CN110623641A - A self-adaptive second and third harmonic joint detection microscopic imaging method and device - Google Patents
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Abstract
本发明涉及一种自适应二次三次谐波联合探测显微成像方法及装置，属于光学显微成像技术领域；本发明在传统谐波显微装置的探测路径上同时设置二次、三次谐波信号探测模块，利用二向色镜和滤光片实现二次谐波信号和三次谐波信号的分离和滤波，采用独立的光电探测器分别对二次谐波、三次谐波信号进行探测；在系统中引入自适应像差校正装置，用于校正对样品进行大深度探测时存在的像差。本发明装置包括激光扫描系统、自适应像差校正系统、谐波信号激发系统、谐波信号探测系统、宽场成像系统、以及图像重建与数据处理系统。本发明实现了二次、三次谐波信号的联合探测以及样品结构信息的互补，且在大深度探测时保持了成像质量。
The invention relates to an adaptive second and third harmonic joint detection microscopic imaging method and device, belonging to the technical field of optical microscopic imaging; the invention simultaneously sets the second and third harmonics on the detection path of the traditional harmonic microscopic device The signal detection module uses a dichroic mirror and an optical filter to separate and filter the second harmonic signal and the third harmonic signal, and uses an independent photodetector to detect the second harmonic and third harmonic signals respectively; An adaptive aberration correction device is introduced into the system to correct the aberrations existing in the large-depth detection of the sample. The device of the invention includes a laser scanning system, an adaptive aberration correction system, a harmonic signal excitation system, a harmonic signal detection system, a wide-field imaging system, and an image reconstruction and data processing system. The invention realizes the joint detection of the second and third harmonic signals and the complementarity of the sample structure information, and maintains the imaging quality in the detection of a large depth.
Description
技术领域technical field
本发明属于光学显微测量领域，主要涉及一种用于活体生物样品的三维微细结构成像的自适应二次三次谐波联合探测显微成像方法及装置。The invention belongs to the field of optical microscopic measurement, and mainly relates to an adaptive second and third harmonic combined detection microscopic imaging method and device for three-dimensional fine structure imaging of living biological samples.
背景技术Background technique
随着科学技术的不断发展，对活体生物样品进行高分辨率、高穿透深度成像已成为系统生物学研究不可或缺的条件。然而，由于光学衍射极限的存在，传统光学显微成像的分辨率受到制约。荧光显微成像能够对生物样品进行超分辨率成像。其中，多光子显微成像技术是最佳的无侵害荧光显微成像方式。双光子激发显微过程存在实际的能量转换，响应时间在纳秒量级。而二次、三次谐波生成过程只有虚拟的能量转换，且响应时间在飞秒量级。因此利用谐波生成过程进行成像可实现高灵敏度，高速响应成像。谐波显微成像方法是一种三维光学成像技术，具有非线性光学成像特有的成像特性。谐波信号的激发需要高强度激光脉冲，因此仅在焦点区域才有谐波信号产生。非线性的强局域效应减少了成像时焦点外的背景噪声干扰，具有很高的信噪比。谐波信号是样品自身产生的非线性效应，不需要外在荧光标记，从而不影响生物样品的活性。谐波信号的激发通常使用近红外的激发光，可以实现很高的探测深度。二次谐波的产生需要样品不具有反转对称性。三次谐波的产生则对样品的特性无要求。二次谐波、三次谐波可以反映样品的不同结构信息，联合探测可以实现样品结构信息的互补。With the continuous development of science and technology, high-resolution and high-penetration depth imaging of living biological samples has become an indispensable condition for systems biology research. However, due to the existence of the optical diffraction limit, the resolution of traditional optical microscopy imaging is restricted. Fluorescence microscopy enables super-resolution imaging of biological samples. Among them, multiphoton microscopic imaging technology is the best non-invasive fluorescence microscopic imaging method. There is actual energy conversion in the microscopic process of two-photon excitation, and the response time is on the order of nanoseconds. The second and third harmonic generation processes only have virtual energy conversion, and the response time is on the order of femtoseconds. Imaging using the harmonic generation process therefore enables high-sensitivity, high-speed response imaging. Harmonic microscopic imaging method is a three-dimensional optical imaging technology, which has the unique imaging characteristics of nonlinear optical imaging. The excitation of harmonic signals requires high-intensity laser pulses, so harmonic signals are generated only in the focal region. The non-linear strong local effect reduces the background noise interference outside the focal point during imaging, and has a high signal-to-noise ratio. The harmonic signal is a nonlinear effect generated by the sample itself, and does not require external fluorescent labels, thus not affecting the activity of biological samples. The excitation of harmonic signals usually uses near-infrared excitation light, which can achieve a high detection depth. The generation of the second harmonic requires that the sample does not have inversion symmetry. The generation of the third harmonic has no requirement on the characteristics of the sample. The second harmonic and the third harmonic can reflect the different structural information of the sample, and the joint detection can realize the complementarity of the sample structural information.
在对活体生物组织进行高深度成像时，由于样品自身光学特性的不均匀性以及折射率失配，成像过程中存在明显的像差。谐波显微成像过程作为高阶非线性光学过程，对像差极其敏感。像差的存在会降低谐波信号的强度以及成像质量。且探测深度越大，像差的影响就越大。一些具有重大研究意义的细节信息会因为像差的存在而无法成像。为了实现大深度高质量的成像，必须对生物样品引起的像差进行校正。When performing high-depth imaging on living biological tissues, there are obvious aberrations in the imaging process due to the inhomogeneity of the optical properties of the sample itself and the mismatch of the refractive index. As a high-order nonlinear optical process, harmonic microscopic imaging is extremely sensitive to aberrations. The existence of aberrations will reduce the intensity of harmonic signals and image quality. And the greater the detection depth, the greater the impact of aberrations. Some detailed information of great research significance cannot be imaged due to the existence of aberrations. In order to achieve high-quality imaging at large depths, the aberrations caused by biological samples must be corrected.
因此，目前需要本领域技术人员迫切解决的一个技术问题就是：如何能够同时对样品进行二次谐波、三次谐波的联合探测，并通过数据处理，实现二次、三次谐波图像的融合和信息的互补。此外，在利用谐波显微成像进行大深度探测时，能够对样品引起的像差进行校正。Therefore, a technical problem that needs to be urgently solved by those skilled in the art is: how to simultaneously detect the second harmonic and the third harmonic of the sample, and realize the fusion and integration of the second and third harmonic images through data processing. complementarity of information. Additionally, sample-induced aberrations can be corrected for large depth probing using harmonic microscopy.
发明内容Contents of the invention
本发明的目的是为了克服已有技术的不足之处，提出一种自适应二次三次谐波联合探测显微成像方法及装置。本发明在谐波显微成像装置中的探测路径上同时设置了二次谐波探测模块和三次谐波探测模块，利用二向色镜和滤光片实现二次谐波信号和三次谐波信号的分离和滤波，采用独立的光电探测器分别对二次谐波、三次谐波信号进行探测；在系统中引入自适应像差校正装置，用于校正对样品大深度探测时存在的像差。本发明克服了传统谐波显微成像探测模式单一以及大深度探测时存在像差的挑战。The object of the present invention is to propose an adaptive second and third harmonic joint detection microscopic imaging method and device in order to overcome the shortcomings of the prior art. In the present invention, a second harmonic detection module and a third harmonic detection module are simultaneously arranged on the detection path of the harmonic microscopic imaging device, and a dichroic mirror and an optical filter are used to realize the second harmonic signal and the third harmonic signal Independent photodetectors are used to detect the second harmonic and third harmonic signals respectively; an adaptive aberration correction device is introduced in the system to correct the aberrations that exist when detecting large depths of samples. The invention overcomes the challenges of single detection mode of traditional harmonic microscopic imaging and aberrations in large depth detection.
为了实现上述目的，本发明采用如下技术方案：In order to achieve the above object, the present invention adopts the following technical solutions:
本发明提出的一种自适应二次三次谐波联合探测显微成像装置，其特征在于所述装置包括：激光扫描系统、自适应像差校正系统、谐波信号激发系统、谐波信号探测系统、宽场成像系统、以及图像重建与数据处理系统；An adaptive second and third harmonic joint detection microscopic imaging device proposed by the present invention is characterized in that the device includes: a laser scanning system, an adaptive aberration correction system, a harmonic signal excitation system, and a harmonic signal detection system , wide-field imaging system, and image reconstruction and data processing system;
其中：所述激光扫描系统包括超短脉冲激光光源、光开关、光束变换单元，以及光束扫描元件；超短脉冲激光光源用于提供产生谐波信号的激发脉冲光，光开关用于激发光的开关控制，光束变换单元用于调整所述激发脉冲光光束尺寸，光束扫描元件用于激发光对样品的扫描。Wherein: the laser scanning system includes an ultrashort pulse laser light source, an optical switch, a beam conversion unit, and a beam scanning element; the ultrashort pulse laser source is used to provide excitation pulse light for generating harmonic signals, and the optical switch is used for excitation light The switch is controlled, the beam conversion unit is used to adjust the beam size of the excitation pulse light, and the beam scanning element is used to scan the sample with the excitation light.
所述自适应像差校正系统置于激光扫描系统之后，包括透镜组、波前传感器和动态光学元件；透镜组用于连接光束扫描元件和动态光学元件，波前传感器用于波前传感，动态光学元件用于调制光路波前从而校正成像过程中的像差。The adaptive aberration correction system is placed behind the laser scanning system, including a lens group, a wavefront sensor and a dynamic optical element; the lens group is used to connect the beam scanning element and the dynamic optical element, the wavefront sensor is used for wavefront sensing, and the dynamic Optical components are used to modulate the wavefront of the optical path to correct for aberrations in the imaging process.
所述谐波信号激发系统置于自适应像差校正系统之后，包括透镜组和显微物镜，由该透镜组连接所述动态光学元件及所述显微物镜后入瞳面，并构成4f系统，用于激发光束的聚焦及谐波信号的激发。The harmonic signal excitation system is placed behind the adaptive aberration correction system, including a lens group and a microscopic objective lens, and the lens group is connected to the dynamic optical element and the rear entrance pupil surface of the microscopic objective lens to form a 4f system , used for the focusing of the excitation beam and the excitation of the harmonic signal.
所述谐波信号探测系统包括三维微位移台、谐波信号探测物镜、透镜、二向色镜、滤光片、以及光电探测器；对于反射式探测，谐波信号探测物镜即为显微物镜；对于透射式探测，谐波信号探测物镜置于样品的透射端；透镜用于谐波信号的聚焦，二向色镜用于二次、三次谐波信号的分离，滤光片用于滤除谐波信号以外的杂散光，光电探测器用于谐波信号的采集。The harmonic signal detection system includes a three-dimensional micro-shift stage, a harmonic signal detection objective lens, a lens, a dichroic mirror, an optical filter, and a photodetector; for reflective detection, the harmonic signal detection objective lens is a microscopic objective lens ; For transmission detection, the harmonic signal detection objective lens is placed at the transmission end of the sample; the lens is used to focus the harmonic signal, the dichroic mirror is used to separate the second and third harmonic signals, and the filter is used to filter out For stray light other than harmonic signals, photodetectors are used to collect harmonic signals.
所述宽场成像系统包括LED光源、透镜组、宽场成像相机；LED光源用于宽场照明，透镜组用于光路调整，宽场成像相机用于成像。The wide-field imaging system includes an LED light source, a lens group, and a wide-field imaging camera; the LED light source is used for wide-field illumination, the lens group is used for optical path adjustment, and the wide-field imaging camera is used for imaging.
所述图像重建与数据处理系统，与所述激光扫描系统中的扫描元件和所述自适应像差校正系统中的动态光学元件以及光电探测器相连，用于对成像器件采集到的扫描图像进行全局图像处理。The image reconstruction and data processing system is connected with the scanning element in the laser scanning system, the dynamic optical element and the photodetector in the adaptive aberration correction system, and is used to perform scanning images collected by the imaging device Global Image Processing.
所有光学元件的光学面中心与入射激光和谐波信号的中心光束形成的光轴重合，所有透镜均垂直于光轴。The centers of the optical surfaces of all optical components coincide with the optical axis formed by the central beam of the incident laser and harmonic signals, and all lenses are perpendicular to the optical axis.
进一步地，所述的动态光学元件为可变形反射镜或空间光调制器，或可变形反射镜、空间光调制器组成的联用系统。Further, the dynamic optical element is a deformable mirror or a spatial light modulator, or a combined system composed of a deformable mirror and a spatial light modulator.
进一步地，所述的光束扫描元件可以是扫描振镜或声光偏转器。Further, the beam scanning element may be a scanning galvanometer or an acousto-optic deflector.
进一步地，所述的超短脉冲激光光源及光束变换系统中，超短脉冲激光光源选用飞秒脉冲激光光源。Further, in the ultrashort pulse laser source and the beam conversion system, the ultrashort pulse laser source is a femtosecond pulse laser source.
进一步地，所述的谐波信号探测系统，采用二次三次谐波同步探测；二向色镜用于分离二次、三次谐波信号；滤光片透过波长分别对应二次、三次谐波信号波长；独立的两个光电探测器分别探测二次谐波和三次谐波信号，探测器灵敏波长分别对应相应的二次、三次谐波波长。Further, the harmonic signal detection system uses the second and third harmonics to detect synchronously; the dichroic mirror is used to separate the second and third harmonic signals; the transmission wavelength of the filter corresponds to the second and third harmonics respectively Signal wavelength: Two independent photodetectors respectively detect the second harmonic and third harmonic signals, and the sensitive wavelengths of the detectors correspond to the corresponding second and third harmonic wavelengths respectively.
一种自适应二次三次谐波联合探测显微成像方法，其特征在于包括下列步骤：An adaptive second and third harmonic joint detection microscopic imaging method is characterized in that it comprises the following steps:
(1)激光扫描系统产生短脉冲激光扫描光束；(1) The laser scanning system generates a short pulse laser scanning beam;
(2)自适应像差校正系统通过调制光路波前，实现像差的自适应校正；(2) The adaptive aberration correction system realizes adaptive correction of aberrations by modulating the wavefront of the optical path;
(3)谐波信号激发系统激发样品，产生谐波信号；(3) The harmonic signal excitation system excites the sample to generate harmonic signals;
(4)谐波信号探测系统收集谐波信号并进行二次、三次谐波信号分离、滤波及同步探测；(4) Harmonic signal detection system collects harmonic signals and performs second and third harmonic signal separation, filtering and synchronous detection;
(5)图像重建与数据处理系统对光电探测器采集到的信号进行图像重建和数据处理，重构出最终的谐波图像。(5) Image reconstruction and data processing The system performs image reconstruction and data processing on the signals collected by the photodetector, and reconstructs the final harmonic image.
本发明所述的自适应二次三次谐波联合探测显微成像方法其特征在于所述的自适应像差校正依照有无波前传感，可选用有波前传感方式或无波前传感方式进行像差校正。The adaptive second and third harmonic joint detection microscopic imaging method described in the present invention is characterized in that the adaptive aberration correction depends on whether there is wavefront sensing, and wavefront sensing or no wavefront sensing can be selected. Sensitive way to correct aberrations.
本发明所述的自适应二次三次谐波联合探测显微成像方法其特征在于谐波信号既可以反射式探测也可以透射式探测。The self-adaptive second and third harmonic joint detection microscopic imaging method described in the present invention is characterized in that the harmonic signal can be either reflected or transmitted.
本发明所述的自适应二次三次谐波联合探测显微成像方法其特征在于所述的图像重建和数据处理可单独形成二次谐波图像或三次谐波图像以及二次、三次谐波融合图像。The adaptive second and third harmonic joint detection microscopic imaging method of the present invention is characterized in that the image reconstruction and data processing can separately form the second harmonic image or the third harmonic image and the second and third harmonic fusion image.
本发明的有益效果在于，通过在传统谐波显微方法中的探测路径上同时设置二次、三次谐波信号探测模块，实现了二次、三次谐波的联合探测以及样品结构信息的互补。此外，通过在成像系统中引入自适应像差校正模块，用于校正大深度成像时样品引起的像差，从而提升了谐波显微成像的成像深度和成像质量。The beneficial effect of the present invention is that, by simultaneously setting the second and third harmonic signal detection modules on the detection path in the traditional harmonic microscopy method, the combined detection of the second and third harmonics and the complementarity of sample structure information are realized. In addition, by introducing an adaptive aberration correction module in the imaging system, it is used to correct the aberration caused by the sample during large-depth imaging, thereby improving the imaging depth and imaging quality of harmonic microscopic imaging.
附图说明Description of drawings
图1是本发明实施例1的自适应二次三次谐波联合探测显微成像方法及装置示意图。Fig. 1 is a schematic diagram of an adaptive second and third harmonic joint detection microscopic imaging method and device according to Embodiment 1 of the present invention.
图2是本发明实施例2的自适应二次三次谐波联合探测显微成像方法及装置示意图。Fig. 2 is a schematic diagram of an adaptive second and third harmonic joint detection microscopic imaging method and device according to Embodiment 2 of the present invention.
其中：1-超短脉冲激光光源、2-光开关、3-光束变换单元、4-光束扫描元件、5-第一透镜组、6-动态光学元件、7-第二透镜组、8-第一二向色镜、9-第三透镜组、10-显微物镜、11-样品、12-三维微位移台、13-谐波信号探测物镜、14-第一透镜、15-第二二向色镜、16-第一滤光片、17-第三二向色镜、18-二次谐波滤光片、19-第一针孔、20-二次谐波光电探测器、21-第二透镜、22-三次谐波滤光片、23-第二针孔、24-三次谐波光电探测器、25-LED光源、26-第四透镜组、27-宽场成像相机、28-第四二向色镜。Among them: 1-ultrashort pulse laser source, 2-optical switch, 3-beam conversion unit, 4-beam scanning element, 5-first lens group, 6-dynamic optical element, 7-second lens group, 8-the first 1 dichroic mirror, 9-third lens group, 10-microscopic objective lens, 11-sample, 12-three-dimensional micro displacement stage, 13-harmonic signal detection objective lens, 14-first lens, 15-second dichroic Chromatic mirror, 16-first filter, 17-third dichroic mirror, 18-second harmonic filter, 19-first pinhole, 20-second harmonic photodetector, 21-th Second lens, 22-third harmonic filter, 23-second pinhole, 24-third harmonic photodetector, 25-LED light source, 26-fourth lens group, 27-wide field imaging camera, 28-the first Four dichroic mirrors.
具体实施方式Detailed ways
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合附图和具体实施实例对本发明做进一步详细的描述。In order to make the above objects, features and advantages of the present invention more comprehensible, the present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation examples.
本发明提出的自适应二次三次谐波联合探测显微成像方法，该方法包括以下步骤：The self-adaptive second and third harmonic joint detection microscopic imaging method proposed by the present invention comprises the following steps:
(1)激光扫描系统产生短脉冲激光扫描光束；(1) The laser scanning system generates a short pulse laser scanning beam;
(2)自适应像差校正系统通过调制光路波前，实现像差的自适应校正；(2) The adaptive aberration correction system realizes adaptive correction of aberrations by modulating the wavefront of the optical path;
(3)谐波信号激发系统激发样品，产生谐波信号；(3) The harmonic signal excitation system excites the sample to generate harmonic signals;
(4)谐波信号探测系统收集谐波信号并进行二次、三次谐波信号分离、滤波及同步探测；(4) Harmonic signal detection system collects harmonic signals and performs second and third harmonic signal separation, filtering and synchronous detection;
(5)图像重建与数据处理系统对光电探测器采集到的信号进行图像重建和数据处理，重构出最终的谐波图像。(5) Image reconstruction and data processing The system performs image reconstruction and data processing on the signals collected by the photodetector, and reconstructs the final harmonic image.
本发明还根据上述方法提出一种自适应二次三次谐波联合探测显微成像装置。The present invention also proposes an adaptive second and third harmonic joint detection microscopic imaging device according to the above method.
实施例1：Example 1:
本实施例1的一种自适应二次三次谐波联合探测显微成像装置示意图如图1所示。该装置包括超短脉冲激光光源1、光开关2、光束变换单元3、光束扫描元件4、第一透镜组5、动态光学元件6、第二透镜组7、第一二向色镜8、第三透镜组9、显微物镜10、样品11、三维微位移台12、谐波信号探测物镜13、第一透镜14、第二二向色镜15、第一滤光片16、第三二向色镜17、二次谐波滤光片18、第一针孔19、二次谐波光电探测器20、第二透镜21、三次谐波滤光片22、第二针孔23、三次谐波光电探测器24、LED光源25、第四透镜组26、宽场成像相机27。上述各元器件的连接关系为：所有光学元件的光学面中心与入射激光和谐波信号的中心光束形成的光轴重合，所有透镜均垂直于光轴；其中，超短脉冲激光光源1、光开关2、光束变换单元3、光束扫描元件4组成激光扫描系统产生短脉冲激光扫描光束；第一透镜组5、动态光学元件6、第二透镜组7组成自适应像差校正系统，置于激光扫描系统之后，调制光路波前，实现像差的自适应校正；第三透镜组9、显微物镜10、样品11、三维微位移台12组成谐波信号激发系统，置于自适应像差校正系统之后，用于激发样品，产生谐波信号；谐波信号探测物镜13、第一透镜14、第二二向色镜15、第一滤光片16、第三二向色镜17、二次谐波滤光片18、第一针孔19、二次谐波光电探测器20、第二透镜21、三次谐波滤光片22、第二针孔23、三次谐波光电探测器24组成谐波信号探测系统，收集谐波信号并进行二次、三次谐波信号分离、滤波及同步探测；第一二向色镜8、LED光源25、第四透镜组26、宽场成像相机27组成宽场成像系统。A schematic diagram of an adaptive second and third harmonic joint detection microscopic imaging device in Embodiment 1 is shown in FIG. 1 . The device includes an ultrashort pulse laser light source 1, an optical switch 2, a beam conversion unit 3, a beam scanning element 4, a first lens group 5, a dynamic optical element 6, a second lens group 7, a first dichroic mirror 8, a second lens group Three-lens group 9, microscope objective lens 10, sample 11, three-dimensional micro-displacement stage 12, harmonic signal detection objective lens 13, first lens 14, second dichroic mirror 15, first filter 16, third dichroic Color mirror 17, second harmonic filter 18, first pinhole 19, second harmonic photodetector 20, second lens 21, third harmonic filter 22, second pinhole 23, third harmonic A photodetector 24 , an LED light source 25 , a fourth lens group 26 , and a wide-field imaging camera 27 . The connection relationship of the above-mentioned components is as follows: the center of the optical surface of all optical components coincides with the optical axis formed by the incident laser and the central beam of the harmonic signal, and all lenses are perpendicular to the optical axis; among them, the ultrashort pulse laser source 1, the light The switch 2, the beam conversion unit 3, and the beam scanning element 4 form a laser scanning system to generate a short-pulse laser scanning beam; the first lens group 5, the dynamic optical element 6, and the second lens group 7 form an adaptive aberration correction system, which is placed in the laser After scanning the system, the wavefront of the optical path is modulated to realize adaptive correction of aberrations; the third lens group 9, the microscope objective lens 10, the sample 11, and the three-dimensional micro-shift stage 12 form a harmonic signal excitation system, which is placed in the adaptive aberration correction After the system, it is used to excite the sample and generate harmonic signals; harmonic signal detection objective lens 13, first lens 14, second dichroic mirror 15, first optical filter 16, third dichroic mirror 17, secondary Harmonic filter 18, first pinhole 19, second harmonic photodetector 20, second lens 21, third harmonic filter 22, second pinhole 23, third harmonic photodetector 24 form harmonic The wave signal detection system collects harmonic signals and performs second and third harmonic signal separation, filtering and synchronous detection; the first dichroic mirror 8, LED light source 25, fourth lens group 26, and wide-field imaging camera 27 form a wide field imaging system.
本实施例中，谐波信号的探测方式为透射式探测；In this embodiment, the detection mode of the harmonic signal is transmission detection;
所述的像差校正系统采用无波前传感方式进行像差校正，动态光学元件采用空间光调制器；The aberration correction system uses a non-wavefront sensing method for aberration correction, and the dynamic optical element uses a spatial light modulator;
所述的光束扫描元件是扫描振镜；The beam scanning element is a scanning galvanometer;
所述的宽场成像系统采用背光式照明。The wide-field imaging system uses backlight illumination.
实施例2：Example 2:
本实施例2的一种自适应二次三次谐波联合探测显微成像装置示意图如图2所示。该装置包括超短脉冲激光光源1、光开关2、光束变换单元3、光束扫描元件4、第一透镜组5、动态光学元件6、第二透镜组7、第一二向色镜8、第三透镜组9、显微物镜10、样品11、三维微位移台12、第一透镜14、第一滤光片16、第三二向色镜17、二次谐波滤光片18、第一针孔19、二次谐波光电探测器20、第二透镜21、三次谐波滤光片22、第二针孔23、三次谐波光电探测器24、LED光源25、第四透镜组26、宽场成像相机27、第四二向色镜28。上述各元器件的连接关系为：所有光学元件的光学面中心与入射激光和谐波信号的中心光束形成的光轴重合，所有透镜均垂直于光轴；其中，超短脉冲激光光源1、光开关2、光束变换单元3、光束扫描元件4组成激光扫描系统产生短脉冲激光扫描光束；第一透镜组5、动态光学元件6、第二透镜组7组成自适应像差校正系统，置于激光扫描系统之后，调制光路波前，实现像差的自适应校正；第三透镜组9、显微物镜10、样品11、三维微位移台12组成谐波信号激发系统，置于自适应像差校正系统之后，用于激发样品，产生谐波信号；显微物镜10、第一透镜14、第一滤光片16、第三二向色镜17、二次谐波滤光片18、第一针孔19、二次谐波光电探测器20、第二透镜21、三次谐波滤光片22、第二针孔23、三次谐波光电探测器24、第四二向色镜28组成谐波信号探测系统，收集谐波信号并进行二次、三次谐波信号分离、滤波及同步探测；第一二向色镜8、LED光源25、第四透镜组26、宽场成像相机27组成宽场成像系统。A schematic diagram of an adaptive second and third harmonic joint detection microscopic imaging device in Embodiment 2 is shown in FIG. 2 . The device includes an ultrashort pulse laser light source 1, an optical switch 2, a beam conversion unit 3, a beam scanning element 4, a first lens group 5, a dynamic optical element 6, a second lens group 7, a first dichroic mirror 8, a second lens group Three-lens group 9, microscope objective lens 10, sample 11, three-dimensional micro-displacement stage 12, first lens 14, first filter 16, third dichroic mirror 17, second harmonic filter 18, first pinhole 19, second harmonic photodetector 20, second lens 21, third harmonic filter 22, second pinhole 23, third harmonic photodetector 24, LED light source 25, fourth lens group 26, Wide-field imaging camera 27 , fourth dichroic mirror 28 . The connection relationship of the above-mentioned components is as follows: the center of the optical surface of all optical components coincides with the optical axis formed by the incident laser and the central beam of the harmonic signal, and all lenses are perpendicular to the optical axis; among them, the ultrashort pulse laser source 1, the light The switch 2, the beam conversion unit 3, and the beam scanning element 4 form a laser scanning system to generate a short-pulse laser scanning beam; the first lens group 5, the dynamic optical element 6, and the second lens group 7 form an adaptive aberration correction system, which is placed in the laser After scanning the system, the wavefront of the optical path is modulated to realize adaptive correction of aberrations; the third lens group 9, the microscope objective lens 10, the sample 11, and the three-dimensional micro-shift stage 12 form a harmonic signal excitation system, which is placed in the adaptive aberration correction After the system, it is used to excite the sample and generate harmonic signals; the microscope objective lens 10, the first lens 14, the first filter 16, the third dichroic mirror 17, the second harmonic filter 18, the first needle A hole 19, a second harmonic photodetector 20, a second lens 21, a third harmonic filter 22, a second pinhole 23, a third harmonic photodetector 24, and a fourth dichroic mirror 28 form a harmonic signal The detection system collects harmonic signals and performs second and third harmonic signal separation, filtering and synchronous detection; the first dichroic mirror 8, LED light source 25, fourth lens group 26, and wide-field imaging camera 27 form wide-field imaging system.
本实施例中，谐波信号的探测方式为反射式探测；In this embodiment, the detection mode of the harmonic signal is reflective detection;
所述的显微物镜既是激发光的聚焦物镜也是谐波信号的探测物镜；The microscopic objective lens is not only a focusing objective lens for excitation light but also a detection objective lens for harmonic signals;
所述的像差校正系统采用无波前传感方式进行像差校正，动态光学元件采用可变形反射镜；The aberration correction system uses a non-wavefront sensing method for aberration correction, and the dynamic optical element uses a deformable mirror;
所述的光束扫描元件是扫描振镜；The beam scanning element is a scanning galvanometer;
所述的宽场成像系统采用背光式照明。The wide-field imaging system uses backlight illumination.
以上对本发明所提出的自适应共焦线扫描谐波显微成像方法及装置进行了详细介绍，本文中应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及核心思想；同时，对于本领域的一般技术人员，依据本发明的思想，在具体实施方式及应用范围上均会有改变之处，这些改变都应属于本发明所附的权利要求的保护范围。综上所述，本说明书内容不应理解为对本发明的限制。The adaptive confocal line scanning harmonic microscopic imaging method and device proposed by the present invention have been introduced in detail above. The principles and implementation methods of the present invention have been explained by using specific examples in this paper. The description of the above embodiments is only used To help understand the method and core idea of the present invention; at the same time, for those of ordinary skill in the art, according to the idea of the present invention, there will be changes in the specific implementation and application scope, and these changes should belong to the scope of the present invention. The scope of protection of the appended claims. In summary, the contents of this specification should not be construed as limiting the present invention.
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