (7), the second beam expander (8), and the second scanning module ( 9), first dichroic mirror (10), scanning lens (11), sleeve lens (12), second dichroic mirror (13), microscope objective lens (14), third dichroic mirror (15 ), the first narrow-band filter (16), the first photomultiplier tube (17), the second narrow-band filter (18), the second photomultiplier tube (19), signal control/acquisition equipment (20) and computer (21), where:
所述第一飞秒脉冲激发光源（1）出射的激光经所述第一光电调制器（2）调制后经所述第一扩束装置（3）进行扩束，然后由所述第一反射镜（4）反射后垂直向下进入所述第一扫描模块（5），形成的扫描光束透射通过所述第一二向色镜（10）后经所述扫描透镜（11）、所述套筒透镜（12）和所述第二二向色镜（13）后，经所述显微镜物镜（14）聚焦于样品上，激发第一种成像染料后产生的发射光被所述显微镜物镜（14）收集后由所述第二二向色镜（13）反射至所述第三二向色镜（15），透射通过所述第三二向色镜（15）后经所述第一窄带滤光片（16）滤光后被所述第一光电倍增管（17）探测，并经所述信号控制/采集设备（20）和电脑（21）进行采集和处理，得到由第一种成像染料标记的细胞或组织的结构图像；The laser beam emitted by the first femtosecond pulse excitation light source (1) is modulated by the first photoelectric modulator (2), then expanded by the first beam expander (3), and then reflected by the first After being reflected by the mirror (4), it enters the first scanning module (5) vertically downwards, and the formed scanning beam transmits through the first dichroic mirror (10) and then passes through the scanning lens (11), the set After the tube lens (12) and the second dichroic mirror (13), it is focused on the sample by the microscope objective lens (14), and the emitted light generated after exciting the first imaging dye is captured by the microscope objective lens (14 ) is collected by the second dichroic mirror (13) and reflected to the third dichroic mirror (15), transmitted through the third dichroic mirror (15) and then passed through the first narrow-band filter The light sheet (16) is filtered and detected by the first photomultiplier tube (17), and is collected and processed by the signal control/acquisition device (20) and computer (21) to obtain the first imaging dye Structural images of labeled cells or tissues;
同时，所述第二飞秒脉冲激发光源（6）出射的激光经所述第二光电调制器（7）调制后经所述第二扩束装置（8）进行扩束，然后进入所述第二扫描模块（9），形成的扫描光束经所述第一二向色镜（10）反射后垂直向下传播，扫描光经所述扫描透镜（11）、所述套筒透镜（12）和所述第二二向色镜（13）后，经所述显微镜物镜（14）聚焦于样品上，激发第二种染料后产生的发射光被所述显微镜物镜（14）收集后并由所述第二二向色镜（13）反射至所述第三二向色镜（15），被所述第三二向色镜（15）反射后经所述第二窄带滤光片（18）滤光后被所述第二光电倍增管（19）探测，并经所述信号控制/采集设备（20）和电脑（21）进行采集和处理，得到由第二种染料标记的细胞或组织的结构图像；At the same time, the laser emitted by the second femtosecond pulse excitation light source (6) is modulated by the second photoelectric modulator (7) and then expanded by the second beam expander (8), and then enters the first Two scanning modules (9), the scanning beam formed is reflected by the first dichroic mirror (10) and propagates vertically downward, and the scanning light passes through the scanning lens (11), the sleeve lens (12) and After the second dichroic mirror (13), it is focused on the sample by the microscope objective lens (14), and the emitted light generated after exciting the second dye is collected by the microscope objective lens (14) and then emitted by the microscope objective lens (14). The second dichroic mirror (13) is reflected to the third dichroic mirror (15), and after being reflected by the third dichroic mirror (15), it is filtered by the second narrow-band filter (18) The light is detected by the second photomultiplier tube (19), collected and processed by the signal control/acquisition device (20) and computer (21), and the structure of the cell or tissue marked by the second dye is obtained image;
对上述得到的结构图像进行图像分割，确定两种不同染料标记的细胞或组织的像素位置，然后利用各扫描模块中扫描振镜的像素时钟信号分别控制光电调制器（2）和（7），对扫描模块（5）和（9）的激发光的开/关进行实时控制，只在目标细胞或组织处进行激发，实现双光子激发的双色同步成像，并避免了双色成像中的激发/发射光串扰问题。Carry out image segmentation on the structural image obtained above, determine the pixel positions of cells or tissues labeled with two different dyes, and then use the pixel clock signals of the scanning mirrors in each scanning module to control the photoelectric modulators (2) and (7), respectively, The on/off of the excitation light of the scanning modules (5) and (9) is controlled in real time, and only the target cells or tissues are excited to realize two-color simultaneous imaging of two-photon excitation and avoid excitation/emission in two-color imaging Optical crosstalk problem.
在其中一些实施例中，所述两个扫描模块（5）和（9）都由一个检流计振镜和一个高速共振振镜组成。In some of these embodiments, the two scanning modules (5) and (9) are both composed of a galvanometer vibrating mirror and a high-speed resonant vibrating mirror.
在其中一些实施例中，所述两个扫描模块（5）和（9），一组由两个检流计振镜组成，另一组由一个检流计振镜和一个高速共振振镜组成。In some of these embodiments, one group of the two scanning modules (5) and (9) consists of two galvanometer vibrating mirrors, and the other group consists of one galvanometer vibrating mirror and a high-speed resonant vibrating mirror .
有益效果Beneficial effect
本申请采用上述技术方案，其有益效果如下：The application adopts the above-mentioned technical scheme, and its beneficial effects are as follows:
本申请提供的双色双光子显微成像技术，包括第一飞秒脉冲激发光源（1）、第一光电调制器（2）、第一扩束装置（3）、第一反射镜（4）、第一扫描模块（5）、第二飞秒脉冲激发光源（6）、第二光电调制器（7）、第二扩束装置（8）、第二扫描模块（9）、第一二向色镜（10）、扫描透镜（11）、套筒透镜（12）、第二二向色镜（13）、显微镜物镜（14）、第三二向色镜（15）、第一窄带滤光片（16）、第一光电倍增管（17）、第二窄带滤光片（18）、第二光电倍增管（19）、信号控制/采集设备（20）及电脑（21）。两个不同波长的激发光通过扫描模块（5）和（9）对两种不同的成像染料或探针分别进行激发并形成图像，根据图像确定两种不同染料标记的细胞或组织的像素位置，然后利用各扫描模块中扫描振镜的像素时钟信号分别控制光电调制器（2）和（7），对扫描模块（5）和（9）的激发光的开/关进行实时控制，只在目标细胞或组织处进行激发，实现双光子激发的双色同步成像，并避免了双色成像中的激发/发射光串扰问题。The two-color two-photon microscopic imaging technology provided by this application includes a first femtosecond pulse excitation light source (1), a first photoelectric modulator (2), a first beam expander (3), a first mirror (4), The first scanning module (5), the second femtosecond pulse excitation light source (6), the second photoelectric modulator (7), the second beam expander (8), the second scanning module (9), the first dichroic mirror (10), scanning lens (11), tube lens (12), second dichroic mirror (13), microscope objective lens (14), third dichroic mirror (15), first narrow-band filter (16), a first photomultiplier tube (17), a second narrow-band filter (18), a second photomultiplier tube (19), a signal control/acquisition device (20) and a computer (21). The excitation light of two different wavelengths excites two different imaging dyes or probes respectively through the scanning modules (5) and (9) and forms an image, and determines the pixel position of the cells or tissues labeled by the two different dyes according to the image, Then use the pixel clock signal of the scanning galvanometer in each scanning module to control the photoelectric modulators (2) and (7) respectively, and control the on/off of the excitation light of the scanning modules (5) and (9) in real time. The cell or tissue is excited to realize two-color simultaneous imaging of two-photon excitation, and avoid the excitation/emission light crosstalk problem in two-color imaging.
附图说明Description of drawings
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。In order to illustrate the technical solutions of the embodiments of the present application more clearly, the following briefly introduces the drawings that are required in the embodiments of the present application or the description of the prior art. Apparently, the drawings described below are only some embodiments of the present application, and those skilled in the art can also obtain other drawings based on these drawings without creative efforts.
图1为本申请实施例提供的在体双色双光子显微成像***原理图。Fig. 1 is a schematic diagram of an in vivo two-color two-photon microscopic imaging system provided in an embodiment of the present application.
图2中a为本申请实施例提供的血管和神经细胞结构图。A in Fig. 2 is a structural diagram of blood vessels and nerve cells provided in the embodiment of the present application.
图2中b为本申请实施例提供的扫描振镜（检流计振镜或共振振镜）驱动电路在扫描换行时产生的电压信号示意图，该信号用于产生像素时钟。b in FIG. 2 is a schematic diagram of the voltage signal generated by the scanning vibrating mirror (galvanometer vibrating mirror or resonant vibrating mirror) driving circuit provided by the embodiment of the present application when scanning and changing lines, and the signal is used to generate a pixel clock.
本发明的实施方式Embodiments of the present invention
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。Embodiments of the present application are described in detail below, examples of which are shown in the drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the figures are exemplary, and are intended to explain the present application, and should not be construed as limiting the present application.
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。In the description of the present application, it should be understood that the orientation or positional relationship indicated by the terms "upper", "lower", "horizontal", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings , is only for the convenience of describing the present application and simplifying the description, but does not indicate or imply that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and thus should not be construed as limiting the present application.
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。In addition, the terms "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. In the description of the present application, "plurality" means two or more, unless otherwise specifically defined.
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详细说明。In order to make the purpose, technical solution and advantages of the present application clearer, the present application will be further described in detail below in conjunction with the accompanying drawings and embodiments.
本申请以活体鼠脑皮层神经和血管的功能性成像为例，详细描述该技术的工作原理。钙荧光指示剂GCaMP6f可用于标记神经钙活动，其在920 nm有较好的双光子吸收截面，发射波长峰值在~510 nm；磷光氧探针Oxyphor2P的发光寿命对氧浓度敏感，可用于测量血氧分压，其在950 nm有较好的双光子吸收截面，发射波长峰值在~758 nm。然而，双光子神经钙活动成像与双光子血氧分压检测的原理不同：神经活动引起细胞内钙离子浓度的变化并使钙指示剂的荧光强度发生改变，需要以每秒数十帧的扫描速度来记录荧光信号的变化；双光子血氧分压检测是基于基态氧猝灭三重态分子发光，改变其发光寿命，需要在离散的像素点处多次重复（如：500-2000次）单次时长为300 μs的激发/采集过程以记录完整的高信噪比的磷光衰减信号，最后，根据预先确定的化学标定将磷光寿命值转化为氧分压。在神经钙活动与血氧分压同步测量过程中，920 nm的激发光在激发GCaMP6f的同时也会激发Oxyphor2P分子，从而对氧分压测量产生磷光信号干扰，影响其准确性。本申请提供的双色双光子显微成像技术能有效地解决此问题，具体过程如下：This application takes the functional imaging of cortical nerves and blood vessels in living rats as an example to describe the working principle of this technology in detail. Calcium fluorescent indicator GCaMP6f can be used to label neural calcium activity, which is at 920 nm has a good two-photon absorption cross-section, and the peak emission wavelength is at ~510 nm; the luminescence lifetime of the phosphorescent oxygen probe Oxyphor2P is sensitive to oxygen concentration, and can be used to measure blood oxygen partial pressure, which has a good two-photon absorption at 950 nm Cross-section, the emission wavelength peaks at ~758 nm. However, the principle of two-photon neural calcium activity imaging is different from that of two-photon blood oxygen partial pressure detection: neural activity causes changes in the concentration of calcium ions in cells and changes the fluorescence intensity of calcium indicators, requiring scanning at tens of frames per second speed to record changes in fluorescence signals; two-photon blood oxygen partial pressure detection is based on ground state oxygen quenching triplet molecular luminescence, changing its luminescence lifetime, which needs to be repeated multiple times (eg: 500-2000 times) at discrete pixel points A 300 μs excitation/acquisition sequence is used to record the complete phosphorescence decay signal with high signal-to-noise ratio, and finally, the phosphorescence lifetime value is converted to oxygen partial pressure according to a predetermined chemical calibration. During the simultaneous measurement of neural calcium activity and blood oxygen partial pressure, the 920 nm excitation light excited GCaMP6f and Oxyphor2P molecules at the same time, which would interfere with the phosphorescence signal of oxygen partial pressure measurement and affect its accuracy. The two-color two-photon microscopic imaging technology provided by this application can effectively solve this problem, and the specific process is as follows:
请参阅图1，为本申请实施例提供的在体双色双光子显微成像***，包括第一飞秒脉冲激发光源（1）、第一光电调制器（2）、第一扩束装置（3）、第一反射镜（4）、第一扫描模块（5）、第二飞秒脉冲激发光源（6）、第二光电调制器（7）、第二扩束装置（8）、第二扫描模块（9）、第一二向色镜（10）、扫描透镜（11）、套筒透镜（12）、第二二向色镜（13）、显微镜物镜（14）、第三二向色镜（15）、第一窄带滤光片（16）、第一光电倍增管（17）、第二窄带滤光片（18）、第二光电倍增管（19）、信号控制/采集设备（20）及电脑（21）。在该实施例中，第一扫描模块（5）由两个检流计振镜组成，第二扫描模块（9）由一个检流计振镜和一个共振振镜组成。具体操作如下：Please refer to Figure 1, the in vivo two-color two-photon microscopic imaging system provided by the embodiment of the present application includes a first femtosecond pulse excitation light source (1), a first photoelectric modulator (2), and a first beam expander (3 ), the first mirror (4), the first scanning module (5), the second femtosecond pulse excitation light source (6), the second photoelectric modulator (7), the second beam expander (8), the second scanning Module (9), first dichroic mirror (10), scan lens (11), tube lens (12), second dichroic mirror (13), microscope objective (14), third dichroic mirror (15), first narrow-band filter (16), first photomultiplier tube (17), second narrow-band filter (18), second photomultiplier tube (19), signal control/acquisition device (20) and computers (21). In this embodiment, the first scanning module (5) is composed of two galvanometer vibration mirrors, and the second scanning module (9) is composed of a galvanometer vibration mirror and a resonant vibration mirror. The specific operation is as follows:
结构成像：第一飞秒脉冲激发光源（1）出射的950 nm的激光经第一光电调制器（2）调制后经第一扩束装置（3）进行扩束，然后由第一反射镜（4）反射后垂直向下进入第一扫描模块（5），第一扫描模块（5）由两个检流计振镜组成，形成的扫描光束透射通过第一二向色镜（10）后通过扫描透镜（11）、套筒透镜（12）和第二二向色镜（13），经显微镜物镜（14）聚焦于鼠脑皮层某一深度处，激发溶解在血液中的氧探针（Oxyphor2P）分子，并发射出以758 nm为中心波长的磷光。发射出的磷光被显微镜物镜（14）收集后由第二二向色镜（13）反射至第三二向色镜（15），透射通过第三二向色镜（15）后经第一窄带滤光片（16）滤光后被第一光电倍增管（17）探测，并经信号控制/采集设备（20）和电脑（21）进行采集和处理，得到血管结构图像（如图2a中的血管图像）；Structural imaging: the 950 nm laser emitted by the first femtosecond pulse excitation light source (1) is modulated by the first photoelectric modulator (2) and then expanded by the first beam expander (3), and then the beam is expanded by the first mirror ( 4) After reflection, it enters the first scanning module (5) vertically downwards. The first scanning module (5) is composed of two galvanometer vibrating mirrors, and the formed scanning beam is transmitted through the first dichroic mirror (10) and then passes through the The scanning lens (11), the sleeve lens (12) and the second dichroic mirror (13) are focused on a certain depth of the mouse brain cortex by the microscope objective lens (14), and the oxygen probe (Oxyphor2P) dissolved in the blood is excited. ) molecule, and emits a 758 nm is the central wavelength of phosphorescence. The emitted phosphorescence is collected by the microscope objective lens (14), reflected by the second dichroic mirror (13) to the third dichroic mirror (15), transmitted through the third dichroic mirror (15), and passed through the first narrow band After the filter (16) is filtered, it is detected by the first photomultiplier tube (17), and is collected and processed by the signal control/acquisition device (20) and the computer (21) to obtain an image of the blood vessel structure (as shown in Figure 2a blood vessel image);
同时，第二飞秒脉冲激发光源（6）出射的920 nm的激光经第二光电调制器（7）调制后经第二扩束装置（8）进行扩束后进入第二扫描模块（9），第二扫描模块（9）由一个检流计振镜和一个共振振镜组成，形成的扫描光束经第一二向色镜（10）反射垂直向下传播，扫描光束经扫描透镜（11）、套筒透镜（12）和第二二向色镜（13）后，经显微镜物镜（14）聚焦于鼠脑皮层同一深度处，标记神经钙活动的荧光指示剂GCaMP6f受920 nm的激光激发后发射出以510 nm为中心波长的荧光。发射出的荧光被显微镜物镜（14）收集后由第二二向色镜（13）反射至第三二向色镜（15），经第三二向色镜（15）反射后经第二窄带滤光片（18）滤光后被第二光电倍增管（19）探测，并经信号控制/采集设备（20）和电脑（21）进行采集和处理，得到神经细胞结构图像（如图2a中的神经细胞）；At the same time, the 920 nm laser emitted by the second femtosecond pulse excitation light source (6) is modulated by the second photoelectric modulator (7) and then expanded by the second beam expander (8) before entering the second scanning module (9) , the second scanning module (9) is composed of a galvanometer vibrating mirror and a resonant vibrating mirror, the formed scanning beam is reflected by the first dichroic mirror (10) and propagates vertically downward, and the scanning beam passes through the scanning lens (11) , the sleeve lens (12) and the second dichroic mirror (13), the microscope objective lens (14) is focused on the same depth of the mouse cerebral cortex, and the fluorescent indicator GCaMP6f, which marks neural calcium activity, is excited by a 920 nm laser Fluorescence with a center wavelength of 510 nm is emitted. The emitted fluorescence is collected by the microscope objective lens (14), reflected by the second dichroic mirror (13) to the third dichroic mirror (15), reflected by the third dichroic mirror (15), and passed through the second narrow band After the filter (18) is filtered, it is detected by the second photomultiplier tube (19), collected and processed by the signal control/acquisition device (20) and the computer (21), and the image of the nerve cell structure is obtained (as shown in Figure 2a nerve cells);
利用图像分割算法分别对血管结构图像和神经细胞结构图像进行二进制分割，确定待测的血管和神经细胞的像素位置信息；Use the image segmentation algorithm to perform binary segmentation on the image of the blood vessel structure and the image of the nerve cell structure, and determine the pixel position information of the blood vessel and nerve cells to be tested;
神经钙活动与血氧分压同步测量：保持被测物不动，通过第二扫描模块（9）中的共振振镜的像素时钟信号（如图2b所示）控制光电调制器（7），使扫描模块（9）的激发光（920 nm，用于激发GCaMP6f）仅在预先选择的神经细胞的像素位置处打开，在其他像素位置处关闭（图2a中白线代表扫描模块（9）的扫描路径，图左所示为光电调制器的电压控制信号，图中圆圈内为预先选择的神经细胞），从而避免激发Oxyphor2P分子产生磷光；同时，根据待测血管的像素位置控制扫描模块（5），在待测血管像素位置处进行血氧分压测量，不受钙成像激发光的干扰，从而实现神经钙活动与血氧分压的同步成像。Synchronous measurement of nerve calcium activity and blood oxygen partial pressure: keep the measured object still, and control the photoelectric modulator (7) through the pixel clock signal (as shown in Figure 2b) of the resonant galvanometer in the second scanning module (9), The excitation light (920 nm, used to excite GCaMP6f) of the scanning module (9) is only turned on at the pixel positions of the pre-selected neurons, and turned off at other pixel positions (the white line in Figure 2a represents the scanning module (9) The scanning path, the left side of the figure is the voltage control signal of the photoelectric modulator, the circle in the figure is the pre-selected nerve cell), so as to avoid the excitation of Oxyphor2P molecules to generate phosphorescence; at the same time, the scanning module is controlled according to the pixel position of the blood vessel to be measured (5 ), the blood oxygen partial pressure is measured at the pixel position of the blood vessel to be measured, without interference from the calcium imaging excitation light, so as to realize the synchronous imaging of nerve calcium activity and blood oxygen partial pressure.
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。The above are only preferred embodiments of the present application, and only specifically describe the technical principles of the present application. These descriptions are only for explaining the principles of the present application, and cannot be interpreted as limiting the protection scope of the present application in any way. Based on the explanations here, any modifications, equivalent replacements and improvements made within the spirit and principles of the application, and those skilled in the art who can think of other specific implementation methods of the application without creative work are all It should be included within the scope of protection of this application.

Claims (3)

  1. 一种在体双色双光子显微成像***，其特征在于，包括第一飞秒脉冲激发光源（1）、第一光电调制器（2）、第一扩束装置（3）、第一反射镜（4）、第一扫描模块（5）、第二飞秒脉冲激发光源（6）、第二光电调制器（7）、第二扩束装置（8）、第二扫描模块（9）、第一二向色镜（10）、扫描透镜（11）、套筒透镜（12）、第二二向色镜（13）、显微镜物镜（14）、第三二向色镜（15）、第一窄带滤光片（16）、第一光电倍增管（17）、第二窄带滤光片（18）、第二光电倍增管（19）、信号控制/采集设备（20）及电脑（21），其中： An in vivo two-color two-photon microscopic imaging system, characterized in that it includes a first femtosecond pulse excitation light source (1), a first photoelectric modulator (2), a first beam expander (3), and a first reflector (4), the first scanning module (5), the second femtosecond pulse excitation light source (6), the second photoelectric modulator (7), the second beam expander (8), the second scanning module (9), the second One dichroic mirror (10), scanning lens (11), sleeve lens (12), second dichroic mirror (13), microscope objective lens (14), third dichroic mirror (15), first Narrowband filter (16), first photomultiplier tube (17), second narrowband filter (18), second photomultiplier tube (19), signal control/acquisition device (20) and computer (21), in:
    所述第一飞秒脉冲激发光源（1）出射的激光经所述第一光电调制器（2）调制后经所述第一扩束装置（3）进行扩束，然后由所述第一反射镜（4）反射后垂直向下进入所述第一扫描模块（5），形成的扫描光束透射通过所述第一二向色镜（10）后经所述扫描透镜（11）、所述套筒透镜（12）和所述第二二向色镜（13）后，经所述显微镜物镜（14）聚焦于样品上，激发第一种成像染料后产生的发射光被所述显微镜物镜（14）收集后由所述第二二向色镜（13）反射至所述第三二向色镜（15），透射通过所述第三二向色镜（15）后经所述第一窄带滤光片（16）滤光后被所述第一光电倍增管（17）探测，并经所述信号控制/采集设备（20）和电脑（21）进行采集和处理，得到由第一种成像染料标记的细胞或组织的结构图像；The laser beam emitted by the first femtosecond pulse excitation light source (1) is modulated by the first photoelectric modulator (2), then expanded by the first beam expander (3), and then reflected by the first After being reflected by the mirror (4), it enters the first scanning module (5) vertically downwards, and the formed scanning beam transmits through the first dichroic mirror (10) and then passes through the scanning lens (11), the set After the tube lens (12) and the second dichroic mirror (13), it is focused on the sample by the microscope objective lens (14), and the emitted light generated after exciting the first imaging dye is captured by the microscope objective lens (14 ) is collected by the second dichroic mirror (13) and reflected to the third dichroic mirror (15), transmitted through the third dichroic mirror (15) and then passed through the first narrow-band filter The light sheet (16) is filtered and detected by the first photomultiplier tube (17), and is collected and processed by the signal control/acquisition device (20) and computer (21) to obtain the first imaging dye Structural images of labeled cells or tissues;
    同时，所述第二飞秒脉冲激发光源（6）出射的激光经所述第二光电调制器（7）调制后经所述第二扩束装置（8）进行扩束，然后进入所述第二扫描模块（9），形成的扫描光束经所述第一二向色镜（10）反射后垂直向下传播，扫描光经所述扫描透镜（11）、所述套筒透镜（12）和所述第二二向色镜（13）后，经所述显微镜物镜（14）聚焦于样品上，激发第二种成像染料后产生的发射光被所述显微镜物镜（14）收集后并由所述第二二向色镜（13）反射至所述第三二向色镜（15），被所述第三二向色镜（15）反射后经所述第二窄带滤光片（18）滤光后被所述第二光电倍增管（19）探测，并经所述信号控制/采集设备（20）和电脑（21）进行采集和处理，得到由第二种成像染料标记的细胞或组织的结构图像；At the same time, the laser emitted by the second femtosecond pulse excitation light source (6) is modulated by the second photoelectric modulator (7) and then expanded by the second beam expander (8), and then enters the first Two scanning modules (9), the scanning beam formed is reflected by the first dichroic mirror (10) and propagates vertically downward, and the scanning light passes through the scanning lens (11), the sleeve lens (12) and After the second dichroic mirror (13), it is focused on the sample by the microscope objective lens (14), and the emitted light generated after exciting the second imaging dye is collected by the microscope objective lens (14) and then emitted by the microscope objective lens (14). The second dichroic mirror (13) is reflected to the third dichroic mirror (15), and after being reflected by the third dichroic mirror (15), it passes through the second narrow-band filter (18) After the light is filtered, it is detected by the second photomultiplier tube (19), collected and processed by the signal control/acquisition device (20) and computer (21), and the cells or tissues labeled by the second imaging dye are obtained the structural image of
    对上述得到的结构图像进行图像分割，确定两种不同染料标记的细胞或组织的像素位置，然后利用各扫描模块中扫描振镜的像素时钟信号分别控制光电调制器（2）和（7），对扫描模块（5）和（9）的激发光的开/关进行实时控制，只在目标细胞或组织处进行激发，实现双光子激发的双色同步成像，并避免了双色成像中的激发/发射光串扰问题。Carry out image segmentation on the structural image obtained above, determine the pixel positions of cells or tissues labeled with two different dyes, and then use the pixel clock signals of the scanning mirrors in each scanning module to control the photoelectric modulators (2) and (7), respectively, The on/off of the excitation light of the scanning modules (5) and (9) is controlled in real time, and only the target cells or tissues are excited to realize two-color simultaneous imaging of two-photon excitation and avoid excitation/emission in two-color imaging Optical crosstalk problem.
  2. 如权利要求1所述的在体双色双光子显微成像***，其特征在于，所述两个扫描模块（5）和（9）都由一个检流计振镜和一个高速共振振镜组成。 The in vivo two-color two-photon microscopic imaging system according to claim 1, wherein the two scanning modules (5) and (9) are composed of a galvanometer vibrating mirror and a high-speed resonant vibrating mirror.
  3. 如权利要求1所述的在体双色双光子显微成像***，其特征在于，所述两个扫描模块（5）和（9），一组由两个检流计振镜组成，另一组由一个检流计振镜和一个高速共振振镜组成。 The in vivo two-color two-photon microscopic imaging system according to claim 1, characterized in that, one group of the two scanning modules (5) and (9) consists of two galvanometer vibrating mirrors, and the other group It consists of a galvanometer mirror and a high-speed resonant mirror.
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