WO2020215802A1 - 一种用于病理检查的光学成像系统 - Google Patents

一种用于病理检查的光学成像系统 Download PDF

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WO2020215802A1
WO2020215802A1 PCT/CN2019/130607 CN2019130607W WO2020215802A1 WO 2020215802 A1 WO2020215802 A1 WO 2020215802A1 CN 2019130607 W CN2019130607 W CN 2019130607W WO 2020215802 A1 WO2020215802 A1 WO 2020215802A1
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pulse laser
sample
nlm
ultrashort pulse
imaging system
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李慧
余佳
夏先园
陈廷爱
郑炜
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Shenzhen Institute of Advanced Technology of CAS
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Shenzhen Institute of Advanced Technology of CAS
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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
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/84—Systems specially adapted for particular applications

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  • This application relates to the technical field of medical equipment, and in particular to an optical imaging system for pathological examinations.
  • Intraoperative evaluation of tumor resection boundary is of great significance for optimizing the surgical plan, implementing radical resection and avoiding reoperation, and determines the prognosis and recurrence rate of the tumor. Histopathological examination is the gold standard for evaluating tumor resection boundaries.
  • this technique involves complicated sample processing steps, including fixation, dehydration, embedding, sectioning, staining, etc., which usually takes 3-5 days, so it cannot be used intraoperatively.
  • the rapid frozen section technology is based on the rapid freezing of the surgically resected tissue to achieve section processing, avoiding time-consuming operations such as fixation and embedding, and generally only takes 15-30 minutes to complete.
  • the sensitivity, specificity, and detection range of the rapid frozen section technique are very limited, and its ability to assess surgical boundaries is not good.
  • some tissues with special composition such as breast tissue with fat as the main component, are not easy to freeze, which further limits the application range of rapid cryosection technology.
  • the purpose of this application is to provide an optical imaging system for pathological examinations, which can increase the speed of NLM (Nonlinear Optical Microscopy, nonlinear optical microscopy) on samples, and use fast optical slice imaging to avoid complex tissue imaging. Processing and physical sectioning to increase the speed of pathological examination.
  • NLM Networkar Optical Microscopy, nonlinear optical microscopy
  • the embodiment of the application provides an optical imaging system for pathological examination, including a pulsed laser output device, a dispersive component, and an NLM signal excitation and detection device;
  • the pulse laser output device generates a first ultrashort pulse laser, and irradiates the first ultrashort pulse laser to the dispersive component;
  • the dispersion component spatially separates the different monochromatic light components contained in the first ultrashort pulse laser, and disperses into multiple second ultrashort pulse lasers;
  • the NLM signal excitation and detection device irradiates a sample with the multiple second ultrashort pulse lasers to excite the sample to generate an NLM signal, and reconstruct an NLM image of the sample according to the NLM signal.
  • the dispersive component is a diffraction grating
  • the diffraction grating disperses the first ultrashort pulse laser light into the multiple second ultrashort pulse laser light through diffraction.
  • the pulse laser output device includes an ultrashort pulse laser and a power regulator
  • the ultrashort pulse laser generates the first ultrashort pulse laser
  • the power regulator After the power regulator attenuates the power of the first ultrashort pulse laser, it irradiates the dispersive component.
  • the pulse laser output device further includes a reflector
  • the reflector reflects the attenuated first ultrashort pulse laser to the dispersive component.
  • the pulse laser output device further includes a beam expander
  • the beam expander expands the first ultrashort pulse laser whose power is attenuated, and then irradiates the dispersion component.
  • the NLM signal excitation and detection device includes a tube lens, a dichroic lens, an objective lens, an imaging lens, a first photosensitive element, and a computer control processing unit;
  • the objective lens After the multiple second ultrashort pulse lasers pass through the tube lens and the dichroic mirror, the objective lens focuses and illuminates the sample to excite the sample to generate the NLM signal;
  • the NLM signal passes through the objective lens, it is reflected by the dichroic mirror and transmitted through the imaging lens, and then collected by the first photosensitive element and converted into a first electrical signal;
  • the computer-controlled processing unit is electrically connected to the first photosensitive element, and receives the first electrical signal output by the first photosensitive element and converted from the NLM signal, and reconstructs based on the first electrical signal The NLM image of the sample.
  • the NLM signal excitation and detection device further includes an axial drive device
  • the axial drive device drives the objective lens closer to or away from the sample.
  • the NLM signal excitation and detection device further includes a translation stage
  • the translation stage carries the sample, and selectively drives the sample to move horizontally with the objective lens.
  • the first photosensitive element is a color CCD.
  • the NLM signal excitation and detection device further includes a first reflector, a white light source, and a second photosensitive element;
  • the white light source starts to work to generate white light to illuminate the sample
  • the sample reflects the white light, and the white light reflected by the sample passes through the objective lens and is collected by the second photosensitive element and converted into a second electrical signal after being reflected by the first reflector;
  • the computer-controlled processing unit receives the second electrical signal output by the second photosensitive element, and reconstructs a white light image of the sample based on the second electrical signal;
  • the first mirror is not located in the optical path where the first ultrashort pulse laser or the multiple second ultrashort pulse lasers are located, and the white light source does not work.
  • an optical imaging system for pathological examinations generates a first ultrashort pulse laser by a pulse laser output device, and irradiates the first ultrashort pulse laser on the dispersive component.
  • the dispersive component separates the different monochromatic light components of the first ultrashort pulse laser spatially, and disperses into multiple second ultrashort pulse lasers, and the NLM signal excitation and detection device performs the multiple second ultrashort pulse laser After converging, illuminate the sample, and cooperate with the dispersive component to form a time-domain focused wide-field illumination, which increases the area where the sample is excited to generate NLM signals, which can increase the speed of NLM imaging of the sample, and use fast optical slice imaging to avoid The tissue undergoes complex processing and physical sectioning to increase the speed of pathological examinations.
  • Fig. 1 shows a schematic structural diagram of an optical imaging system provided by an embodiment of the present application
  • Fig. 2 shows another schematic structural diagram of an optical imaging system provided by an embodiment of the present application.
  • 10-optical imaging system 20-sample; 100-pulse laser output device; 110-ultra-short pulse laser; 120-power regulator; 130-reflector; 131-second mirror; 132-third reflection Mirror; 140-beam expander; 141-first beam expander; 142-second beam expander; 200-dispersion component; 300-NLM signal excitation and detection device; 310-tube mirror; 320-dichroic mirror; 330-objective lens; 340-imaging lens; 350-first photosensitive element; 360-computer control processing unit; 370-axial drive device; 380-translation stage; 390-white light source; 391-first mirror; 392 -The second photosensitive element.
  • Optical slice tomography imaging technology can avoid physical slices of tissues, simplify sample processing steps, and have great application potential in the intraoperative evaluation of tumor resection boundaries.
  • OCT Optical Coherence Tomography, Optical Coherence Tomography
  • SRS Stimulated Raman Scattering, Stimulated Raman Scattering
  • other technologies that rely on endogenous contrast imaging have a certain structure and conventional pathological examination results. The gap cannot be directly interpreted by pathologists, so it is difficult to be widely accepted.
  • SIM Structure Illumination Microscopy
  • LSM Light-Sheet Microscopy
  • MUSE Mesospect with Ultraviolet Surface Excitation, UV surface excitation
  • CFM Confocal Fluorescence Microscopy, confocal fluorescence microscopy
  • NLM technology has significant advantages such as superior tissue penetration, inherent tomographic capability, low photobleaching and phototoxicity, but it also has the disadvantage of slow imaging speed due to the same point scanning imaging mode.
  • FIG. 1 shows a schematic structural diagram of an optical imaging system 10 provided by an embodiment of the present application.
  • the optical imaging system 10 can be used for pathological examinations to increase the speed of pathological examinations.
  • the optical imaging system 10 includes a pulsed laser output device 100, a dispersive component 200, and an NLM signal excitation and detection device 300.
  • the pulsed laser output device 100 when the optical imaging system 10 is working, the pulsed laser output device 100 generates the first ultrashort pulse laser, and irradiates the first ultrashort pulse laser to the dispersive component 200, and the dispersive component 200 contains the first ultrashort pulse laser.
  • the different monochromatic light components are separated in space, and the dispersion is divided into multiple second ultrashort pulse lasers, and the NLM signal excitation and detection device 300 irradiates the sample 20 with multiple second ultrashort pulse lasers to excite the sample
  • the dispersion component 200 is used to disperse a first ultrashort pulse laser to obtain multiple second ultrashort pulse lasers
  • the mode of illuminating a region of the sample 20 for wide-field imaging after time-domain focusing replaces the mode of spot scanning imaging by illuminating a point of the sample 20 after spatial focusing by a first ultrashort pulse laser, which improves the NLM imaging of the sample 20 speed.
  • the dispersive component 200 may adopt a diffraction grating.
  • the diffraction grating disperses the first ultrashort pulse laser into multiple second ultrashort pulses through diffraction. Short pulse laser.
  • the dispersive component 200 may also adopt some other structures or devices besides diffraction gratings.
  • a dispersive prism may also be used as the dispersive component 200, as long as it is used as The structure or equipment of the dispersion component 200 can disperse the first ultrashort pulse laser into multiple second ultrashort pulse lasers.
  • the pulse laser output device 100 includes an ultrashort pulse laser 110 and a power regulator 120.
  • the ultrashort pulse laser 110 generates the first ultrashort pulse laser, and after the power of the first ultrashort pulse laser is attenuated by the power regulator 120, it irradiates the dispersive component 200 so that the first ultrashort pulse laser of the dispersive component 200 is incident
  • the optical power complies with laser safety application standards, such as ANSI (AMERICAN NATIONAL STANDARDS INSTITUTE, American National Standards Institute) standards, while ensuring that the optical power irradiated on the surface of the sample 20 will not cause light damage or phototoxicity to the sample 20.
  • the ultrashort pulse laser 110 can be an ultrashort pulse laser with a high repetition rate, such as a Ti:Sapphire femtosecond laser with a repetition rate of 80MHz, or other high-repetition rate lasers. Femtosecond, picosecond lasers, or other lasers that can be used for sample 20 two-photon excitation fluorescence imaging and second harmonic imaging.
  • the ultrashort pulse laser 110 that generates the first ultrashort pulse laser may be wavelength tunable, such as the aforementioned Ti:Sapphire femtosecond laser covering the 680-1020nm band and a repetition frequency of 80MHz, or a single wavelength, such as A specific combination of fluorescent dyes, acridine orange and sulforhodamine 101, select ultra-short pulse lasers with wavelengths of 780nm or 1040nm as the light source of the optical imaging system 10 for NLM imaging, and obtain similar to H&E (Hematoxylin-Eosin, The histological image of hematoxylin-eosin) stained sections can reduce the cost compared with the wavelength tunable femtosecond pulse laser commonly used in NLM imaging.
  • wavelength tunable femtosecond laser such as the aforementioned Ti:Sapphire femtosecond laser covering the 680-1020nm band and a repetition frequency of 80MHz
  • a single wavelength such as A
  • the pulse laser output device 100 further includes a reflector 130 that reflects the first ultrashort pulse laser attenuated by the power regulator 120 to the dispersion component 200, thereby making
  • the positions of the ultrashort pulse laser 110 and the power regulator 120 need not be fixed, but can be coordinated according to the specific architectural design requirements of the optical imaging system 10.
  • the reflector 130 can be composed of a second reflector 131 and a third reflector 132, and the second reflector 131 and the third reflector 132 can attenuate the power.
  • An ultrashort pulse laser is reflected to the dispersion component 200.
  • the reflector 130 in FIG. 1 includes the second reflector 131 and the third reflector 132 for illustration only. In some other possible implementations of the embodiment of the present application, the reflector 130 may also include more or Fewer reflectors, such as one reflector, or three, four or more reflectors, as long as the reflector 130 can reflect the first ultrashort pulse laser to the dispersive component 200.
  • the pulse laser output device 100 further includes a beam expander 140 that expands the first ultrashort pulse laser with attenuated power and irradiates the dispersive component 200. Furthermore, the beam of the first ultrashort pulse laser irradiated on the dispersive component 200 is sufficiently large, so that the dispersive component 200 can sufficiently disperse the first ultrashort pulse laser to obtain multiple second ultrashort pulse lasers.
  • the beam expander 140 may be composed of a first beam expander 141 and a second beam expander 142, and is composed of a first beam expander 141 and a second beam expander 142.
  • the first ultrashort pulse laser beam is expanded and then irradiated to the dispersion component 200.
  • the beam expander 140 in FIG. 1 includes the first beam expander 141 and the second beam expander 142 for illustration only. In some other possible implementations of the embodiment of the present application, the beam expander 140 may also Including more beam expanders, such as four beam expanders, as long as the beam expander 140 can expand the beam of the first ultrashort pulse laser and irradiate the dispersive component 200.
  • the NLM signal excitation and detection device 300 includes a tube lens 310, a dichroic mirror 320, an objective lens 330, an imaging lens 340, a first photosensitive element 350, and a computer Control processing unit 360.
  • the dichroic mirror 320 can transmit the second ultrashort pulse laser light, and cause the NLM signal to be reflected.
  • each second ultrashort pulse laser is adjusted to parallel light, and recombined at the focal plane of the objective lens 330, convergent and irradiated to the sample 20, forming time-domain focused wide-field illumination to excite the sample 20 generates NLM signals, such as two-photon excitation fluorescence signals, second-harmonic signals, etc.; and the NLM signal generated by exciting the sample 20 passes through the objective lens 330 and irradiates on the dichroic mirror 320, and is irradiated by the dichroic mirror 320 After being reflected and transmitted through the imaging lens 340, it is collected by the first photosensitive element 350 and converted into a first electrical signal.
  • NLM signals such as two-photon excitation fluorescence signals, second-harmonic signals, etc.
  • the computer control processing unit 360 electrically connected to the first photosensitive element 350 receives the NLM output from the first photosensitive element 350.
  • the first electrical signal is converted from the signal, and the NLM image of the sample 20 is reconstructed based on the first electrical signal.
  • a single second ultrashort pulse laser illuminates an area of the sample 20 in the form of parallel light for wide-field imaging, which is compared with a single first ultrashort pulse laser
  • spatial focusing only one point of the sample 20 is irradiated for point scan imaging, which increases the speed of NLM imaging of the sample 20, thereby saving time in the imaging process of pathological examinations, and using NLM optical section technology to avoid pathological examinations Perform physical sectioning at the same time, thereby avoiding complicated sample processing before physical sectioning, and improving the speed of pathological examination.
  • the first photosensitive element 350 may adopt a color CCD (Charge-Coupled Device, charge coupled device). Since it is usually necessary to use a variety of dyes to stain and mark different structural components of the sample 20 to obtain a histological image close to the clinical pathological examination, the NLM signal obtained by the first photosensitive element 350 usually contains a variety of fluorescent signals. Using a color CCD, the first photosensitive element 350 can directly obtain a true color image of the sample 20, and the pathologist can distinguish different types of fluorescent signals through color differences, thereby avoiding the use of spectroscopy, filter devices and multiple optical filters in the detection light path of the imaging system. The detector simplifies the system structure.
  • CCD Charge-Coupled Device, charge coupled device
  • the focal plane of the objective lens 330 is conjugated to the dispersive element 200, that is, the dispersive element 200 is placed at the focal plane of the tube lens 310, and the distance between the tube lens 310 and the objective lens 330 is Equal to the focal length of the tube lens 310 plus the focal length of the objective lens 330, so that each second ultra-short pulse laser illuminates the sample 20 in the form of parallel light to form a wide-field illumination, thereby realizing wide-field NLM imaging and improving the performance of the sample 20 The speed of NLM imaging.
  • the objective lens 330 may include multiple objective lens units that can be freely switched and have different magnifications for realizing pathological imaging of multiple scales (ie, multiple resolutions and multiple field of view sizes).
  • the NLM signal excitation and detection device 300 further includes an axial drive device 370, which drives the objective lens 330 to approach or move away from the sample 20 to
  • the multiple second ultrashort pulse lasers are adjusted to converge at different depths of the sample 20, so that when the sample 20 is imaged, the information of the different depths of the sample 20 can be obtained to realize three-dimensional imaging.
  • the axial drive device 370 can be implemented by a stepping motor, and a corresponding control program can be configured in the computer control processing unit 360 to control the operation of the stepping motor, and then bring the animal lens 330 close to Or stay away from sample 20.
  • the NLM signal excitation and detection device 300 further includes a translational stage 380 that carries the sample 20 and selectively drives the sample 20 and the objective lens 330 are horizontally shifted.
  • the translation stage 380 can be realized by an electric stage, and a corresponding control program can be configured in the computer control processing unit 360 to control the operation of the electric stage, and then when needed At this time, the motorized stage can be controlled to move, thereby driving the sample 20 and the objective lens 330 to move horizontally, so that when performing NLM imaging on the sample 20, information of different regions of the sample 20 can be obtained to realize large-area imaging.
  • FIG. 2 shows another schematic structural diagram of the optical imaging system 10 according to an embodiment of the present application.
  • the NLM signal excitation and detection device 300 also It includes a white light source 390, a first reflector 391, and a second photosensitive element 392.
  • the sample 20 when the pulse laser output device 100 is not working, the sample 20 will not be excited by the laser to generate an NLM signal.
  • the white light source 390 starts working to generate white light.
  • the sample 20 is irradiated by the white light and reflects the white light signal.
  • the white light signal passes through the objective lens 330, is reflected by the first mirror 391, and is collected by the second photosensitive element 392 and converted into a second electrical signal.
  • the computer control processing unit 360 electrically connected to the second photosensitive element 392 receives the second electrical signal.
  • the second electrical signal converted from the reflected light signal output by the photosensitive element 392, and based on the second electrical signal, a white light image of the sample 20 is reconstructed to facilitate pathologists to observe the sample 20.
  • an optical imaging system 10 is in white light imaging mode.
  • the second photosensitive element 392 can also be replaced by an eyepiece, that is, the eyepiece is directly used in conjunction with the first reflector 391 and the objective lens 330 to observe the sample 20, without the need for the second
  • the photosensitive element 392 combines with the computer control processing unit 360 to collect white light images in real time for observation.
  • the first mirror 391 when the pulse laser output device 100 is working, the first mirror 391 is not located in the optical path where the first ultrashort pulse laser or multiple second ultrashort pulse lasers are located, so as to avoid the first mirror 391
  • the NLM imaging is blocked and the white light source 390 does not work.
  • the optical imaging system 10 is in the NLM imaging mode.
  • the above-mentioned white light imaging mode can be used to quickly locate the sample 20 before NLM imaging, including the selection and focus adjustment of the region of interest.
  • the doctor can observe the sample 20 before the NLM imaging, and control the axial drive device 370 to drive the objective lens 330 close to or away from the sample 20, so that the sample 20 is in the objective lens At the focal plane of 330; or control the translation stage 380 to move the region of interest in the sample 20 to the center of the field of view.
  • the first mirror 391 can also be fixed to a mechanical device, such as a pull rod, a one-dimensional translation stage, etc., or other mechanical devices used to control the movement of the first mirror 391.
  • the optical imaging system 10 When the optical imaging system 10 is in the white light imaging mode, the first mirror 391 is pushed into the optical path, it cooperates with the objective lens 330 and the second photosensitive element 392 for the doctor to observe the sample 20; and when the optical imaging system 10 is in the NLM imaging mode, it moves The first mirror 391 exits the optical path where the first ultrashort pulse laser or the second ultrashort pulse laser is located, so as to switch between the white light imaging mode and the NLM imaging mode.
  • optical imaging system 10 provided in the embodiment of the present application will be exemplarily described below in conjunction with operational application examples.
  • a low-power objective such as a 5x objective
  • acridine orange and sulforhodamine 101 can be selected here 780nm excitation wavelength, rapid imaging of tissue samples to obtain structural information of tissue samples; according to the imaging results, further determine the area that needs to observe fine structure; then switch the objective lens to a high-power lens, such as a 10x objective lens, for this area
  • a high-power lens such as a 10x objective lens
  • pathologists can comprehensively evaluate the tumor resection boundary and make a diagnosis.
  • the imaging plan can be flexibly adjusted according to actual needs, and three-dimensional imaging or large-volume imaging of the tissue can also be performed to obtain the three-dimensional structure information of the tissue to assist diagnosis. From processing tissue samples to imaging observation, it only takes a few minutes to ten minutes to complete, which greatly improves the speed of pathological examinations. After imaging, the tissue sample can be immediately put into formalin for fixation for subsequent routine pathological examinations.

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Abstract

一种用于病理检查的光学成像系统(10)，涉及医疗设备技术领域，通过由脉冲激光输出装置(100)产生第一超短脉冲激光，并将第一超短脉冲激光照射在色散组件(200)，由色散组件(200)将第一超短脉冲激光包含的不同单色光组分在空间上分离开，色散为多束第二超短脉冲激光，进而由物镜(330)聚焦，在物镜(330)的焦平面重新组合在一起，形成时域聚焦，同时由物镜(330)与色散组件(200)相配合，使每束第二超短脉冲激光都以平行光的形式照射样本(20)，形成宽场照明，增大激发样本(20)产生非线性光学信号的区域，能够提升对样本(20)进行非线性光学显微成像NLM的速度，利用快速的光学切片成像避免对组织进行复杂的处理及物理切片，以提升病理检查的速度。

Description

一种用于病理检查的光学成像系统 技术领域
本申请涉及医疗设备技术领域，具体而言，涉及一种用于病理检查的光学成像系统。
背景技术
肿瘤切除边界的术中评估对于优化手术方案、实施根治性切除以及避免再次手术意义重大，决定着肿瘤的预后和复发几率。组织病理学检查是用于评估肿瘤切除边界的金标准。然而，该技术涉及复杂的样品处理步骤，包括固定、脱水、包埋、切片、染色等，通常耗时3-5天，因而无法用于术中。
在实际的术中，取而代之的是快速冰冻切片技术，该技术基于对手术切除组织的快速冰冻实现切片处理，避免了固定、包埋等耗时操作，一般只需15-30分钟即可完成。然而，相对于常规组织病理学检查，快速冰冻切片技术的灵敏度、特异性，以及检测范围都非常有限，对于手术边界的评估能力欠佳。此外，一些组成特殊的组织，如以脂肪为主要成分的乳腺组织，是不易冻结的，这进一步限制了快速冰冻切片技术的应用范围。
发明内容
本申请的目的在于提供一种用于病理检查的光学成像系统，能够提升对样本进行NLM(Nonlinear Optical Microscopy，非线性光学显微成像)的速度，利用快速的光学切片成像避免对组织进行复杂的处理及物理切片，以提升病理检查的速度。
为了实现上述目的，本申请实施例采用的技术方案如下：
本申请实施例提供一种用于病理检查的光学成像系统，包括脉冲激光输出装置、色散组件及NLM信号激发与探测装置；
所述脉冲激光输出装置产生第一超短脉冲激光，并将所述第一超短脉冲激光照射至所述色散组件；
所述色散组件将所述第一超短脉冲激光包含的不同单色光组分在空间上分离开，色散为多束第二超短脉冲激光；
所述NLM信号激发与探测装置利用所述多束第二超短脉冲激光照射样本，以激发所述样本产生NLM信号，并根据所述NLM信号重建出所述样本的NLM图像。
进一步地，所述色散组件为衍射光栅；
所述衍射光栅通过衍射将所述第一超短脉冲激光色散为所述多束第二超短脉冲激光。
进一步地，所述脉冲激光输出装置包括超短脉冲激光器和功率调节器；
所述超短脉冲激光器产生所述第一超短脉冲激光；
所述功率调节器将所述第一超短脉冲激光的功率衰减后，照射至所述色散组件。
进一步地，所述脉冲激光输出装置还包括反射件；
所述反射件将所述功率衰减的第一超短脉冲激光反射至所述色散组件。
进一步地，所述脉冲激光输出装置还包括扩束件；
所述扩束件将所述功率衰减的第一超短脉冲激光扩束后，照射至所述色散组件。
进一步地，所述NLM信号激发与探测装置包括管镜、二向色镜、物镜、成像透镜、第一感光元件及计算机控制处理单元；
所述多束第二超短脉冲激光透过所述管镜及所述二向色镜后，由所述物镜聚焦照射所述样本，以激发所述样本产生所述NLM信号；
所述NLM信号透过所述物镜后，由所述二向色镜反射并透过所述成像透镜后被所述第一感光元件采集并转化为第一电信号；
所述计算机控制处理单元与所述第一感光元件电连接，且接收所述第一感光元件输出的由所述NLM信号转化而来的第一电信号，并基于所述第一电信号重建出所述样本的所述NLM图像。
进一步地，所述NLM信号激发与探测装置还包括轴向驱动设备；
所述轴向驱动设备驱动所述物镜靠近或远离所述样本。
进一步地，所述NLM信号激发与探测装置还包括平移载物台；
所述平移载物台承载所述样本，并选择性地带动所述样本与所述物镜水平错动。
进一步地，所述第一感光元件为彩色CCD。
进一步地，所述NLM信号激发与探测装置还包括第一反射镜、白光光源及第二感光元件；
在所述脉冲激光输出装置不工作时，所述白光光源启动工作，产生白光，以照亮所述样本；
所述样本反射所述白光，所述经样本反射的白光透过所述物镜，并经所述第一反射镜反射后被所述第二感光元件采集并转化为第二电信号；
所述计算机控制处理单元接收所述第二感光元件输出的所述第二电信号，并基于所述第二电信号，重建出所述样本的白光图像；
在所述脉冲激光输出装置启动工作时，所述第一反射镜不位于所述第一超短脉冲激光或所述多束第二超短脉冲激光所在的光路，且所述白光光源不工作。
相对于现有技术，本申请实施例提供的一种用于病理检查的光学成像系统，由脉冲激光输出装置产生第一超短脉冲激光，并将第一超短脉冲激光照射在色 散组件，由色散组件将第一超短脉冲激光的不同单色光组分在空间上分离开，色散为多束第二超短脉冲激光，进而由NLM信号激发与探测装置对多束第二超短脉冲激光进行会聚后照射样本，并通过与色散组件相配合，形成时域聚焦宽场照明，增大激发样本产生NLM信号的区域，能够提升对样本进行NLM成像的速度，利用快速的光学切片成像避免对组织进行复杂的处理及物理切片，以提升病理检查的速度。
附图说明
图1示出了本申请实施例所提供的光学成像系统的一种示意性结构图；
图2示出了本申请实施例提供的光学成像系统的另一种示意性结构图。
图中：10-光学成像系统；20-样本；100-脉冲激光输出装置；110-超短脉冲激光器；120-功率调节器；130-反射件；131-第二反射镜；132-第三反射镜；140-扩束件；141-第一扩束镜；142-第二扩束镜；200-色散组件；300-NLM信号激发与探测装置；310-管镜；320-二向色镜；330-物镜；340-成像透镜；350-第一感光元件；360-计算机控制处理单元；370-轴向驱动设备；380-平移载物台；390-白光光源；391-第一反射镜；392-第二感光元件。
具体实施方式
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。应当理解，此处所描述的具体实施例仅用以解释本申请，并不用于限定本申请。
下面结合附图及具体实施例对本发明作进一步详细的说明。
光学切片层析显微成像技术能够避免对组织进行物理切片，简化样品处理步骤，在肿瘤切除边界的术中评估方面具有较大的应用潜力。其中，OCT (Optical Coherence Tomography，光学相干断层成像)、SRS(Stimulated Raman Scattering，受激拉曼散射显微成像)等依赖内源性对比度成像的技术所显示的结构与常规病理检查结果存在一定的差距，无法直接由病理医生解读，因而难以被广泛接受。依赖外源性对比度成像的技术中，SIM(Structure Illumination Microscopy，结构光照明显微成像)、LSM(Light-Sheet Microscopy，光片照明显微成像)及MUSE(Microscopy with Ultraviolet Surface Excitation，紫外表面激发显微成像)虽然成像速度快，但只能获得组织的浅表信息，CFM(Confocal Fluorescence Microscopy，共聚焦荧光显微成像)采用点扫描成像模式，成像速度慢，并且穿透深度有限，存在光漂白、光毒性等问题。
NLM技术具有超强的组织穿透能力、固有的层析能力、低的光漂白和光毒性等显著优势，但因同样采用点扫描成像模式，也具有成像速度慢的缺陷。
请参阅图1，图1示出了本申请实施例所提供的光学成像系统10的一种示意性结构图，该光学成像系统10可以用于病理检查，以提升病理检查的速度。该光学成像系统10包括脉冲激光输出装置100、色散组件200及NLM信号激发与探测装置300。
其中，该光学成像系统10在工作时，脉冲激光输出装置100产生第一超短脉冲激光，并将第一超短脉冲激光照射在色散组件200，由色散组件200将第一超短脉冲激光包含的不同单色光组分在空间上分离开，色散为多束第二超短脉冲激光，进而由NLM信号激发与探测装置300在利用多束第二超短脉冲激光照射样本20，以激发样本20产生NLM信号，并利用样本20所产生的NLM信号重建样本20的NLM图像时，采用与色散组件200相配合，将一束第一超短脉冲激光色散得到多束第二超短脉冲激光，时域聚焦后照射样本20的一个区域进行宽场成像的模式，替换由一束第一超短脉冲激光空间聚焦后照 射样本20的一个点进行点扫描成像的模式，提升对样本20进行NLM成像的速度。
可选地，作为一种可能的实现方式，色散组件200可以采用衍射光栅，第一超短脉冲激光照射在衍射光栅时，衍射光栅通过衍射将第一超短脉冲激光色散为多束第二超短脉冲激光。
值得说明的是，在本申请实施例其他的一些实施方式中，色散组件200还可以采用除衍射光栅之外的其他一些结构或设备，比如，还可以采用色散棱镜作为色散组件200，只要用作色散组件200的结构或设备能够将第一超短脉冲激光色散为多束第二超短脉冲激光即可。
可选地，作为一种可能的实现方式，脉冲激光输出装置100包括超短脉冲激光器110和功率调节器120。
超短脉冲激光器110产生第一超短脉冲激光，并由功率调节器120将第一超短脉冲激光的功率衰减后，照射至色散组件200，使得入射色散组件200的第一超短脉冲激光的光功率符合激光安全应用标准，比如ANSI(AMERICAN NATIONAL STANDARDS INSTITUTE，美国国家标准学会)标准，同时确保照射在样本20表面的光功率不会对样本20造成光损伤或光毒性。
其中，可选地，作为一种可能的实现方式，超短脉冲激光器110可以选用高重复频率的超短脉冲激光器，比如重复频率为80MHz的钛宝石飞秒激光器，也可以选用其他高重复频率的飞秒、皮秒激光器，或其他能够用于样本20双光子激发荧光成像和二次谐波成像的激光器。
另外，产生第一超短脉冲激光的超短脉冲激光器110可以是波长可调谐的，比如上述覆盖680–1020nm波段、重复频率为80MHz的钛宝石飞秒激光器，也可以是单一波长的，比如针对特定的荧光染料组合，吖啶橙和磺酰罗丹明 101，选择780nm或者1040nm等波长的超短脉冲激光器，作为该光学成像系统10用于NLM成像的光源，获取近似于H&E(Hematoxylin-Eosin，苏木精-伊红)染色切片的组织学图像，相比NLM成像通常使用的波长可调谐飞秒脉冲激光器，能够降低成本。
可选地，作为一种可能的实现方式，该脉冲激光输出装置100还包括反射件130，该反射件130将经功率调节器120衰减的第一超短脉冲激光反射至色散组件200，进而使超短脉冲激光器110和功率调节器120的位置无需固定不变，而是能够根据光学成像系统10的具体架构设计需求进行协调。
比如，作为一种可能的实现方式，请继续参阅图1，反射件130可由第二反射镜131和第三反射镜132构成，由第二反射镜131和第三反射镜132将功率衰减的第一超短脉冲激光反射至色散组件200。
值得说明的是，图1中反射件130包括第二反射镜131和第三反射镜132仅为示意，在本申请实施例其他的一些可能的实现方式中，反射件130还可以包括更多或更少的反射镜，比如采用一个反射镜实现，或者采用三个、四个甚至更多的反射镜实现，只要反射件130能够将第一超短脉冲激光反射至色散组件200即可。
可选地，作为一种可能的实现方式，该脉冲激光输出装置100还包含扩束件140，该扩束件140将功率衰减的第一超短脉冲激光扩束后，照射至色散组件200。进而使照射在色散组件200上的第一超短脉冲激光的光束足够大，从而使色散组件200能够将第一超短脉冲激光充分色散得到多束第二超短脉冲激光。
比如，作为一种可能的实现方式，请继续参阅图1，扩束件140可由第一扩束镜141和第二扩束镜142构成，由第一扩束镜141和第二扩束镜142相配 合，将第一超短脉冲激光扩束后照射至色散组件200。
值得说明的是，图1中扩束件140包括第一扩束镜141和第二扩束镜142仅为示意，在本申请实施例其他的一些可能的实现方式中，扩束件140还可以包括更多的扩束镜，比如采用四个扩束镜实现，只要扩束件140能够将第一超短脉冲激光扩束后照射至色散组件200即可。
可选地，请继续参阅图1，作为一种可能的实现方式，NLM信号激发与探测装置300包括管镜310、二向色镜320、物镜330、成像透镜340、第一感光元件350及计算机控制处理单元360。
其中，二向色镜320能够使第二超短脉冲激光透过，而使NLM信号被反射。
由此，经过色散组件200色散得到的多束第二超短脉冲激光，透过管镜310后，多束第二超短脉冲激光之间的关系被调整为相互平行，再透过二向色镜320和物镜330后，每束第二超短脉冲激光被调整为平行光，且在物镜330的焦平面重新组合在一起，会聚照射至样本20，形成时域聚焦宽场照明，以激发样本20产生NLM信号，比如双光子激发荧光信号、二次谐波信号等；并且，激发样本20所产生的NLM信号又透过物镜330，照射在二向色镜320上，被二向色镜320反射并透过成像透镜340后被第一感光元件350采集并转化为第一电信号，由此，与第一感光元件350电连接的计算机控制处理单元360接收第一感光元件350输出的由NLM信号转化而来的第一电信号，并基于第一电信号重建出样本20的NLM图像。其中，由于多束第二超短脉冲激光时域聚焦后，单束第二超短脉冲激光以平行光形式照射样本20的一个区域进行宽场成像，相比于单束第一超短脉冲激光空间聚焦后仅照射样本20的一个点进行点扫描成像，提升了对样本20进行NLM成像的速度，从而使病理 检查在成像环节节约了时间，并利用NLM这种光学切片技术避免了在病理检查时进行物理切片，进而避免了复杂的物理切片前样品处理，提升了病理检查的速度。
其中，作为一种可能的实现方式，第一感光元件350可以采用彩色CCD(Charge-Coupled Device，电荷耦合元件)。由于通常需要采用多种染料对样本20的不同结构组分进行染色标记才能获得与临床病理检查接近的组织学图像，第一感光元件350所获得的NLM信号中通常包含有多种荧光信号，如果采用彩色CCD，第一感光元件350就能够直接获取样本20的真彩图像，病理医生可以通过色彩差异区分不同种类的荧光信号，从而避免在成像系统的探测光路使用分光、滤光器件和多个探测器，简化了系统结构。
作为一种可能的实现方式，在本申请实施例中，物镜330的焦平面与色散组件200共轭，即色散组件200放置于管镜310的焦平面处，而管镜310与物镜330的距离等于管镜310的焦距加上物镜330的焦距，以使每束第二超短脉冲激光都以平行光的形式照射样本20，形成宽场照明，进而实现宽场NLM成像，提升对样本20进行NLM成像的速度。
作为一种可能的实现方式，物镜330可以包括有多个可自由切换且放大倍率不同的物镜单元，用于实现多种尺度(即多种分辨率和多种视场大小)的病理成像。
可选地，请继续参阅图1，作为一种可能的实现方式，NLM信号激发与探测装置300还包括有轴向驱动设备370，该轴向驱动设备370驱动物镜330靠近或远离样本20，以调整多束第二超短脉冲激光会聚在样本20的不同深度，使得在对样本20进行成像时，能够获取样本20不同深度的信息，实现三维成像。
比如，作为一种可能的实现方式，轴向驱动设备370可以采用步进电机实现，并且，可在计算机控制处理单元360中配置相应的控制程序，以控制步进电机工作，进而带动物镜330靠近或远离样本20。
可选地，请继续参阅图1，作为一种可能的实现方式，NLM信号激发与探测装置300还包括有平移载物台380，该平移载物台380承载样本20，并选择性地带动样本20与物镜330水平错动。
比如，作为一种可能的实现方式，平移载物台380可以采用电动载物台实现，并且，可在计算机控制处理单元360中配置相应的控制程序，以控制电动载物台工作，进而在需要时，可以控制电动载物台移动，进而带动样本20与物镜330水平错动，使得对样本20进行NLM成像时，能够获取样本20不同区域的信息，实现大面积成像。
可选地，请参阅图2，图2示出了本申请实施例提供的光学成像系统10的另一种示意性结构图，作为一种可能的实现方式，该NLM信号激发与探测装置300还包括白光光源390、第一反射镜391及第二感光元件392。
其中，在脉冲激光输出装置100不工作时，样本20不会受激光激发产生NLM信号，此时，白光光源390启动工作，产生白光，样本20受白光照射反射白光信号，该经样本20反射的白光信号透过物镜330，经过第一反射镜391反射后被第二感光元件392采集并转化为第二电信号，由此，与第二感光元件392电连接的计算机控制处理单元360接收第二感光元件392输出的由反射光信号转化而来的第二电信号，并基于该第二电信号，重建出样本20的白光图像，便于病理医生观察样本20，此时，可以理解为光学成像系统10处于白光成像模式。
值得说明的是，在本申请实施例其他一些可能的应用场景中，还可以采用 目镜替换第二感光元件392，即直接用目镜配合第一反射镜391及物镜330观察样本20，无需由第二感光元件392结合计算机控制处理单元360实时采集白光图像用于观察。
并且，在本申请实施例中，脉冲激光输出装置100在工作时，第一反射镜391不位于第一超短脉冲激光或多束第二超短脉冲激光所在的光路，避免第一反射镜391阻断NLM成像，且白光光源390不工作，此时，可以理解为光学成像系统10处于NLM成像模式。
其中，上述白光成像模式可以用于在NLM成像前对样本20进行快速定位，包括感兴趣区域的选择和调焦。在白光成像模式下，通过目镜观察或实时采集样本图像的方式，医生可在NLM成像前对样本20进行观察，控制轴向驱动设备370驱动物镜330靠近或远离样本20，进而使样本20处于物镜330的焦平面处；或者是控制平移载物台380将样本20中的感兴趣区域移至视场中央。并且，第一反射镜391还可以固定于一机械装置，比如拉杆、一维平移台等，或者是其他用于控制第一反射镜391移动的机械装置，当该光学成像系统10处于白光成像模式时，第一反射镜391被推入光路中，与物镜330及第二感光元件392等相配合，以供医生对样本20进行观察；而当该光学成像系统10处于NLM成像模式时，则移动第一反射镜391退出第一超短脉冲激光或第二超短脉冲激光所在的光路，以实现白光成像模式与NLM成像模式间的切换。
下面结合操作应用实例，对本申请实施例提供的光学成像系统10进行示例性说明。
首先对手术切除得到的组织进行近似于H&E染色的荧光标记，例如采用吖啶橙和磺酰罗丹明101对组织进行约2分钟的染色，然后采用缓冲液漂洗约 20秒；随后，将经过漂洗的组织放置于平移载物台380上的样品盒内，盖上盖玻片，准备成像；接下来，先在低倍物镜下，例如5倍物镜下，采用白光成像模式调整组织样本的横向和纵向位置，确定成像区域后，将该光学成像系统10切换至NLM成像模式，在NLM成像模式下，采用荧光标记物相对应的激发波长，例如这里针对吖啶橙和磺酰罗丹明101可以选择780nm的激发波长，对组织样本进行快速成像，获取组织样本的结构信息；根据成像结果，进一步确定需要观察精细结构的区域；然后将物镜切换至高倍镜下，例如10倍物镜下，对该区域进行高分辨NLM成像，病理医生可综合成像结果对肿瘤切除边界进行评估、作出诊断。其中，可以根据实际需求灵活调整成像方案，也可以对组织进行三维成像或大体积成像获得组织的三维结构信息，用以辅助诊断。从处理组织样品到成像观察，仅需几分钟到十几分钟就能完成，大幅度提升了病理检查的速度。成像结束后，可立刻将组织样品放入福尔马林进行固定，用于后续常规病理检查。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种用于病理检查的光学成像系统，其特征在于，包括脉冲激光输出装置、色散组件及非线性光学显微成像NLM信号激发与探测装置；
    所述脉冲激光输出装置产生第一超短脉冲激光，并将所述第一超短脉冲激光照射至所述色散组件；
    所述色散组件将所述第一超短脉冲激光包含的不同单色光组分在空间上分离开，色散为多束第二超短脉冲激光；
    所述NLM信号激发与探测装置利用所述多束第二超短脉冲激光照射样本，以激发所述样本产生NLM信号，并根据所述NLM信号重建出所述样本的NLM图像。
  2. 如权利要求1所述的光学成像系统，其特征在于，所述色散组件为衍射光栅；
    所述衍射光栅通过衍射将所述第一超短脉冲激光色散为所述多束第二超短脉冲激光。
  3. 如权利要求1所述的光学成像系统，其特征在于，所述脉冲激光输出装置包括超短脉冲激光器和功率调节器；
    所述超短脉冲激光器产生所述第一超短脉冲激光；
    所述功率调节器将所述第一超短脉冲激光的功率衰减后，照射至所述色散组件。
  4. 如权利要求3所述的光学成像系统，其特征在于，所述脉冲激光输出装置还包括反射件；
    所述反射件将所述功率衰减的第一超短脉冲激光反射至所述色散组件。
  5. 如权利要求3所述的光学成像系统，其特征在于，所述脉冲激光输出装置还包括扩束件；
    所述扩束件将所述功率衰减的第一超短脉冲激光扩束后，照射至所述色散组件。
  6. 如权利要求1-5中任一项所述的光学成像系统，其特征在于，所述NLM信号激发与探测装置包括管镜、二向色镜、物镜、成像透镜、第一感光元件及计算机控制处理单元；
    所述多束第二超短脉冲激光透过所述管镜及所述二向色镜后，由所述物镜聚焦照射所述样本，以激发所述样本产生所述NLM信号；
    所述NLM信号透过所述物镜后，由所述二向色镜反射并透过所述成像透镜后被所述第一感光元件采集并转化为第一电信号；
    所述计算机控制处理单元与所述第一感光元件电连接，且接收所述第一感光元件输出的由所述NLM信号转化而来的第一电信号，并基于所述第一电信号重建出所述样本的所述NLM图像。
  7. 如权利要求6所述的光学成像系统，其特征在于，所述NLM信号激发与探测装置还包括轴向驱动设备；
    所述轴向驱动设备驱动所述物镜靠近或远离所述样本。
  8. 如权利要求6所述的光学成像系统，其特征在于，所述NLM信号激发与探测装置还包括平移载物台；
    所述平移载物台承载所述样本，并选择性地带动所述样本与所述物镜水平错动。
  9. 如权利要求6所述的光学成像系统，其特征在于，所述第一感光元件为彩色CCD。
  10. 如权利要求6所述的光学成像系统，其特征在于，所述NLM信号激发与探测装置还包括第一反射镜、白光光源及第二感光元件；
    在所述脉冲激光输出装置不工作时，所述白光光源启动工作，产生白光，以照亮所述样本；
    所述样本反射所述白光，所述经样本反射的白光透过所述物镜，并经所述第一反射镜反射后被所述第二感光元件采集并转化为第二电信号；
    所述计算机控制处理单元接收所述第二感光元件输出的所述第二电信号，并基于所述第二电信号，重建出所述样本的白光图像；
    在所述脉冲激光输出装置启动工作时，所述第一反射镜不位于所述第一超短脉冲激光或所述多束第二超短脉冲激光所在的光路，且所述白光光源不工作。
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