CN115951486A - Double-color fluorescence microscope system - Google Patents

Abstract

The invention relates to a bicolor fluorescence microscope system, which comprises an illumination system light path component, a focusing system light path component, an objective system light path component and an imaging system light path component, wherein the illumination system light path component comprises two illumination light sources with different wavelengths, and a first light path component which combines light beams and selectively transmits the light beams; the focusing system light path component comprises a focusing light source, a second light path component capable of adjusting the angle of a main light beam on the focusing light source axis, a first dichroic mirror and a first tube mirror; the objective system light path component comprises an objective lens which is positioned on a coaxial light path with the focusing light source, a second dichroic mirror which is arranged on a light path between the objective lens and the first tube mirror, and a driving motor which drives the objective lens to move relative to the focusing light source; the imaging system light path component is used for shooting the fluorescence reflected by the second dichroic mirror to form an image. The invention integrates the lighting system, the imaging system and the focusing system into a whole, greatly improves the focusing precision and is beneficial to the small-sized batch production of equipment.

Description

Translated fromChinese

一种双色荧光显微系统A two-color fluorescent microscope system

技术领域technical field

本发明涉及荧光显微成像领域，尤其是一种双色荧光显微系统。The invention relates to the field of fluorescent microscopic imaging, in particular to a two-color fluorescent microscopic system.

背景技术Background technique

荧光显微成像在生物医学研究领域有着重要作用，尤其是在临床诊断等领域日益受到人们的广泛重视。在现有的荧光显微成像系统中人们都是通过对生物芯片的观察来进行研究的，生物芯片自身具有一定的厚度，中间具备流体，呈现一个三明治结构，这样光线经过生物芯片时就会经过多个面的反射，每个面的反射都需要对焦，而现有的荧光显微系统中对焦系统与荧光显微系统是处于分离的状态，不利于对焦，还会造成体积大，不利于设备的小型化，集成化，而且分离的两个系统兼容性不佳，会导致拍照虚焦现象特别严重，影响实验进度。Fluorescence microscopic imaging plays an important role in the field of biomedical research, especially in the fields of clinical diagnosis and other fields, which has been paid more and more attention by people. In the existing fluorescence microscopy imaging system, people conduct research by observing the biochip. The biochip itself has a certain thickness, and there is a fluid in the middle, presenting a sandwich structure, so that when the light passes through the biochip, it will pass through the biochip. The reflection of multiple surfaces, the reflection of each surface needs to be focused, and the focus system and the fluorescence microscope system in the existing fluorescence microscope system are in a separate state, which is not conducive to focusing, and will also cause large volume, which is not conducive to equipment The miniaturization, integration, and poor compatibility of the two separated systems will lead to a particularly serious phenomenon of virtual focus in the photo, which will affect the progress of the experiment.

发明内容Contents of the invention

针对现有的不足，本发明提供一种双色荧光显微系统。Aiming at the existing deficiencies, the invention provides a two-color fluorescence microscope system.

本发明解决其技术问题所采用的技术方案是：一种双色荧光显微系统，包括照明系统光路组件、对焦系统光路组件、物镜系统光路组件和成像系统光路组件，所述照明系统光路组件包括两种具有不同波长的照明光源、将两种照明光源发出的光合成同一光路并使指定波长的光射向对焦系统光路组件的第一光路组件；所述对焦系统光路组件包括用于调焦的对焦光源、能调节对焦光源轴上光束主光线角度的第二光路组件、反射第一光路组件所透射光线并透射透过第二光路组件光线的第一二向色镜、透射经过第一二向色镜反射和透射光线的第一管镜；所述物镜系统光路组件包括与对焦光源处于共轴光路上的物镜、设于物镜和第一管镜之间光路上的第二二向色镜、驱动物镜相对对焦光源移动的驱动电机；所述成像系统光路组件用于拍摄经过第二二向色镜反射的荧光形成图像。The technical solution adopted by the present invention to solve the technical problem is: a two-color fluorescent microscope system, including an optical path assembly of an illumination system, an optical path assembly of a focusing system, an optical path assembly of an objective lens system, and an optical path assembly of an imaging system. The optical path assembly of the illumination system includes two An illumination light source with different wavelengths, combining the light emitted by the two illumination sources into the same optical path and directing the light of the specified wavelength to the first optical path assembly of the optical path assembly of the focusing system; the optical path assembly of the focusing system includes a focusing light source for focusing , the second optical path component capable of adjusting the angle of the chief ray of the light beam on the axis of the focusing light source, the first dichroic mirror that reflects the light transmitted by the first optical path component and transmits the light through the second optical path component, and transmits the light through the first dichroic mirror The first tube mirror that reflects and transmits light; the objective lens system optical path assembly includes an objective lens on the coaxial optical path with the focus light source, a second dichroic mirror on the optical path between the objective lens and the first tube lens, and a driving objective lens A drive motor that moves relative to the focusing light source; the optical path assembly of the imaging system is used to shoot the fluorescent light reflected by the second dichroic mirror to form an image.

作为优选，所述第二光路组件包括设于第一二向色镜和对焦光源光路上的小孔光阑、处于小孔光阑和对焦光源之间的激光衰减片。Preferably, the second optical path assembly includes an aperture stop arranged on the optical path between the first dichroic mirror and the focus light source, and a laser attenuation sheet between the aperture stop and the focus light source.

作为优选，所述对焦光源是半导体激光二极管、固体激光器中的任意一种。Preferably, the focused light source is any one of semiconductor laser diodes and solid-state lasers.

作为优选，所述第一光路组件包括两个聚光准直透镜、两个复眼透镜、一个第三二向色镜和一个激发滤光片；每个所述照明光源射出光线的光路上均依次设置一个聚光准直透镜和一个复眼透镜，所述第三二向色镜设置在两个复眼透镜之后且两个复眼透镜对应于第三二向色镜的反射和透射光路设置，所述激发滤光片设置在第三二向色镜反射光和透射光的光路上，所述激发滤光片透过的光射向第一二向色镜。Preferably, the first optical path assembly includes two condensing and collimating lenses, two fly-eye lenses, a third dichroic mirror and an excitation filter; A condensing collimator lens and a fly-eye lens are set, the third dichroic mirror is arranged behind the two fly-eye lenses and the two fly-eye lenses are set corresponding to the reflection and transmission optical path of the third dichroic mirror, the excitation The optical filter is arranged on the optical path of the reflected light and the transmitted light of the third dichroic mirror, and the light transmitted by the excitation optical filter is directed to the first dichroic mirror.

作为优选，所述第三二向色镜和激发滤光片之间的光路上还设有一个积分聚焦透镜，所述第一二向色镜和激发滤光片之间的光路上还设有一个消杂光光阑。Preferably, an integral focusing lens is also provided on the optical path between the third dichroic mirror and the excitation filter, and an integral focusing lens is also provided on the optical path between the first dichroic mirror and the excitation filter. A stray light reduction diaphragm.

作为优选，两个所述照明光源以射出光线成直角的方式设置。Preferably, the two illumination light sources are arranged in such a way that the outgoing light rays are at right angles to each other.

作为优选，所述成像系统光路组件包括在第二二向色镜反射光路上依次设置的出射滤光片、第二管镜和工业相机。Preferably, the optical path assembly of the imaging system includes an exit filter, a second tube mirror and an industrial camera sequentially arranged on the reflection optical path of the second dichroic mirror.

作为优选，所述主光线角度的调节范围是0-45度。Preferably, the adjustment range of the chief ray angle is 0-45 degrees.

作为优选，所述成像系统光路组件和驱动电机均与一控制主机电性连接。Preferably, the optical path assembly of the imaging system and the driving motor are both electrically connected to a controller.

作为优选，所述照明光源包括红光LED灯和绿光LED灯。Preferably, the illumination light source includes a red LED lamp and a green LED lamp.

本发明的有益效果在于：该发明将照明系统、成像系统、对焦系统集成于一体，使得对焦精度大幅改善，有利于设备的小型化批量化生产，将照明系统和对焦系统光线形成共轴光路，并利用驱动电机对物镜的驱动就可以对生物芯片的每个面进行自动对焦，提高了对焦的精度，使得成像质量得到改善。The beneficial effect of the present invention is that the invention integrates the lighting system, the imaging system, and the focusing system, so that the focusing accuracy is greatly improved, which is conducive to the miniaturization and batch production of equipment, and the light rays of the lighting system and the focusing system form a coaxial optical path, And by using the driving motor to drive the objective lens, each surface of the biological chip can be automatically focused, which improves the precision of focusing and improves the imaging quality.

附图说明Description of drawings

图1是本发明实施例的系统光路结构示意图；Fig. 1 is a schematic diagram of the system optical path structure of an embodiment of the present invention;

图2是生物芯片反射光线的结构示意图；Fig. 2 is a schematic diagram of the structure of the biochip reflecting light;

图中零部件名称及序号：1-红光LED灯2-聚光准直透镜3-复眼透镜4-第三二向色镜5-积分聚焦透镜6-激发滤光片7-消杂光光阑8-激光衰减片9-小孔光阑10-第一二向色镜11-第一管镜12-第二二向色镜13-物镜14-出射滤光片15-第二管镜16-工业相机17-绿光LED灯18-对焦光源19-驱动电机。Parts and serial numbers in the figure: 1-Red LED lamp 2-Concentrating collimating lens 3-Fly eye lens 4-Third dichroic mirror 5-Integral focusing lens 6-Excitation filter 7-Stray light elimination Diaphragm 8-laser attenuator 9-aperture diaphragm 10-first dichroic mirror 11-first tube mirror 12-second dichroic mirror 13-objective lens 14-exit filter 15-second tube mirror 16 -Industrial camera 17-Green LED light 18-Focus light source 19-Drive motor.

具体实施方式Detailed ways

为了更清楚地说明本发明实施例的目的、技术方案和优点，下面将结合实施例对本发明作进一步说明，进行清楚、完整的描述，显然，所描述的实施例是本发明的部分实施例，而不是全部实施例。基于本发明的实施例，本领域普通技术人员在没有付出创造性劳动的前提下所获得的所有其他实施例，都属于本发明的保护范围。In order to more clearly illustrate the purpose, technical solutions and advantages of the embodiments of the present invention, the present invention will be further described below in conjunction with the embodiments, and a clear and complete description will be made. Obviously, the described embodiments are part of the embodiments of the present invention. rather than all examples. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

在本发明中，如图1至图2中所示，一种双色荧光显微系统，包括照明系统光路组件、对焦系统光路组件、物镜系统光路组件和成像系统光路组件，就可以将成像系统光路组件和照明系统光路组件以一种上下结构的布局方式设置在一起，缩减整个系统的高度和尺寸，有利于设备的小型化批量化生产，所述照明系统光路组件包括两种具有不同波长的照明光源、将两种照明光源发出的光合成同一光路并使指定波长的光射向对焦系统光路组件的第一光路组件，即在照明系统光路组件中是有两个照明光源的，一个照明光源发出一种波长的光，两个照明光源发出的光的波长是不同的，如选择照明光源为红光LED灯1和绿光LED灯17，第一光路组件是将两个照明光源发出的光线合束成一个光路，然后再将指定波长的光透过，第一光路组件包括两个聚光准直透镜2、两个复眼透镜3、一个第三二向色镜4和一个激发滤光片6；每个所述照明光源射出光线的光路上均依次设置一个聚光准直透镜2和一个复眼透镜3，所述第三二向色镜4设置在两个复眼透镜3之后且两个复眼透镜3对应于第三二向色镜4的反射和透射光路设置，所述激发滤光片6设置在第三二向色镜4反射光和透射光的光路上，所述激发滤光片6透过的光射向第一二向色镜10，此时选择红光LED灯1是透射光路，绿光LED灯17是反射光路，也就是说红光LED灯1发出的光经过聚光准直透镜2和复眼透镜3后再经过第三二向色镜4透射，绿光LED灯17发出的光经过聚光准直透镜2和复眼透镜3后再经过第三二向色镜4反射，在相同原理下红光LED灯1和绿光LED灯17所处的光路位置可以进行互换，亦即两个照明光源以射出光线成直角的方式设置，第三二向色镜4处在两个光线的交叉处即可，这样整个光线的光路就是，LED灯光源发射的光线经过聚光准直透镜2之后形成类准直光路，两个类准直光路分别入射到复眼透镜3后经过第三二向色镜4合成同一个光路，激发滤光片6则用于过滤红光LED灯1和绿光LED灯17光源的波长，只允许特定波长的光透过，利于激发特定的荧光染料；所述第三二向色镜4和激发滤光片6之间的光路上还设有一个积分聚焦透镜5，这样复眼透镜3对准直光线进行重新划分分布后再经过积分聚焦透镜5后形成光斑形状可调，大小可控的矩形光斑，而光斑的形状(例如方形，三角形、六边形等)，尺寸以及长宽比就依据复眼透镜3和积分聚焦透镜5而定，也就解决了现有照明光斑是一个圆形光斑，在相机端呈现内接圆或外接圆的形式，对于内接圆，相机四个角的像素没得到有效利用，对于外接圆的照明形式，照明光斑变大，相机四个角像素得到有效利用，但是会造成物镜下端视场区域外侧荧光多次激发，不仅造成荧光基团寿命衰减还会生成多余的荧光，造成背景噪音拉高，影响图片的信噪比的问题，对照明光斑做整形处理，提升光效的同时，降低背景噪音，提升信噪比和荧光基团的寿命以及提高测序设备单位时间测序信息的吞吐量，对光束整形，提升了激发光源的光效，提升了信噪比，避免物镜视场之外的荧光基团受到多次激发，提升荧光基团的寿命，解决了现有系统照明方式光效利用率低，相机需要增加曝光时间来弥补光效，影响相机的拍照整体时间，拉长测序设备的测序时间，测序通量大幅降低的问题，所述第一二向色镜10和激发滤光片6之间的光路上还设有一个消杂光光阑7，消杂光光阑7用于放置在激发滤光片6的后面用于抑制多余的杂散光，合束之后的红光和绿光经过消杂光光阑7之后通过第一二向色镜10反射进入第一管镜11后形成无限远光束透过第二二向色镜12进入无限远显微物镜后聚焦在物镜13的后焦面上激发荧光染料基团释放荧光信号；所述对焦系统光路组件包括用于调焦的对焦光源18、能调节对焦光源18轴上光束主光线角度的第二光路组件、反射第一光路组件所透射光线并透射透过第二光路组件光线的第一二向色镜10、透射经过第一二向色镜10反射和透射光线的第一管镜11，对焦光源18是半导体激光二极管、固体激光器中的任意一种，或者是其它在对焦光源端做聚焦微孔处理的LED灯等，第二光路组件包括设于第一二向色镜10和对焦光源18光路上的小孔光阑9、处于小孔光阑9和对焦光源18之间的激光衰减片8，对焦光源18通过激光衰减片8之后入射小孔光阑9，使得对焦光源18的轴上光束主光线角度发生变化，主光线角度的调节范围可选择为0-45度，如从轴上主光线的0角度变成10度、15度、30度等，带有主光线角度偏移的对焦光线入射至第一二向色镜10之后透射和照明系统进行合束形成照明系统和自动对焦系统的共轴光路，进入第一管镜11后形成无限远光束透过第二二向色镜12进入物镜13后聚焦在物镜13的后焦面上，对焦光线在进入生物芯片后会经过多次生物芯片的反射进入物镜13，生物芯片的每一个面都是一个自动对焦的反射面，如图2中所示，给出了三个反射面的反射光线的示意图，就可以对生物芯片的多个面进行自动对焦，也就是说，无论生物芯片在哪一个面上具备生物荧光基团，该对焦系统都可以进行准确的对焦，就确保了物镜13能够准确对焦，在对焦成功之后，红光和绿光依次打开进行拍照；所述物镜系统光路组件包括与对焦光源18处于共轴光路上的物镜13、设于物镜13和第一管镜11之间光路上的第二二向色镜12、驱动物镜13相对对焦光源18移动的驱动电机19，物镜13就可以选择大视场的显微物镜，使得单张拍照的范围达到4.6mm，视野面积最大化，解决现有成像系统视场小，单张拍照的视野太局限，需要增加拍照数量，造成测序时间无限拉长，测序通量大幅降低的问题；所述成像系统光路组件用于拍摄经过第二二向色镜12反射的荧光形成图像，成像系统光路组件包括在第二二向色镜12反射光路上依次设置的出射滤光片14、第二管镜15和工业相机16，红光和绿光经过物镜13聚焦在荧光基团，紧接着荧光基团释放荧光信号被物镜13收集后经过第二二向色镜12反射后进入出射滤光片14，此时出射滤光片14可以小角度的倾斜来保证成像质量，又能避免多次反射造成的鬼像，如倾斜角度在15度以内，出射滤光片14只允许特定的被激发的荧光波段的光通过，激发光以及其他的杂散光均被滤除在外，过滤之后的具备特定波段的荧光信号进入第二管镜15，亦即成像系统管镜，经过第二管镜15后聚焦在工业相机16的光电传感器Sensor上，生物芯片有多少个反射面，在光电传感器Sensor上就会具备多少个光斑信号，这些光斑信号就可用于后期的自动对焦处理，经过自动对焦处理后的位置信息会反馈给驱动电机19驱动物镜13的移动，使得对焦精度大幅改善，而自动对焦处理则是先手动对焦记录物镜13处于最佳焦面时驱动电机19所处的Z轴坐标，然后将物镜13在最佳焦面位置沿Z轴方向上下移动触发拍照建立自动对焦原始模型数据库，最后依据Z轴位置变化和相机自动对焦光斑的移动量形成自动对焦曲线，之后在每次正常拍照前将对焦光源打开，相机记录自动对焦光斑位置信息，然后利用该对焦光斑位置信息和自动对焦曲线就可以得出当前物镜13的Z轴坐标和物镜13处于最佳焦面位置时的偏差量，以此偏差量作为驱动电机19的反馈值指导驱动电机19驱动物镜13移动到最佳焦面位置，整个自动对焦光斑位置的计算以及驱动电机19的反馈运动则均由控制主机直接执行，响应快速，将物镜13移动到最佳焦面，由工业相机16拍摄物镜13下端的荧光基团形成图片后保存用于后期处理。In the present invention, as shown in Fig. 1 to Fig. 2, a kind of two-color fluorescent microscope system comprises illumination system optical path assembly, focusing system optical path assembly, objective lens system optical path assembly and imaging system optical path assembly, just can imaging system optical path assembly The components and the optical path components of the lighting system are arranged together in an up-and-down structure, which reduces the height and size of the entire system and is conducive to the miniaturization and mass production of equipment. The optical path components of the lighting system include two types of lighting with different wavelengths Light source, combining the light emitted by the two lighting sources into the same optical path and directing the light of the specified wavelength to the first optical path component of the optical path component of the focusing system, that is, there are two lighting sources in the optical path component of the lighting system, and one lighting source emits one light of different wavelengths, the wavelengths of the light emitted by the two illumination sources are different, such as selecting the illumination sources asred LED lamp 1 andgreen LED lamp 17, the first optical path component is to combine the light beams emitted by the two illumination sources Form an optical path, and then transmit the light of the specified wavelength. The first optical path assembly includes two light-condensingcollimating lenses 2, two fly-eye lenses 3, a thirddichroic mirror 4 and anexcitation filter 6; A condensingcollimating lens 2 and a fly-eye lens 3 are all arranged in sequence on the optical path of each of the illumination light sources emitting light, and the thirddichroic mirror 4 is arranged behind the two fly-eye lenses 3 and the two fly-eye lenses 3 Corresponding to the reflection and transmission optical path setting of the thirddichroic mirror 4, theexcitation filter 6 is arranged on the optical path of the reflected light and the transmitted light of the thirddichroic mirror 4, and theexcitation filter 6 passes through The light of the red light is directed to the firstdichroic mirror 10. At this time, thered LED light 1 is selected as the transmitted light path, and thegreen LED light 17 is the reflected light path, that is to say, the light emitted by the redlight LED light 1 passes through thecondensing collimator 2 and the fly-eye lens 3 are then transmitted through the thirddichroic mirror 4, and the light emitted by thegreen LED lamp 17 is reflected by the thirddichroic mirror 4 after passing through the condensing and collimatinglens 2 and the fly-eye lens 3. In principle, the positions of the light paths of thered LED light 1 and thegreen LED light 17 can be interchanged, that is, the two light sources are arranged so that the emitted light is at right angles, and the thirddichroic mirror 4 is positioned between the two light beams. In this way, the light path of the entire light is that the light emitted by the LED light source passes through the condensing and collimatinglens 2 to form a quasi-collimated light path, and the two quasi-collimated light paths are respectively incident on the fly-eye lens 3 and pass through the third and second Thechromatic mirror 4 synthesizes the same optical path, and theexcitation filter 6 is used to filter the wavelengths of thered LED light 1 and thegreen LED light 17 light source, only allowing light of a specific wavelength to pass through, which is beneficial to excite specific fluorescent dyes; Anintegral focus lens 5 is also provided on the optical path between the thirddichroic mirror 4 and theexcitation filter 6, so that the fly-eye lens 3 re-divides and distributes the collimated light rays and then passes through theintegral focus lens 5 to form a light spot Adjustable shape, controllable rectangular spot, and the shape (such as square, triangle, hexagon, etc.), size and aspect ratio of the spot is determined according to the fly-eye lens 3 and the integral focusinglens 5, which solves the problem The illuminated spot is a circular spot, which is in the form of an inscribed circle or a circumscribed circle on the camera side. For the inscribed circle, the pixels at the four corners of the camera are not effectively used. For the circumscribed circle illumination form, the illumination spot becomes larger. The four corner pixels of the camera are effectively used, but it will cause multiple excitations of fluorescence outside the field of view area at the lower end of the objective lens, which will not only cause the lifetime of the fluorophore to decay, but also generate redundant fluorescence, which will increase the background noise and affect the signal-to-noise ratio of the image. The problem is to reshape the illumination spot, improve the light efficiency, reduce background noise, improve the signal-to-noise ratio and the life of the fluorophore, and improve the throughput of sequencing information per unit time of the sequencing equipment, and shape the beam to improve the excitation light source The light efficiency improves the signal-to-noise ratio, avoids multiple excitations of the fluorophore outside the field of view of the objective lens, improves the life of the fluorophore, and solves the problem of low light efficiency utilization in the existing system lighting method, and the camera needs to increase the exposure time to To make up for the light effect, affect the overall time of taking pictures of the camera, prolong the sequencing time of the sequencing equipment, and greatly reduce the sequencing throughput, the optical path between the firstdichroic mirror 10 and theexcitation filter 6 is also provided with a Astray light stop 7, thestray light stop 7 is used to place behind theexcitation filter 6 to suppress unnecessary stray light, after the combined red light and green light pass through thestray light stop 7 After being reflected by the firstdichroic mirror 10 and entering thefirst tube mirror 11, an infinite light beam is formed, passes through the seconddichroic mirror 12, enters the infinite microscope objective lens, and then focuses on the rear focal plane of theobjective lens 13 to excite the fluorescent dye group. Release the fluorescent signal; the optical path assembly of the focusing system includes a focusinglight source 18 for focusing, a second optical path assembly capable of adjusting the main ray angle of the light beam on the axis of the focusinglight source 18, reflecting the light transmitted by the first optical path assembly and transmitting through the second optical path assembly The firstdichroic mirror 10 of the two optical path assembly light, thefirst tube mirror 11 that transmits through the firstdichroic mirror 10 reflection and transmission light, and the focusinglight source 18 is any one of semiconductor laser diodes and solid lasers, or It is other LED lamps that do focusing microhole processing at the focusing light source end, and the second optical path assembly includes anaperture diaphragm 9 located on the optical path of the firstdichroic mirror 10 and the focusinglight source 18, theaperture diaphragm 9 and the Thelaser attenuation sheet 8 between thefocus light source 18, thefocus light source 18 passes through thelaser attenuation sheet 8 and enters thepinhole diaphragm 9, so that the on-axis beam chief ray angle of thefocus light source 18 changes, and the adjustment range of the chief ray angle can be selected as 0-45 degrees, such as from the 0 angle of the chief ray on the axis to 10 degrees, 15 degrees, 30 degrees, etc., the focused light with the chief ray angle offset enters the firstdichroic mirror 10 and then transmits and illuminates the system Combine beams to form the coaxial optical path of the illumination system and the auto-focus system. After entering thefirst tube lens 11, an infinite light beam is formed, passes through the seconddichroic mirror 12, enters theobjective lens 13, and then focuses on the rear focal plane of theobjective lens 13. Focusing After the light enters the biochip, it will enter theobjective lens 13 through multiple reflections of the biochip, and each face of the biochip is an autofocus reflective surface, as shown in Figure 2, which provides the reflected light rays of the three reflective surfaces The schematic diagram of the biochip can automatically focus on multiple sides of the biochip, that is to say, no matter which side of the biochip has a bioluminescent group, the focusing system can perform accurate focusing, which ensures that theobjective lens 13 can Accurate focus, after the focus is successful, the red light and the green light are turned on in turn to take pictures; the objective lens system optical path assembly includes theobjective lens 13 on the coaxial optical path with the focusinglight source 18, and is located between theobjective lens 13 and thefirst tube lens 11 The seconddichroic mirror 12 on the optical path, thedrive motor 19 that drives theobjective lens 13 to move relative to the focusinglight source 18, and theobjective lens 13 can choose a microscopic objective lens with a large field of view, so that the range of a single photo can reach 4.6mm, and the field of view is the largest It solves the problem that the field of view of the existing imaging system is small, the field of view of a single photo is too limited, and the number of photos needs to be increased, resulting in an infinitely prolonged sequencing time and a significant decrease in the sequencing throughput; the optical path components of the imaging system are used to photograph the The fluorescence reflected by the twodichroic mirrors 12 forms an image, and the optical path assembly of the imaging system includes anexit filter 14, asecond tube lens 15 and anindustrial camera 16 that are sequentially arranged on the reflected light path of the seconddichroic mirror 12, red light and The green light is focused on the fluorophore through theobjective lens 13, and then the fluorophore releases a fluorescent signal that is collected by theobjective lens 13 and then reflected by the seconddichroic mirror 12 and then enters theexit filter 14. At this time, theexit filter 14 can be small The angle of inclination ensures the imaging quality and avoids ghost images caused by multiple reflections. If the inclination angle is within 15 degrees, theexit filter 14 only allows the light of the specific excited fluorescent band to pass through, the excitation light and other The stray light is all filtered out, and the filtered fluorescent signal with a specific band enters thesecond tube lens 15, that is, the tube lens of the imaging system, and after passing through thesecond tube lens 15, it is focused on the photoelectric sensor Sensor of theindustrial camera 16. As many reflective surfaces as there are on the chip, there will be as many spot signals on the photoelectric sensor Sensor. These spot signals can be used for later autofocus processing, and the position information after autofocus processing will be fed back to the drivingmotor 19 to drive theobjective lens 13. move, so that the focus accuracy is greatly improved, and the automatic focus processing is to manually focus and record the Z-axis coordinates of thedrive motor 19 when theobjective lens 13 is in the best focus plane, and then place theobjective lens 13 in the best focus plane position along the Z-axis direction Move up and down to trigger the camera to establish the autofocus original model database, and finally form the autofocus curve according to the Z-axis position change and the movement of the camera's autofocus spot. After that, the focus light source is turned on before each normal photo shoot, and the camera records the position information of the autofocus spot. Then, the Z-axis coordinates of the currentobjective lens 13 and the deviation amount when theobjective lens 13 is in the best focal plane position can be obtained by utilizing the focusing spot position information and the autofocus curve, and the deviation amount is used as the feedback value of the drivingmotor 19 to guide the drivingmotor 19 drives theobjective lens 13 to move to the best focal plane position, the calculation of the entire auto-focus spot position and the feedback movement of thedrive motor 19 are all directly executed by the control host, and the response is fast, and theobjective lens 13 is moved to the best focal plane. 16 Take pictures of the fluorescent groups at the lower end of theobjective lens 13 and save them for later processing.

进一步的改进，所述成像系统光路组件和驱动电机19均与一控制主机电性连接，整个双色荧光显微系统的工作方式是在每一次绿光和红光的激发光源打开之前都会先将自动对焦的光源打开判断当前的物镜13位置是否在最佳焦面，如果在最佳焦面则打开红光和绿光激发光，对两种激发光进行相机拍照，如果控制主机依据成像系统光路组件也就是工业相机所成图像以及自动对焦曲线判断当前物镜13位置不在最佳焦面，就根据自动对焦处理和自动对焦曲线给出驱动电机19需要反馈的位移量，将物镜13移动至最佳焦面后方可进行红光和绿光的拍照，控制主机就作为接收处理成像光路组件产生的图像并将依据相应信息去调节驱动电机19的动作来实现自动对焦。As a further improvement, the optical path components of the imaging system and the drivingmotor 19 are electrically connected to a control main unit, and the working mode of the entire two-color fluorescence microscope system is to automatically switch on each time before the excitation light sources of green light and red light are turned on. Turn on the focused light source to judge whether the current position of theobjective lens 13 is on the best focal plane. If it is on the best focal plane, turn on the red and green excitation lights, and take pictures of the two excitation lights. If the control host is based on the imaging system optical path components That is to say, the image formed by the industrial camera and the auto-focus curve judge that the current position of theobjective lens 13 is not in the best focus plane, and the displacement amount that thedrive motor 19 needs to feedback is given according to the auto-focus processing and the auto-focus curve, and theobjective lens 13 is moved to the best focus plane. The red light and green light can be photographed behind the camera, and the control host is used to receive and process the images generated by the imaging optical path components and adjust the action of thedrive motor 19 according to the corresponding information to achieve autofocus.

应当理解的是，对本领域普通技术人员来说，可根据上述说明加以改进或变换，而所有这些改进和变换都应属于本发明所附权利要求的保护范围。It should be understood that those skilled in the art can make improvements or changes based on the above description, and all these improvements and changes should fall within the protection scope of the appended claims of the present invention.

Claims (10)

Translated fromChinese

1.一种双色荧光显微系统，其特征在于:包括照明系统光路组件、对焦系统光路组件、物镜系统光路组件和成像系统光路组件，所述照明系统光路组件包括两种具有不同波长的照明光源、将两种照明光源发出的光合成同一光路并使指定波长的光射向对焦系统光路组件的第一光路组件；所述对焦系统光路组件包括用于调焦的对焦光源、能调节对焦光源轴上光束主光线角度的第二光路组件、反射第一光路组件所透射光线并透射透过第二光路组件光线的第一二向色镜、透射经过第一二向色镜反射和透射光线的第一管镜；所述物镜系统光路组件包括与对焦光源处于共轴光路上的物镜、设于物镜和第一管镜之间光路上的第二二向色镜、驱动物镜相对对焦光源移动的驱动电机；所述成像系统光路组件用于拍摄经过第二二向色镜反射的荧光形成图像。1. a two-color fluorescence microscope system, is characterized in that: comprise illumination system optical path assembly, focusing system optical path assembly, objective lens system optical path assembly and imaging system optical path assembly, described illumination system optical path assembly comprises two kinds of illumination sources with different wavelengths 1. Combining the light emitted by the two lighting sources into the same optical path and directing the light of the specified wavelength to the first optical path assembly of the focusing system optical path assembly; the focusing system optical path assembly includes a focusing light source for focusing, and can adjust the axis of the focusing light source The second optical path component of the beam chief ray angle, the first dichroic mirror that reflects the light transmitted by the first optical path component and transmits the light through the second optical path component, and the first dichroic mirror that transmits the reflected and transmitted light of the first dichroic mirror tube lens; the optical path assembly of the objective lens system includes an objective lens on the coaxial optical path with the focusing light source, a second dichroic mirror located on the optical path between the objective lens and the first tube lens, and a drive motor that drives the objective lens to move relative to the focusing light source ; The optical path assembly of the imaging system is used to shoot the fluorescence reflected by the second dichroic mirror to form an image.2.根据权利要求1所述双色荧光显微系统，其特征在于:所述第二光路组件包括设于第一二向色镜和对焦光源光路上的小孔光阑、处于小孔光阑和对焦光源之间的激光衰减片。2. according to the described two-color fluorescent microscope system of claim 1, it is characterized in that: described second optical path assembly comprises the pinhole diaphragm that is located at the first dichroic mirror and the focusing light source light path, is positioned at pinhole diaphragm and Laser attenuation sheet between focused light sources.3.根据权利要求1所述双色荧光显微系统，其特征在于:所述对焦光源是半导体激光二极管、固体激光器中的任意一种。3. two-color fluorescent microscope system according to claim 1, is characterized in that: described focus light source is any one in semiconductor laser diode, solid-state laser.4.根据权利要求1所述双色荧光显微系统，其特征在于:所述第一光路组件包括两个聚光准直透镜、两个复眼透镜、一个第三二向色镜和一个激发滤光片；每个所述照明光源射出光线的光路上均依次设置一个聚光准直透镜和一个复眼透镜，所述第三二向色镜设置在两个复眼透镜之后且两个复眼透镜对应于第三二向色镜的反射和透射光路设置，所述激发滤光片设置在第三二向色镜反射光和透射光的光路上，所述激发滤光片透过的光射向第一二向色镜。4. two-color fluorescence microscope system according to claim 1, is characterized in that: described first optical path assembly comprises two light-condensing collimating lenses, two fly-eye lenses, a 3rd dichroic mirror and an excitation filter A condensing collimating lens and a fly-eye lens are arranged in turn on the optical path of each of the light emitted by the illumination light source, and the third dichroic mirror is arranged behind the two fly-eye lenses and the two fly-eye lenses correspond to the first The reflection and transmission optical paths of the three dichroic mirrors are set, the excitation filter is arranged on the optical path of the reflected light and the transmitted light of the third dichroic mirror, and the light transmitted by the excitation filter is directed to the first and second dichroic mirrors. Chromatic mirror.5.根据权利要求4所述双色荧光显微系统，其特征在于:所述第三二向色镜和激发滤光片之间的光路上还设有一个积分聚焦透镜，所述第一二向色镜和激发滤光片之间的光路上还设有一个消杂光光阑。5. according to the described two-color fluorescent microscope system of claim 4, it is characterized in that: the optical path between described 3rd dichroic mirror and excitation filter is also provided with an integral focusing lens, and described first dichroic There is also a stray light stop on the optical path between the color mirror and the excitation filter.6.根据权利要求1所述双色荧光显微系统，其特征在于:两个所述照明光源以射出光线成直角的方式设置。6. The two-color fluorescence microscope system according to claim 1, characterized in that: two said illumination light sources are arranged in a right-angled mode with emitted light.7.根据权利要求1所述双色荧光显微系统，其特征在于:所述成像系统光路组件包括在第二二向色镜反射光路上依次设置的出射滤光片、第二管镜和工业相机。7. two-color fluorescent microscope system according to claim 1, is characterized in that: described imaging system optical path assembly comprises the exit filter, the second tube mirror and industrial camera that are arranged successively on the second dichroic mirror reflection optical path .8.根据权利要求1所述双色荧光显微系统,其特征在于:所述主光线角度的调节范围是0-45度。8. The two-color fluorescence microscope system according to claim 1, characterized in that: the adjustment range of the chief ray angle is 0-45 degrees.9.根据权利要求1所述双色荧光显微系统，其特征在于:所述成像系统光路组件和驱动电机均与一控制主机电性连接。9. The two-color fluorescence microscope system according to claim 1, wherein: the optical path assembly of the imaging system and the driving motor are all electrically connected with a control host.10.根据权利要求1所述双色荧光显微系统,其特征在于:所述照明光源包括红光LED灯和绿光LED灯。10. The two-color fluorescence microscope system according to claim 1, wherein the illumination light source comprises a red LED light and a green LED light.

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