WO2023010718A1 - 椭半球曲面大视野高通量双光子显微镜 - Google Patents

椭半球曲面大视野高通量双光子显微镜 Download PDF

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WO2023010718A1
WO2023010718A1 PCT/CN2021/133173 CN2021133173W WO2023010718A1 WO 2023010718 A1 WO2023010718 A1 WO 2023010718A1 CN 2021133173 W CN2021133173 W CN 2021133173W WO 2023010718 A1 WO2023010718 A1 WO 2023010718A1
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sub
beam combining
laser
scanning
level
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PCT/CN2021/133173
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English (en)
French (fr)
Chinese (zh)
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唐玉国
周镇乔
李敏
吕晶
王艳
刘勤颖
陈月岩
贾宏博
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中国科学院苏州生物医学工程技术研究所
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Priority to DE112021008078.4T priority Critical patent/DE112021008078T5/de
Publication of WO2023010718A1 publication Critical patent/WO2023010718A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
    • G01N21/359—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using near infrared light
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/02—Objectives
    • G02B21/04—Objectives involving mirrors
    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/19—Dichroism
    • 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
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0068—Optical details of the image generation arrangements using polarisation
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/008—Details of detection or image processing, including general computer control
    • G02B21/0084—Details of detection or image processing, including general computer control time-scale detection, e.g. strobed, ultra-fast, heterodyne detection
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/0092—Polarisation microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B2207/00—Coding scheme for general features or characteristics of optical elements and systems of subclass G02B, but not including elements and systems which would be classified in G02B6/00 and subgroups
    • G02B2207/114—Two photon or multiphoton effect

Definitions

  • the invention relates to the field of microscopic imaging instruments, in particular to an elliptical hemispherical curved surface with a large field of view and a high-throughput two-photon microscope.
  • the resolution of neurons needs to be achieved, and the optical resolution is required to be 1-2 microns, corresponding to a numerical aperture of 0.3-0.5;
  • the image refresh rate needs to be at least 5 frames per second in order to effectively capture the calcium function signal of neurons;
  • a larger field of view can be imaged, so that the number of neurons can be recorded in a single time.
  • the maximum field of view diameter of two-photon imaging in the world is about 5mm, but the imaging speed of the whole field of view is less than 1 frame per second, which cannot meet the requirements of functional signal detection.
  • the field of view of two-photon imaging is only 1mm ⁇ 1mm, and it only covers a single brain functional area for mice.
  • the mouse's whole cerebral cortex area is about 200mm2, and the brain surface is uneven, close to an elliptical hemisphere, which is a great challenge for optical microscopic imaging.
  • Parallel scanning and detection is a common method to improve the throughput of laser scanning and fluorescence detection.
  • a large field of view is divided into several areas, multiple laser focal points are scanned independently, and multi-channel detectors or area array detectors are combined to detect each area. Fluorescent signal, which can significantly increase imaging throughput.
  • the laser energy loss is large when multiple beams are combined to stitch the field of view. Every time the laser energy passes through the depolarization beam combining prism, the laser energy is reduced by half; multi-channel parallel detection has signal crosstalk caused by fluorescence scattering. Specifically, The fluorescence scattering area is large, and the fluorescence falls on several adjacent areas at the same time, causing signal crosstalk between channels.
  • the technical problem to be solved by the present invention is to provide a high-throughput two-photon microscope with an ellipsoidal curved surface, a large field of view, and a high-throughput two-photon microscope for the above-mentioned deficiencies in the prior art.
  • the technical solution adopted in the present invention is: an ellipsoidal curved surface with a large field of view and high-throughput two-photon microscope, including: a near-infrared femtosecond pulse laser group, a beam splitting delay module group, and a scanning unit group.
  • the laser light emitted by the near-infrared femtosecond pulsed laser group passes through the dichroic film after passing through the beam splitting and delay module group, the scanning unit group, and the beam combining and splicing module in sequence, and then passes through the ellipsoidal surface
  • the large field of view reflective objective lens irradiates the sample, and the fluorescence generated by the sample is collected by the ellipsoidal surface large field of view reflective objective lens, and then reflected by the dichroic plate to the photomultiplier tube detection array;
  • the ellipsoidal curved large field of view reflective objective lens includes a primary mirror, a secondary mirror and three mirrors arranged in sequence along the optical path, the optical surface of the primary mirror is a hyperboloid, the optical surface of the secondary mirror is an oblate ellipse, and the The optical surface of the three mirrors is a flat ellipse, and the laser light entering the large field of view reflective objective lens on the ellipsoid curved surface is reflected by the primary mirror, the secondary mirror and the third mirror in turn and then irradiates the sample;
  • the near-infrared femtosecond pulsed laser group includes 4 near-infrared femtosecond pulsed lasers
  • the beam splitting delay module group includes 4 beam splitting delay modules, and the beam splitting delay module includes a delay optical path and a beam splitting optical path;
  • the scanning unit group includes 4 scanning units, each scanning unit includes 4 independent scanning modules, and each scanning module independently realizes the two-dimensional scanning of a rectangular sub-scanning area;
  • Each of the near-infrared femtosecond pulsed lasers corresponds to one of the beam splitting delay modules, and each of the beam splitting delay modules corresponds to one of the scanning units;
  • the lasers emitted by the four near-infrared femtosecond pulse lasers pass through the delayed optical path to form four delayed lasers with a time interval of T/4 in turn, and each delayed laser passes through the beam splitting optical path, etc.
  • the 4 sub-lasers from the same delay laser enter the same scanning unit, and each sub-laser corresponds to a scanning module to realize the scanning of 1 sub-scanning area, so that through 16 sub-lasers and 16 One-to-one correspondence of the scanning modules realizes the scanning of 16 sub-scanning areas;
  • the beam combining and splicing module is used to realize the beam combining and splicing of 16 sub-lasers emitted by 16 scanning modules.
  • a rectangular scanning field distributed in a 4 ⁇ 4 array is formed, and the laser beams located at the same time position are combined and spliced.
  • the borders of the four sub-scanning areas of the point are not adjacent, but any four areas with adjacent borders are located at different time points in time.
  • the optical surface of the primary mirror is an eighth-order hyperboloid
  • the optical surface of the secondary mirror is a quadratic oblate ellipse
  • the optical surface of the third mirror is a sixth-order oblate ellipse.
  • the numerical aperture of the ellipsoidal curved large-field reflective objective lens ranges from 0.3 to 0.5.
  • the imaging field of view of the reflective objective lens is an ellipsoidal surface, the radius of curvature of the major axis is 9-12mm, the radius of curvature of the minor axis is 6-9mm, and the plane projection size of the field of view is 6mm ⁇ 6mm.
  • the delay optical path includes a first reflector and a second reflector, and the laser light emitted by the near-infrared femtosecond pulse laser is sequentially reflected by the first reflector and the second reflector to form a delayed laser output, by adjusting the distance between the first reflector and the second reflector so that the output delay laser produces different delay amounts;
  • the beam splitting optical path includes a first beam splitting element, a second beam splitting element and a third beam splitting element, and the delayed laser light output by the second reflector enters the first beam splitting element and is equally divided into two paths, and one path is transmitted through the
  • the first light-splitting element reaches the second light-splitting element, and is divided into two paths by the second light-splitting element, and the other path is reflected by the first light-splitting element and reaches the third light-splitting element, and is divided into two paths by the second light-splitting element.
  • the third light splitting element is equally divided into two paths, so that the time-delayed laser light is evenly divided into four paths.
  • the first light-splitting element, the second light-splitting element and the third light-splitting element are all depolarized light-splitting prisms or 50/50 light-splitting plates.
  • the scanning module includes a fast axis resonant scanning mirror and a slow axis galvanometer vibrating mirror.
  • the beam combining and splicing module includes 4 primary beam combining and splicing modules and 1 secondary beam combining and splicing module, and each primary beam combining and splicing module corresponds to one of the scanning units, so that one The 4 sub-lasers emitted by the 4 scanning modules in the scanning unit are combined and spliced;
  • the secondary beam combining and splicing module is used for recombining and splicing the 4 sets of lasers emitted by the 4 primary beam combining and splicing modules, and then inputting them into the dichroic film.
  • the first-level beam combining and splicing module includes two first-level beam combining and splicing sub-optical paths, a first-level polarization beam combining prism, a first-level lens, a first-level combining half-wave plate and a second-level lens,
  • the laser beams emitted from the two first-level beam combining splicing sub-optical paths are combined by the first-level polarization beam combining prism, and then sequentially pass through the first-level lens, the first-level combined half-wave plate and the second-level lens before being output.
  • the one-stage combined half-wave plate includes 4 different half-wave plates;
  • the two first-level beam combining and splicing sub-optical paths have the same structure, and both include a first-level sub-half-wave plate, a second-level sub-half-wave plate, a first-level sub-polarization beam combining prism, a first-level sub-lens, a A first-level combined half-wave plate and a second-level sub-lens, the first-level combined half-wave plate includes two different half-wave plates, and the two first-level beam-combining and splicing sub-optical paths are respectively denoted as the first The first level of beam combining and splicing sub-optical paths and the second level of beam combining and splicing sub-optical paths;
  • the four sub-lasers emitted by the four scanning modules in the same scanning unit are respectively recorded as: the first sub-laser, the second sub-laser, the third sub-laser, and the fourth sub-laser,
  • the first-pass laser beam and the second-pass laser beam are transmitted through the first-order polarization beam combining prism after being combined by the first-order beam combining and splicing sub-optical paths, specifically:
  • the first-pass laser beam passes through the first-stage sub-half-wave plate and transmits the first-stage sub-polarization beam combining prism, and the second-pass sub-laser is polarized by the first-stage sub-half-wave plate after passing through the second-stage sub-half-wave plate Reflected by the beam combining prism, then combine with the first laser beam transmitted through the first-level sub-polarization beam-combining prism, and then pass through the first-level sub-lens together, at the back focal plane of the first-level sub-lens
  • Two sub-scanning areas are formed on two different half-wave plates of the upper-level sub-combined half-wave plate, and the distance between the two sub-scanning areas is one sub-scanning area; the two sub-lasers after beam combining pass through the After the second level of sub-lenses, the collimated laser is changed back, and then transmitted through the first-level polarization beam combining prism, wherein the polarization states of the two laser beam
  • the third-pass sub-laser and the fourth-pass sub-laser are combined by the second-level beam combining splicing sub-optical path and then reflected by the first-level polarization beam combining prism, and then combined with the beam-combining laser transmitted through the first-level polarization beam combining prism ; then pass through the first-level lens together, and form 4 sub-scanning areas on four different half-wave plates of the first-level combined half-wave plate on the back focal plane of the first-level lens, and 4 sub-scanning areas
  • the scanning areas are separated by a distance of a sub-scanning area, and finally pass through the second-level lens and turn back into a collimated laser, which is output to the second-level beam combining and splicing module, wherein, after the first-level combined half-wave
  • the polarization states of the 4 lasers behind the chip are the same.
  • the secondary beam combining and splicing module includes 2 secondary beam combining and splicing sub-optical paths, a secondary polarizing beam combining prism and a secondary lens, and the laser light emitted from the two secondary beam combining and splicing sub-optical paths passes through the two secondary beam combining and splicing sub-optical paths.
  • the beam After being combined by the primary polarization beam combining prism, the beam passes through the secondary lens, and then enters the dichroic plate;
  • the two secondary beam combining and splicing sub-optical paths have the same structure, and both include a secondary sub-polarizing beam combining prism, a first secondary sub-lens and a second secondary sub-lens, and the two secondary beam combining and splicing sub-optical paths are respectively Recorded as the first-level beam combining and splicing sub-optical path and the second-level beam combining and splicing sub-optical path;
  • the beam combining lasers of the 4 sub-lasers emitted by the 4 first-level beam combining and splicing modules are respectively recorded as: the first beam combining laser, the second beam combining laser, the third beam combining laser, and the fourth beam combining laser ;
  • the beam combining laser light of the first path and the beam combining laser light of the second path are transmitted through the secondary polarization beam combining prism after being combined by the first secondary beam combining splicing sub-optical path, specifically:
  • the first beam combining laser beam After the first beam combining laser beam is transmitted through the secondary sub-polarization beam combining prism, it is combined with the second beam combining laser beam reflected by the secondary sub-polarization beam combining prism, and then passes through the first secondary sub-polarization beam combining prism together.
  • the lens forms 8 sub-scanning areas on the rear focal plane of the first and secondary sub-lenses, and the 8 sub-scanning areas are formed by splicing the 4 sub-scanning areas of the first beam combining laser and the second beam combining laser respectively
  • the two-line scanning area is evenly divided, and the four sub-scanning areas in each line of scanning area are adjacent to each other in sequence, and the distance between the two-line scanning area is one line of scanning area;
  • the third beam combining laser and the fourth beam combining laser are reflected by the secondary polarization beam combining prism after being combined by the second secondary beam combining splicing sub-optical path, and then transmitted through the secondary polarization beam combining prism
  • the combined beams of laser beams are combined, and finally pass through the secondary lens together to form a complete rectangular scanning field in a 4 ⁇ 4 array formed by 16 sub-scanning areas on the rear focal plane of the secondary lens.
  • the photomultiplier tube detection array is an array composed of 4 ⁇ 4 photomultiplier tubes, and each photomultiplier tube corresponds to the fluorescence detection of the imaging field of view of one scanning module.
  • the present invention realizes a large field of view of an elliptical curved surface through the reflective objective lens design, and can convert a flat rectangular laser scanning field into an elliptical hemispherical laser scanning field, thereby adapting to the curved shape of the animal brain cortex.
  • the present invention adopts multi-channel laser parallel scanning and multi-channel fluorescence channel parallel detection to realize high-throughput scanning.
  • the four-color principle is used to combine 16 sub-fields of 4X4 arrays Divided into 4 groups, the boundaries of the 4 sub-regions of the same group are not adjacent.
  • This method maximizes the energy transmittance of multiple polarization beam combining, and the efficiency of each beam combining is better than 90%, instead of reducing the laser energy by half every time it passes through the depolarization beam combining prism in the traditional method.
  • the beam combining and splicing between different groups adopts the traditional beam combining method.
  • each beam combining process by adjusting the incident angle to the polarizing beam splitter or depolarizing beam splitting prism, the relative position between the scanning sub-views is controlled, so as to achieve the designed position distribution effect after splicing.
  • the laser beams corresponding to the 4 sub-regions of the same group are at the same time point in time, and the laser beams of different groups are different in time, and the time delay between two adjacent groups is 3.125ns. It is equivalent to dividing the 12.5ns pulse period into 4 parts, and each group of lasers is at one of the time points.
  • the photomultiplier tube detection array is an array composed of 4 ⁇ 4 photomultiplier tubes, and each array element photomultiplier tube corresponds to the fluorescence detection of the imaging field of view of one scanning unit. Due to the scattering of fluorescence, when the laser scans to the boundary of the field of view, the generated fluorescence not only enters the detection array element, but also has a certain probability of falling into the adjacent detection array elements. Due to the difference in time delay between scanning lasers in adjacent areas, the resulting fluorescence also has the same time difference. Therefore, the source area of fluorescence can be distinguished in time, thereby reducing the signal crosstalk caused by fluorescence scattering.
  • the present invention can effectively distinguish the source area of the fluorescent signal in time and space by making the area of the scanning area not adjacent at the same time, and the adjacent area is not at the same time, and greatly avoids the inter-area signal crosstalk.
  • the large field of view high-throughput two-photon microscope with an elliptical hemispherical surface realizes the large field of view of an elliptical curved surface through the design of a reflective objective lens, and can convert a flat rectangular laser scanning field into a laser scanning field of an elliptical hemispherical surface, thereby adapting to Match the curved shape of the animal brain cortex;
  • the present invention adopts multi-channel laser parallel scanning and multi-channel fluorescent parallel detection to realize high-throughput scanning, and uses the four-color principle to divide non-adjacent areas into a group in the process of splicing and combining multiple sub-fields of view. , using the area gap and combined half-wave plate to maximize the energy efficiency of multiple polarization beam combining; and without introducing delay between groups of lasers, through the scheme that the areas of simultaneous points are not adjacent and the adjacent areas are not at the same time, in The source area of the fluorescent signal can be effectively distinguished in time and space, so that the signal crosstalk between the areas caused by the fluorescence scattering can be largely avoided.
  • FIG. 1 is a schematic diagram of the general principle structure of the large-field-of-view high-throughput two-photon microscope with an ellipsoidal curved surface in an embodiment of the present invention.
  • Figure 2 is an optical path diagram of the beam splitting delay module.
  • FIG. 3 is a schematic diagram of the time distribution of each laser beam on the time axis after passing through the delayed optical path.
  • FIG. 4 is a schematic diagram of the distribution of the scanning field of view after the scanning and beam combining and splicing of each laser beam.
  • Fig. 5 is an optical path diagram of the primary beam combining and splicing module.
  • Fig. 6 is an optical path diagram of the secondary beam combining and splicing module.
  • FIG. 7 is a schematic diagram of the principle of an ellipsoidal curved large-field reflective objective lens.
  • Fig. 8 is a schematic diagram of the distribution of fluorescence excited by each laser at the photomultiplier tube detection array.
  • Beam splitting delay module group 2.1 ⁇ 2.4—Beam splitting delay module; 2.11—First mirror; 2.12—Second mirror; 2.13—First light splitting element; 2.14—Second light splitting element; 2.15— the third light splitting element;
  • A, B, C, D laser
  • A1 first laser
  • A2 second laser
  • A3 third laser
  • A4 fourth laser
  • an ellipsoid curved surface large-field-of-view high-throughput two-photon microscope in this embodiment includes: near-infrared femtosecond pulsed laser group 1, beam splitting delay module group 2, scanning unit group 3, combination Beam splicing module 4, ellipsoidal curved surface large field of view reflective objective lens 5, dichroic film 6 and photomultiplier tube detection array 7;
  • the laser light emitted by the near-infrared femtosecond pulsed laser group 1 passes through the beam splitting and delay module group 2, the scanning unit group 3, and the beam combining and splicing module 4 in sequence, and then transmits the dichroic film 6, and then is reflected by the ellipsoidal surface with a large field of view
  • the type objective lens 5 is irradiated onto the sample, and the fluorescence generated by the sample is collected by the ellipsoidal curved surface large field of view reflection type objective lens 5, and then reflected by the dichroic film 6 to the photomultiplier tube detection array 7.
  • the near-infrared femtosecond pulsed laser group 1 includes 4 near-infrared femtosecond pulsed lasers
  • the beam splitting delay module group 2 includes 4 beam splitting delay modules, and the beam splitting delay module includes a delay optical path and a split optical path;
  • Scanning unit group 3 includes 4 scanning units, each scanning unit includes 4 independent scanning modules, and each scanning module independently realizes two-dimensional scanning of a rectangular sub-scanning area;
  • Each near-infrared femtosecond pulsed laser corresponds to a beam splitting delay module, and each beam splitting delay module corresponds to a scanning unit;
  • the lasers emitted by 4 near-infrared femtosecond pulsed lasers go through the delay optical path to form 4 delay lasers with a time interval of T/4 in turn.
  • Each delay laser is divided into 4 sub-lasers after passing through the beam splitting optical path.
  • 4 sub-lasers from the same delay laser enter the same scanning unit, and each sub-laser corresponds to a scanning module to realize the scanning of a sub-scanning area, so that 16 sub-lasers are in one-to-one correspondence with 16 scanning modules.
  • T is the pulse period of the laser emitted by the near-infrared femtosecond pulse laser; the sub-scanning areas scanned by each scanning module in the same scanning unit are at the same time point, different scanning The sub-scanning areas scanned by the scanning module in the unit are located at different time points;
  • the beam combining and splicing module 4 is used to realize the beam combining and splicing of 16 sub-lasers emitted by 16 scanning modules.
  • a rectangular scanning field with a 4 ⁇ 4 array distribution is formed, and it is located at the same time point
  • the boundaries of the 4 sub-scanning areas are not adjacent, but any 4 areas with adjacent boundaries are located at different time points in time.
  • near-infrared femtosecond pulsed laser group 11 comprises four near-infrared femtosecond pulsed lasers 1.1, 1.2, 1.3 and 1.4, and their models are consistent, and emit femtosecond laser A, B, respectively.
  • C and D enter their respective beam splitting delay modules 2.1-2.4.
  • the beam splitting delay module group 2 includes 4 beam splitting delay modules 2.1 to 2.4, and the beam splitting delay module includes a delay optical path and a beam splitting optical path, as shown in Figure 2 (only 2.1, 2.2 are shown ⁇ 2.4 same);
  • Delay optical path comprises the first reflector 2.11 and the second reflector 2.12, and the laser A that the near-infrared femtosecond pulse laser sends is formed delay laser after being reflected by the first reflector 2.11 and the second reflector 2.12 successively Output, by adjusting the distance L between the first reflector 2.11 and the second reflector 2.12 so that the output delay laser produces a different delay amount L/c, where c is the speed of light;
  • the beam-splitting optical path includes a first light-splitting element 2.13, a second light-splitting element 2.14, and a third light-splitting element 2.15.
  • the delayed laser output from the second reflector 2.12 enters the first light-splitting element 2.13 and is divided into two paths, and one path transmits the first light-splitting element. After the element 2.13 reaches the second light-splitting element 2.14, it is equally divided into two paths by the second light-splitting element 2.14, and the other path is reflected by the first light-splitting element 2.13 and reaches the third light-splitting element 2.15, and is equally divided into two paths by the third light-splitting element 2.15. Two channels, so that the delay laser is divided into four channels.
  • the first light splitting element 2.13, the second light splitting element 2.14 and the third light splitting element 2.15 are all depolarizing light splitting prisms or 50/50 light splitting plates.
  • the repetition frequency of the femtosecond laser pulse is generally 80 MHz, that is, the period is 12.5 ns.
  • the four groups of laser pulses A to D are separated in time, and the time delays between two adjacent channels are separated by 3.125 ns, as shown in Figure 3 , which is equivalent to dividing the 12.5ns pulse period into four equal parts, and each group of lasers is at one of the time points.
  • each laser is equally divided into 4 channels, so a total of 16 laser channels are divided.
  • Each laser beam further enters the parallel scanning unit group 33 and is incident on the corresponding scanning unit.
  • A1-A4 lasers correspond to scanning units 3.11-3.14
  • B1-B4 lasers correspond to scanning units 3.21-3.24
  • C1-C4 lasers correspond to scanning units 3.31-3.34
  • D1-D4 lasers correspond to scanning units 3.41-3.44.
  • Each scanning module 3.11-3.14 includes a fast-axis 8kHz resonant scanning mirror and a slow-axis galvanometer galvanometer to realize two-dimensional fast scanning of each individual sub-region, and the scanning field of view of each sub-region is rectangular.
  • the 16 sub-area scanning fields of view need to be combined and spliced by the beam combining and splicing module 4 to form a complete scanning field before entering the objective lens 5, as shown in Figure 4, which is a 4 ⁇ 4 scanning field of view array.
  • the arrangement characteristics of the sub-areas formed by laser scanning of each channel are: the boundaries of the sub-areas of the same group of lasers are not adjacent, such as A1-A4 are not adjacent; the sub-areas with adjacent boundaries come from different groups of lasers, such as A1 , B1, C1, and D1 are adjacent to each other and come from four groups of lasers; detailed description will be given below.
  • the beam combining and splicing module 4 includes four primary beam combining and splicing modules 4.1-4.4 and one secondary beam combining and splicing module 4.5, and each primary beam combining and splicing module corresponds to a scanning unit respectively, so that the beam combining and splicing modules in one scanning unit The 4 sub-lasers emitted by the 4 scanning modules are combined and spliced;
  • the secondary beam combining and splicing module 4.5 is used to combine and splice the 4 sets of lasers emitted by the 4 primary beam combining and splicing modules again, and then input them to the dichroic film 6 .
  • the 16 scanning branches perform beam combining and field of view stitching through the beam combining and stitching module 44 to finally form a scan field as shown in FIG. 4 .
  • the four scanning branches of the same group of laser beams are first combined and spliced through the first-level beam combining and splicing module. 4.3 and 4.4 perform bundle splicing.
  • the following takes the first-level beam combining and splicing module 4.1 as an example to describe how to perform beam combining and splicing, and 4.2 to 4.4 are the same.
  • the first-level beam combining and splicing module includes two first-level beam combining and splicing sub-optical paths 4.1.01 and 4.1.02, a first-level polarization beam combining prism 4.1.13, a first-level lens 4.1.14, and a first-level combining Formula half-wave plate 4.1.15 and the second-level lens 4.1.16, the laser light emitted by the two first-level beam combining splicing sub-optical paths 4.10 passes through the first level after being combined by the first-level polarization beam combining prism 4.1.13
  • the lens 4.1.14, the first-level combined half-wave plate 4.1.15 and the second-level lens 4.1.16 are output, and the first-level combined half-wave plate 4.1.15 includes 4 different half-wave plates;
  • the two first-level beam combining sub-optical paths 4.1.01 and 4.1.02 have the same structure, including the first-level sub-half-wave plate 4.1.1, the second-level sub-half-wave plate 4.1.2, and the first-level sub-polarization combining Beam prism 4.1.3, first-level sub-lens 4.1.4, first-level sub-combined half-wave plate 4.1.5 and second-level sub-lens 4.1.6, first-level sub-combined half-wave plate 4.1.5 includes Two different half-wave plates, two first-level beam combining and splicing sub-optical paths are respectively recorded as the first-level beam combining and splicing sub-optical path 4.1.01 and the second-level beam combining and splicing sub-optical path 4.1.02;
  • the four sub-lasers emitted by the four scanning modules 3.11 to 3.14 in the same scanning unit are respectively recorded as: the first sub-laser A1, the second sub-laser A2, the third sub-laser A3, and the fourth sub-laser A4,
  • the first-pass laser A1 and the second-pass laser A2 pass through the first-stage polarization beam-combining prism 4.1.13 after being combined by the first-stage beam combining and splicing sub-optical path 4.1.01, specifically:
  • the first sub-laser A1 passes through the first sub-half-wave plate 4.1.1 and transmits the sub-polarization beam combining prism 4.1.3
  • the second sub-laser A2 passes through the second sub-half-wave plate 4.1.2 and is transmitted by a sub-polarization beam combining prism 4.1.3
  • the first-level sub-polarization beam combining prism 4.1.3 reflects, then combines with the first-pass laser beam A1 that transmits the first-level sub-polarization beam combining prism 4.1.3, and then passes through the first-level sub-lens 4.1.4 together, in the first one
  • Two different half-wave plates of the first-level sub-merging half-wave plate 4.1.5 on the rear focal plane of the first-level sub-lens 4.1.4 form 2 sub-scanning areas, and the interval between the 2 sub-scanning areas is one sub-scanning area Distance; adjust the first-level sub-half-wave plate 4.1.1 and the second-level sub-half-wave plate 4.1.2 to maximize the beam combining efficiency of the
  • the two sub-lasers pass through the second-level sub-lens 4.1.6 and then become collimated lasers, and then pass through the first-level polarization beam combining prism 4.1.13, wherein, after passing through the first-level sub-combining half-wave plate 4.1. After 5, the polarization states of the two lasers are the same;
  • the polarization states of A1 and A2 after polarization combining are different, and they are exactly perpendicular to each other. After subsequent polarization combining, they will separate again and lose the beam combining effect. Choose suitable different directions through the crystal orientation of the two half-wave plates glued together in 4.1.5 of the first-stage combined half-wave plate, so that the laser polarization states in A1 and A2 areas become consistent after passing through 4.1.5, and exactly Adjust to the best polarization state required for the next polarization beam combining.
  • the third-pass sub-laser A3 and the fourth-pass sub-laser A4 are combined through the second-level beam-combining and splicing sub-optical path 4.1.02, and then reflected by the first-level polarization beam combining prism 4.1.13, so that A3, A4 and the transmitted one
  • the beam combining laser beams A1 and A2 of the first-order polarization beam combining prism 4.1.13 combine together, then pass through the first-order lens 4.1.14 together, and the first-order combined semi-polarization on the rear focal plane of the first-order lens 4.1.14
  • Four different half-wave plates of the wave plate 4.1.15 form four sub-scanning areas, and the distance between the four sub-scanning areas is one sub-scanning area, and finally pass through the second-level lens 4.1.16 to return to the quasi-scanning area.
  • the straight laser light is output to the secondary beam combining and splicing module 4.5, wherein the polarization states of
  • the efficiency of beam combining is maximized through the first-stage combined half-wave plates 4.1.5 and 4.1.11.
  • the scanning field of view of the four scanning sub-areas is within the first-stage lens 4.1.
  • the mutual position on the back focal plane of 14 is a designed suitable position.
  • the boundaries of these four sub-fields are still non-adjacent, so they can also completely fall on four different half-wave plates.
  • Four half-wave plates are glued together to form a one-stage combined half-wave plate 4.1.15.
  • the polarization states of the four subviews after passing through 4.1.15 are the same, which is the best polarization state required for the next polarization beam combining.
  • the laser light is changed back to collimated laser light through the second-level lens 4.1.16.
  • A1-A4 have completed the beam combining and field of view stitching through the first-level beam combining and stitching module.
  • the beam combining methods of groups B, C, and D are the same and will not be described again.
  • the following four groups of laser beams A, B, C, and D are combined and spliced through the secondary beam combining and splicing module 4.5.
  • the secondary beam combining and splicing module 4.5 includes 2 secondary beam combining and splicing sub-optical paths 4.5.01 and 4.5.02, the secondary polarizing beam combining prism 4.5.7 and the secondary lens 4.5.9, and 2 secondary beam combining and splicing sub-optical paths
  • the laser light emitted from the optical path 4.5.0 is combined by the secondary polarization beam combining prism 4.5.7, then passes through the secondary lens 4.5.9, and then enters the dichroic film 6;
  • the two secondary beam combining sub-optical paths 4.5.01 and 4.5.02 have the same structure, and both include the secondary sub-polarizing beam combining prism 4.5.1, the first secondary sub-lens 4.5.2 and the second secondary sub-lens 4.5. 3.
  • the two secondary beam combining and splicing sub-optical paths 4.5.0 are respectively recorded as the first secondary beam combining and splicing sub-optical path 4.5.01 and the second secondary beam combining and splicing sub-optical path 4.5.02;
  • the beam combining lasers of the 4 sub-lasers emitted by the 4 first-level beam combining and splicing modules 4.1 ⁇ 4.4 are respectively recorded as: the first beam combining laser A, the second beam combining laser B, the third beam combining laser C, and the fourth beam combining laser Luzi combined beam laser D;
  • the beam combining laser A of the first path and the beam combining laser B of the second path pass through the secondary polarization beam combining prism 4.5.7 after being combined by the first secondary beam combining splicing sub-optical path 4.5.01, specifically:
  • the first beam combining laser A transmits the secondary sub-polarization beam combining prism 4.5.1, it combines with the second beam combining laser B reflected by the secondary sub-polarization beam combining prism 4.5.1, and then passes through the first and second beams together.
  • the first-level sub-lens 4.5.2 forms 8 sub-scanning areas on the rear focal plane of the first-level sub-lens 4.5.2, and the 8 sub-scanning areas are respectively composed of the first beam combining laser A and the second beam combining laser B
  • the 4 sub-scanning areas of the 4 sub-scanning areas are spliced to form an evenly divided two-line scanning area, and the 4 sub-scanning areas in each line of scanning area are adjacent in turn, and the distance between the two-line scanning area is the distance of one line of scanning area;
  • the third beam combining laser C and the fourth beam combining laser D are combined by the second secondary beam combining sub-optical path 4.5.02 and then reflected by the secondary polarization beam combining prism 4.5.7, and then combined with the transmitted secondary polarization
  • the combined laser beams of the beam prism 4.5.7 are finally combined through the secondary lens 4.5.9 to form a complete 4 ⁇ 4 array distribution formed by 16 sub-scanning areas on the rear focal plane of the secondary lens 4.5.9 Rectangular scanning field, as shown in Figure 4.
  • the laser beams of group A and group B are combined through the secondary polarization beam combining prism 4.5.7.
  • the respective polarization states have been adjusted to the best through the combined half-wave plate, ensuring The combining efficiency here.
  • the combination and splicing of group C and D lasers is the same.
  • the secondary beam combining and splicing module 4.5 further includes a baffle 4.5.8, and the baffle 4.5.8 is used to block half of the laser light lost by depolarization and beam combining.
  • the baffle plate 4.5.8 can also be replaced with a camera, which is used to monitor the current splicing situation of each field of view in real time. If the position of the field of view in a certain sub-area deviates, the maintenance personnel can find out in time and carry out Adjustment.
  • the secondary polarization beam combining prism 4.5.7 can also be replaced by a 50/50 beam splitting plate.
  • the dichroic film 6 can efficiently transmit the laser waveband, and reflect the visible fluorescent waveband, with high reflectivity.
  • the laser light after scanning field of view will pass through the dichroic film 6 and then reach the reflective objective lens to form an excitation scanning field in the sample.
  • the excited fluorescence in the sample reaches the dichroic film 6 through the reflective objective lens and is reflected to the photomultiplier tube
  • the detection array performs photoelectric signal conversion.
  • planar scanning field ( FIG. 4 ) spliced by multiple scanning sub-regions is converted into a curved scanning field of view of an elliptical hemisphere through an ellipsoidal curved large-field reflective objective lens 55 .
  • the ellipsoidal curved large field of view reflective objective lens 5 comprises a primary mirror 5.1, a secondary mirror 5.2 and a third mirror 5.3 arranged in sequence along the optical path, the optical surface of the primary mirror 5.1 is an eighth-order hyperboloid, and the optical surface of the secondary mirror 5.2 It is a quadratic flat ellipse, and the optical surface of the third mirror 5.3 is a sixth-order flat ellipse.
  • the laser light entering the ellipsoidal curved surface wide field of view reflective objective lens 5 is reflected by the primary mirror 5.1, the secondary mirror 5.2, and the third mirror 5.3 in turn, and then irradiates the sample. superior.
  • the range of the numerical aperture of the ellipsoidal curved large field of view reflective objective lens 5 is 0.3-0.5.
  • the imaging field of view of the reflective objective lens is an ellipsoidal surface, the radius of curvature of the major axis is 9-12mm, the radius of curvature of the minor axis is 6-9mm, and the plane projection size of the field of view is 6mm ⁇ 6mm.
  • AA1 ⁇ AA4 are the fluorescence excited by A1 ⁇ A4 lasers respectively
  • BB1 ⁇ BB4 are the fluorescences excited by B1 ⁇ B4 lasers respectively
  • CC1 ⁇ CC4 are the fluorescences excited by C1 ⁇ C4 lasers respectively
  • DD1 ⁇ DD4 are the fluorescences excited by D1 ⁇ D4 lasers respectively fluorescence.
  • the photomultiplier tube detection array 7 is an array composed of 4 ⁇ 4 photomultiplier tubes, each element photomultiplier tube corresponds to the fluorescence detection of the imaging field of view of one scanning unit, as shown in FIG. 8 . After the fluorescence from the curved field of view is converted by the scanning field of the reflective objective lens 5, the distribution of the fluorescence in the detection array 7 is changed back to a plane matrix.
  • the generated fluorescence not only enters the detection array element, but also has a certain probability of falling into the adjacent detection array elements. Due to the difference in time delay between scanning lasers in adjacent areas, the resulting fluorescence also has the same time difference. Therefore, the source area of fluorescence can be distinguished in time, thereby reducing the signal crosstalk caused by fluorescence scattering.
  • the present invention can effectively distinguish the source area of the fluorescent signal in time and space through the idea that the areas of simultaneous points are not adjacent, and the adjacent areas are not at the same time, and the signal crosstalk between areas is largely avoided. .
  • the embodiment of the present invention describes the scanning and detection method of the 16-way 4 ⁇ 4 array, and according to the four-color principle, for any number of areas distributed in the plane, these areas can always be divided into four types, and the areas of the same type are different. Adjacent, the energy transmittance of combined beams can be improved through the unique beam combination and splicing method provided by the present invention, and the crosstalk of fluorescent signals can be reduced through the space-time distinction method provided by the present invention, so the implementation of the present invention can be extended to any number of parallel Scanning and probing.

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