CN102566048A - Astigmatism-based sample axial drift compensating method and device - Google Patents
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Abstract
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Description
Translated fromChinese技术领域technical field
本发明属于高精度、超分辨显微领域，特别涉及一种基于象散的样品轴向漂移补偿方法和装置。The invention belongs to the field of high-precision and super-resolution microscopy, and in particular relates to a sample axial drift compensation method and device based on astigmatism.
背景技术Background technique
在显微系统的工作过程当中，由于热漂移和应力漂移等因素的影响，待测样品不可避免地会随时间发生轴向漂移，形成离焦，从而对显微系统的成像精度造成影响。特别是对于需要多次对同一像素点进行重复扫描的显微方法来说，这种轴向漂移所带来的影响将更为明显，因为轴向漂移将导致多次重复扫描的并非为同一像素点。因此，一种可以实时地测量显微样品的轴向漂移并进行补偿的方法，对于提高显微系统的精度将具有十分重要的作用。During the working process of the microscopic system, due to the influence of factors such as thermal drift and stress drift, the sample to be tested will inevitably drift axially over time, resulting in defocus, which will affect the imaging accuracy of the microscopic system. Especially for microscopic methods that require multiple repeated scans of the same pixel, the impact of this axial drift will be more obvious, because the axial drift will cause multiple repeated scans to not be the same pixel point. Therefore, a method that can measure and compensate the axial drift of the microscopic sample in real time will play a very important role in improving the precision of the microscopic system.
随着科学技术的发展，科研工作者们提出了多种测量样品轴向漂移的方法，其中以光学非接触测量方法的应用最为广泛。目前，光学非接触测量方法多是基于共焦系统。这种系统虽然具有较好的测量精度，但是构造比较复杂，在一定程度上限制了方法的适用范围。With the development of science and technology, researchers have proposed a variety of methods to measure the axial drift of the sample, among which the optical non-contact measurement method is the most widely used. Currently, optical non-contact measurement methods are mostly based on confocal systems. Although this system has good measurement accuracy, its structure is relatively complicated, which limits the scope of application of the method to a certain extent.
发明内容Contents of the invention
本发明提供了一种基于象散的样品轴向漂移补偿方法和装置，装置结构简单，方法使用方便，而且具有较高的测量灵敏度。该种方法和装置可以广泛应用于共聚焦显微镜(Confocal Microscopy)，受激发射损耗显微镜(STED：Stimulated Emission Depletion Microscopy)等高精度超分辨显微设备当中，用以对待测样品的轴向位置漂移进行补偿，从而保证显微系统的精度。The invention provides a sample axial drift compensation method and device based on astigmatism, the device has a simple structure, the method is convenient to use, and has high measurement sensitivity. This method and device can be widely used in high-precision super-resolution microscopy equipment such as confocal microscopy (Confocal Microscopy), stimulated emission depletion microscopy (STED: Stimulated Emission Depletion Microscopy), to measure the axial position drift of the sample to be tested. Compensation is performed to ensure the precision of the microscope system.
一种基于象散的样品轴向漂移补偿方法，包括以下步骤：A sample axial drift compensation method based on astigmatism, comprising the following steps:
(1)由激光器发出的激光光束经过单模光纤和准直透镜进行准直；(1) The laser beam emitted by the laser is collimated through a single-mode fiber and a collimating lens;
(2)经过准直后的光线经过分光棱镜的作用后，分解为第一反射光线和第一透射光线；(2) The collimated light is decomposed into the first reflected light and the first transmitted light after being acted on by the dichroic prism;
(3)所述第一透射光线经过由两个焦距相同且横截面相互正交的柱面镜所构成的象散处理器形成第一象散光束；(3) The first transmitted light passes through an astigmatic processor composed of two cylindrical mirrors with the same focal length and mutually orthogonal cross-sections to form a first astigmatic light beam;
(4)所述第一象散光束通过显微物镜聚焦后投射到置于三维纳米扫描平台上的待测样品之上，经待测样品反射后的光束沿原光路逆向返回，由所述显微物镜收集后，再次经过所述象散处理器形成第二象散光束；(4) The first astigmatic light beam is projected onto the sample to be measured placed on the three-dimensional nano-scanning platform after being focused by a microscope objective lens, and the light beam reflected by the sample to be measured returns along the original optical path in reverse, and is determined by the microscope. After being collected by the micro-objective lens, the second astigmatic beam is formed through the astigmatism processor again;
(5)第二象散光束经所述分光棱镜分解为第二透射光线和第二反射光线；(5) the second astigmatic light beam is decomposed into a second transmitted ray and a second reflected ray by the dichroic prism;
(6)所述第二反射光线作为监控光束经聚焦透镜聚焦，再经过光衰减器后由光电感应器件接收，得到监控光束聚焦光斑的特征参数，标定监控光束聚焦光斑的特征参数与待测样品的轴向漂移量的关系，把这个关系式作为系统标定函数输入计算机，完成系统的标定；(6) The second reflected light is focused by the focusing lens as the monitoring beam, and then received by the photoelectric sensor after passing through the optical attenuator to obtain the characteristic parameters of the focused spot of the monitoring beam, and calibrate the characteristic parameter of the focused spot of the monitoring beam and the sample to be tested The relationship between the axial drift amount, this relationship is input into the computer as a system calibration function to complete the calibration of the system;
(7)系统标定好后，用于样品轴向漂移补偿：当待测样品发生轴向漂移时，计算机根据由所述光电感应器件输出的监控光束聚焦光斑的特征参数在标定好的系统内跟踪查询到相应的待测样品的轴向漂移量，并据此发出指令调整三维纳米扫描平台的轴向位置，实现对待测样品的轴向漂移补偿。(7) After the system is calibrated, it is used to compensate for the axial drift of the sample: when the sample to be tested drifts axially, the computer tracks in the calibrated system according to the characteristic parameters of the monitoring beam focus spot output by the photoelectric sensor The axial drift of the corresponding sample to be tested is queried, and an instruction is issued to adjust the axial position of the three-dimensional nano-scanning platform to realize axial drift compensation of the sample to be tested.
所述的光电感应器件为高速电荷耦合器件(CCD：Charge CoupleDevice)或者四象限探测器(QD：Quadrant Detector)，所述的四象限探测器垂直放置。The photoelectric sensing device is a high-speed charge-coupled device (CCD: Charge Couple Device) or a quadrant detector (QD: Quadrant Detector), and the quadrant detector is placed vertically.
当使用CCD作为光电感应器件时，所述监控光束聚焦光斑的特征参数为监控光束在CCD上所成聚焦光斑强度分布曲线的半高全宽值。When a CCD is used as the photoelectric sensing device, the characteristic parameter of the focused spot of the monitoring beam is the full width half maximum value of the intensity distribution curve of the focused spot formed by the monitoring beam on the CCD.
当使用QD作为光电感应器件时，QD垂直放置，所述监控光束聚焦光斑的特征参数为根据四象限探测器四象限上的输出电流计算得到的四象限输出电流差分值。When the QD is used as the photoelectric sensing device, the QD is placed vertically, and the characteristic parameter of the focused spot of the monitoring beam is the four-quadrant output current differential value calculated according to the output current on the four quadrants of the four-quadrant detector.
本发明还提供了一种基于象散的样品轴向漂移补偿装置，包括：The present invention also provides a sample axial drift compensation device based on astigmatism, including:
激光器；laser;
在所述激光器的出射光光路的光轴上依次设置的单模光纤、准直透镜、分光棱镜、象散处理器、显微物镜和用于放置待测样品的三维纳米扫描平台；其中，所述单模光纤的出射端面位于准直透镜的物方焦点处，所述象散处理器由焦距相同且横截面相互正交的两个柱面镜构成；A single-mode optical fiber, a collimating lens, a beam splitting prism, an astigmatism processor, a microscopic objective lens and a three-dimensional nano-scanning platform for placing a sample to be measured are sequentially arranged on the optical axis of the outgoing light path of the laser; wherein, the The exit end face of the single-mode optical fiber is located at the focal point of the collimator lens, and the astigmatism processor is composed of two cylindrical mirrors with the same focal length and mutually orthogonal cross-sections;
在监控光束光路的光轴上依次设置的聚焦透镜、光衰减器和光电感应器件，所述光电感应器件位于所述聚焦透镜的像方焦点处；其中，所述监控光束光路的光轴与所述激光器的出射光光路的光轴垂直，所述监控光束为由待测样品反射的光依次被所述显微物镜收集和所述象散处理器进行象散后再由所述分光棱镜分光得到的反射光线；A focusing lens, an optical attenuator and a photoelectric sensing device are arranged in sequence on the optical axis of the monitoring beam optical path, and the photoelectric sensing device is located at the image focus of the focusing lens; wherein, the optical axis of the monitoring beam optical path is in line with the The optical axis of the outgoing light path of the laser is vertical, and the monitoring beam is obtained by sequentially collecting the light reflected by the sample to be measured and being astigmatized by the astigmatism processor and then split by the beam splitting prism the reflected light;
以及同时与所述三维纳米扫描平台和光电感应器件均相连的计算机。And a computer connected to the three-dimensional nano-scanning platform and the photoelectric sensing device at the same time.
本发明装置中，所述激光器用于发出激光光束，所述单模光纤和准直透镜用于对所述激光光束进行准直，所述分光棱镜用于分光，所述象散处理器用于对入射光束的光斑形状进行调制得到象散光束，所述显微物镜用于入射的光线聚焦投射到待测样品上并用于收集经待测样品反射的光束，所述聚焦透镜用于将监控光束聚焦到光电感应器件的感应面上，所述光衰减器用于进行光衰减以避免光电感应器件出现光强饱和，所述光电感应器件用于接收监控光束聚焦光斑并根据接收信号输出相应的监控信息(监控光束聚焦光斑的特征参数)，所述计算机用于接收光电感应器件反馈的监控信息并进行分析处理后发出调整控制信号，所述三维纳米扫描平台用于放置待测样品并根据所述计算机发出的调整控制信号来调整扫描平台上待测样品的轴向位置。In the device of the present invention, the laser is used to emit a laser beam, the single-mode fiber and collimating lens are used to collimate the laser beam, the beam splitting prism is used to split light, and the astigmatism processor is used to The spot shape of the incident beam is modulated to obtain an astigmatic beam. The microscopic objective lens is used to focus and project the incident light onto the sample to be tested and to collect the beam reflected by the sample to be tested. The focusing lens is used to focus the monitoring beam On the sensing surface of the photoelectric sensing device, the optical attenuator is used for light attenuation to avoid light intensity saturation of the photoelectric sensing device, and the photoelectric sensing device is used for receiving the focused spot of the monitoring beam and outputting corresponding monitoring information according to the received signal ( monitor the characteristic parameters of the focused light spot of the light beam), the computer is used to receive the monitoring information fed back by the photoelectric sensing device and send out an adjustment control signal after analyzing and processing, and the three-dimensional nano-scanning platform is used to place the sample to be tested and send out an adjustment signal according to the computer. The adjustment control signal is used to adjust the axial position of the sample to be measured on the scanning platform.
优选的技术方案中，所述象散处理器中，第一柱面镜横截面与水平面的夹角为45°，第二柱面镜横截面与水平面的夹角为135°。In a preferred technical solution, in the astigmatism processor, the angle between the cross section of the first cylindrical mirror and the horizontal plane is 45°, and the angle between the cross section of the second cylindrical mirror and the horizontal plane is 135°.
优选的技术方案中，所述象散处理器中，两个柱面镜焦距为150mm。In a preferred technical solution, in the astigmatism processor, the focal length of the two cylindrical mirrors is 150mm.
优选的技术方案中，所述光电感应器件为高速电荷耦合器件(CCD)或者四象限探测器(QD)。In a preferred technical solution, the photoelectric sensing device is a high-speed charge-coupled device (CCD) or a four-quadrant detector (QD).
本发明原理如下：Principle of the present invention is as follows:
对于一个柱面镜来说，其横截面(即径向截面)可以看成球面透镜，而纵截面(即轴向截面)则可以看成平板。因此，当两条平行光线在柱面镜的横截面内入射时，出射光线会相交于柱面镜焦线上的一点；而当两条平行光线在柱面镜的纵截面内入射时，出射光线将仍然保持平行。所以，柱面镜成像具有象散特性。For a cylindrical lens, its cross section (that is, the radial section) can be regarded as a spherical lens, while the longitudinal section (that is, the axial section) can be regarded as a flat plate. Therefore, when two parallel rays are incident in the cross section of the cylindrical mirror, the outgoing rays will intersect at a point on the focal line of the cylindrical mirror; and when two parallel rays are incident in the longitudinal section of the cylindrical mirror, the outgoing rays The rays will still remain parallel. Therefore, the imaging of the cylindrical mirror has astigmatism.
根据以上所述柱面镜的成像性质，当高斯光束通过单个柱面镜之后，出射光束的光斑将会成椭圆形，且椭圆光斑的长轴与柱面镜的横截面垂直。According to the above-mentioned imaging properties of the cylindrical mirror, when the Gaussian beam passes through a single cylindrical mirror, the spot of the outgoing beam will be elliptical, and the long axis of the elliptical spot is perpendicular to the cross section of the cylindrical mirror.
当高斯光束通过由两个焦距相同且横截面互相正交的柱面镜所构成的象散处理器之后，出射光束的椭圆光斑形状将会随着出射光束的传播而发生变化：在第一柱面镜的像方焦点处，出射光束椭圆光斑的长轴与第一柱面镜的横截面垂直；而在第二柱面镜的像方焦点处，出射光束椭圆光斑的长轴与第二柱面镜的横截面垂直。由于两柱面镜的横截面相互正交，因此当出射光束由第一柱面镜的像方焦点传播到第二柱面镜的像方焦点时，光束椭圆光斑的指向(椭圆的长轴方向)转过了90°。在此过程中，光束椭圆光斑在与第一柱面镜横截面垂直方向上的长度将逐渐变短，在与第二柱面镜横截面垂直方向上的长度将逐渐变长。After the Gaussian beam passes through the astigmatism processor composed of two cylindrical mirrors with the same focal length and cross-sections orthogonal to each other, the shape of the elliptical spot of the outgoing beam will change as the outgoing beam propagates: in the first cylinder At the focal point of the image side of the surface mirror, the long axis of the elliptical spot of the outgoing beam is perpendicular to the cross section of the first cylindrical mirror; while at the focal point of the image square of the second cylindrical mirror, the long axis of the elliptical spot of the outgoing beam is perpendicular to the cross section of the second cylindrical mirror The cross-section of the mirror is vertical. Since the cross-sections of the two cylindrical mirrors are orthogonal to each other, when the outgoing beam propagates from the image focal point of the first cylindrical mirror to the image focal point of the second cylindrical mirror, the direction of the elliptical spot of the beam (the direction of the long axis of the ellipse) ) turned by 90°. During this process, the length of the beam elliptical spot in the direction perpendicular to the cross-section of the first cylindrical mirror gradually becomes shorter, and the length in the direction perpendicular to the cross-section of the second cylindrical mirror gradually becomes longer.
当高斯光束依次通过由两个焦距相同且横截面互相正交的柱面镜所构成的象散处理器和显微物镜之后，出射光束的椭圆光斑的形状同样会随着出射光束的传播而发生变化：在第一柱面镜和显微物镜的组合像方焦点处，出射光束椭圆光斑的长轴与第一柱面镜的横截面垂直；而在第二柱面镜和显微物镜的组合像方焦点处，出射光束椭圆光斑的长轴与第二柱面镜的横截面垂直。由于两柱面镜的横截面相互正交，因此当出射光束由第一柱面镜和显微物镜的组合像方焦点传播到第二柱面镜和显微物镜的组合像方焦点时，光束椭圆光斑的指向(椭圆的长轴方向)转过了90°。在此过程中，光束椭圆光斑在与第一柱面镜横截面垂直方向上的长度将逐渐变短，在与第二柱面镜横截面垂直方向上的长度将逐渐变长。When the Gaussian beam sequentially passes through the astigmatism processor and the microscopic objective lens composed of two cylindrical lenses with the same focal length and orthogonal cross-sections, the shape of the elliptical spot of the outgoing beam will also change with the propagation of the outgoing beam. Change: At the focal point of the combined image side of the first cylindrical lens and the microscopic objective lens, the long axis of the elliptical spot of the outgoing beam is perpendicular to the cross section of the first cylindrical lens; while at the combination of the second cylindrical lens and the microscopic objective lens At the focal point of the image side, the long axis of the elliptical spot of the outgoing beam is perpendicular to the cross section of the second cylindrical mirror. Since the cross-sections of the two cylindrical mirrors are orthogonal to each other, when the outgoing beam propagates from the combined image focal point of the first cylindrical mirror and the microscopic objective lens to the combined image focal point of the second cylindrical mirror and the microscopic objective lens, the light beam The direction of the elliptical light spot (the direction of the long axis of the ellipse) has been rotated by 90°. During this process, the length of the beam elliptical spot in the direction perpendicular to the cross-section of the first cylindrical mirror gradually becomes shorter, and the length in the direction perpendicular to the cross-section of the second cylindrical mirror gradually becomes longer.
第一柱面镜和显微物镜的组合像方焦点与第二柱面镜和显微物镜的组合像方焦点之间的区域视为等效焦区，等效焦区的轴向长度称为等效焦深。等效焦深可按如下公式计算：The area between the combined image focal point of the first cylindrical lens and the microscopic objective lens and the combined image focal point of the second cylindrical lens and the microscopic objective lens is regarded as the equivalent focal area, and the axial length of the equivalent focal area is called Equivalent depth of focus. The equivalent focal depth can be calculated according to the following formula:
其中Δ为等效焦深，f为两柱面镜的焦距，f′为显微物镜的焦距，d1为两柱面镜的间距，d2为显微物镜与第二柱面镜之间的间距。Where Δ is the equivalent focal depth, f is the focal length of the two cylindrical lenses, f′ is the focal length of the microscopic objective lens, d1 is the distance between the two cylindrical lenses, and d2 is the distance between the microscopic objective lens and the second cylindrical lens Pitch.
当待测样品在等效焦区内发生轴向漂移时，第一象散光束经显微物镜聚焦在待测样品上所成椭圆光斑的形状会随之发生改变，其相应的监控光束(由待测样品反射的光依次被所述显微物镜收集和象散处理器进行象散后再由所述分光棱镜分光得到的反射光线为监控光束)聚焦后在光电感应器件上所成椭圆光斑的形状也会随之发生改变。因此，可以标定能反映在光电感应器件上所成椭圆光斑的形状变化的监控光束聚焦光斑的特征参数与样品轴向漂移量的关系并用于样品轴向漂移的实时补偿。When the sample to be measured axially drifts in the equivalent focal area, the shape of the elliptical spot formed by the first astigmatic beam focusing on the sample to be measured through the microscope objective lens will change accordingly, and the corresponding monitoring beam (by The light reflected by the sample to be measured is sequentially collected by the microscopic objective lens and astigmatized by the astigmatism processor, and then the reflected light obtained by the light splitting by the dichroic prism is the monitoring light beam) and is focused on the photoelectric sensor to form an elliptical spot. The shape will also change accordingly. Therefore, it is possible to calibrate the relationship between the characteristic parameters of the monitoring beam focusing spot and the axial drift of the sample, which can reflect the shape change of the elliptical spot formed on the photoelectric sensing device, and use it for real-time compensation of the axial drift of the sample.
本发明的基于象散的样品轴向漂移补偿装置还可以作为一个独立的模块应用于高精度超分辨显微镜的测量光路中，实时地对待测样品的轴向漂移进行补偿。The astigmatism-based sample axial drift compensation device of the present invention can also be used as an independent module in the measurement optical path of a high-precision super-resolution microscope to compensate the axial drift of the sample to be measured in real time.
相对于现有技术，本发明具有以下有益的技术效果：Compared with the prior art, the present invention has the following beneficial technical effects:
(1)装置结构简单，操作方便；(1) The device has a simple structure and is easy to operate;
(2)具有较高的测量灵敏度和较大的测量范围；(2) It has high measurement sensitivity and large measurement range;
(3)调整过程快速准确，且不会对显微镜工作光路形成干扰。(3) The adjustment process is fast and accurate, and will not interfere with the working optical path of the microscope.
附图说明Description of drawings
图1为本发明的基于象散的样品轴向漂移补偿装置示意图；Fig. 1 is a schematic diagram of the sample axial drift compensation device based on astigmatism of the present invention;
图2为本发明的基于象散的样品轴向漂移补偿装置作为一个独立模块应用于显微镜工作光路时的示意图；2 is a schematic diagram of the astigmatism-based sample axial drift compensation device of the present invention when it is applied to the working optical path of a microscope as an independent module;
图3为本发明采用CCD作为光电感应器件时，所测得的半高全宽值与待测样品轴向位置漂移量的对应曲线；Fig. 3 is when the present invention adopts CCD as photoelectric induction device, the corresponding curve of measured full width at half maximum value and axial position drift of the sample to be measured;
图4为本发明采用QD作为光电感应器件时，四象限输出电流差分值与待测样品轴向位置漂移量的对应曲线。Fig. 4 is a corresponding curve of the four-quadrant output current differential value and the axial position drift of the sample to be measured when QD is used as the photoelectric sensing device in the present invention.
具体实施方式Detailed ways
下面结合实施例和附图来详细说明本发明，但本发明并不仅限于此。The present invention will be described in detail below in conjunction with the embodiments and accompanying drawings, but the present invention is not limited thereto.
一种基于象散的样品轴向漂移补偿装置，如图1所示，包括：激光器1、单模光纤2、准直透镜3、分光棱镜4、第一柱面镜5、第二柱面镜6、显微物镜7、三维纳米扫描平台8、聚焦透镜9、光衰减器10、光电感应器件11和计算机12。A sample axial drift compensation device based on astigmatism, as shown in Figure 1, comprising: a
激光器1发射出激光光束，单模光纤2、准直透镜3、分光棱镜4、第一柱面镜5、第二柱面镜6、显微物镜7和三维纳米扫描平台8依次位于激光器1的出射光光路的光轴上，其中，单模光纤2的出射端面位于准直透镜3的物方焦点，第一柱面镜5的横截面与水平面的夹角为45°，第二柱面镜6的横截面与水平面的夹角为135°，第一柱面镜5和第二柱面镜6焦距相同，显微物镜7将光线聚焦到位于三维纳米扫描平台8的待测样品上，三维纳米扫描平台8可以调整待测样品的轴向位置。The
聚焦透镜9、光衰减器10和光电感应器件11依次位于监控光束R2光路的光轴上，光电感应器件11位于聚焦透镜9的像方焦点处；其中，监控光束R2光路的光轴与激光器1的出射光光路的光轴垂直，监控光束R2为由待测样品反射的光依次被显微物镜7收集和象散处理器(由第一柱面镜5和第二柱面镜6构成)进行象散后再由分光棱镜4分光得到的反射光线；The focusing lens 9, the
计算机12同时连接三维纳米扫描平台8和光电感应器件11。The
上述装置中，光衰减器10的作用是为避免光电感应器件11出现光强饱和。光电感应器件11为高速电荷耦合器件(CCD)或者四象限探测器(QD)。In the above device, the function of the
采用图1所示的装置进行基于象散的样品轴向漂移补偿方法如下：Using the device shown in Figure 1 to compensate the axial drift of the sample based on astigmatism is as follows:
从激光器1发出的激光光束，首先被导入单模光纤2，从单模光纤2出射的激光光束，经过准直透镜3完成准直。The laser beam emitted from the
准直后的光束通过分光棱镜4后分解为第一透射光线和第一反射光线，第一透射光线通过象散处理器(由第一柱面镜5和第二柱面镜6构成)后形成第一象散光束R1。The collimated light beam is decomposed into the first transmitted light and the first reflected light after passing through the
第一象散光束R1经显微物镜7聚焦后投射到位于三维纳米扫描平台8上的待测样品之上，经待测样品反射后的光束沿原光路逆向返回，由显微物镜7收集后，再次经过该象散处理器(由第一柱面镜5和第二柱面镜6组成)形成第二象散光束，第二象散光束经分光棱镜4作用分解为第二透射光线和第二反射光线，其中第二反射光线为监控光束R2。The first astigmatic beam R1 is focused by the microscope
监控光束R2经聚焦透镜9聚焦，再经过光衰减器10后由光电感应器件11接收，得到监控光束聚焦光斑的特征参数，标定监控光束聚焦光斑的特征参数与待测样品的轴向漂移量的关系，把这个关系式作为系统标定函数输入计算机12，完成系统的标定。The monitoring beam R2 is focused by the focusing lens 9, and then received by the
系统标定好后，用于样品轴向漂移补偿：当待测样品发生轴向漂移时，计算机12根据由所述光电感应器件输出的监控光束聚焦光斑的特征参数在标定好的系统内跟踪查询到相应的待测样品的轴向漂移量，并据此发出指令调整三维纳米扫描平台8的轴向位置，实现对待测样品的轴向漂移补偿。After the system is calibrated, it is used to compensate the axial drift of the sample: when the sample to be tested drifts axially, the
具体来说：Specifically:
当使用CCD作为光电感应器件11时，将待测样品放在三维纳米扫描平台8上，通过调整三维纳米扫描平台8的轴向位置，并实时获取监控光束R2在CCD上所成聚焦光斑强度分布曲线的半高全宽值，从而得到聚焦光斑的半高全宽值与待测样品的轴向漂移量的关系，曲线如图3所示，把这个关系式作为系统标定函数输入计算机12，完成系统的标定。When using a CCD as the
系统标定好后，用于样品轴向漂移补偿：当待测样品发生轴向漂移时，CCD将聚焦光斑的半高全宽值传送给计算机12，计算机12在标定好的系统内跟踪查询到相应的待测样品的轴向位置漂移量，并据此发出指令调整三维纳米扫描平台8的轴向位置，实现待测样品轴向漂移补偿。当所测聚焦光斑的半高全宽值与样品无轴向漂移时的标定值相等时，即完成了对待测样品轴向位置漂移补偿。After the system is calibrated, it is used to compensate the axial drift of the sample: when the sample to be measured drifts axially, the CCD transmits the full width at half maximum value of the focused spot to the
上述提及的聚焦光斑的半高全宽值是通过以下方式得到的：过CCD光电感应面的中心，在感应面内作一条与水平方向夹角为45°的直线，计算该直线上各点光强分布曲线的半高全宽值。由于根据聚焦光斑的半高全宽值可以唯一确定聚焦光斑的形状，因此这样情况下监控光束在所述光电感应器件上所成聚焦光斑强度分布曲线的半高全宽值可以作为监控光束聚焦光斑的特征参数。The full width at half maximum of the focus spot mentioned above is obtained by the following method: through the center of the CCD photoelectric sensing surface, draw a straight line with an angle of 45° with the horizontal direction in the sensing face, and calculate the light intensity of each point on the straight line The full width at half maximum value of the distribution curve. Since the shape of the focused spot can be uniquely determined according to the full width at half maximum of the focused spot, the full width at half maximum of the intensity distribution curve of the focused spot formed by the monitoring beam on the photoelectric sensing device can be used as a characteristic parameter of the focused spot of the monitoring beam.
当使用QD作为光电感应器件时，将QD垂直放置。根据QD的探测原理，其四象限上的输出电流分别与照射在各个象限上的光斑能量呈线性关系，因此，可以通过对各象限的输出电流进行差分运算来表征监控光束所成椭圆光斑的形状，具体公式如下：When using QDs as photo-sensing devices, place the QDs vertically. According to the detection principle of QD, the output current on the four quadrants is linearly related to the energy of the spot irradiated on each quadrant. Therefore, the shape of the elliptical spot formed by the monitoring beam can be characterized by performing a differential operation on the output current of each quadrant. , the specific formula is as follows:
Δr＝I1+I3-I2-I4Δr=I1 +I3 -I2 -I4
其中，I1、I2、I3、I4分别为光束在QD四象限上的输出电流，Δr为四象限输出电流差分值，作为监控光束聚焦光斑的特征参数用以表征聚焦光斑的形状。Among them, I1 , I2 , I3 , and I4 are the output currents of the beam on the four quadrants of the QD respectively, and Δr is the differential value of the output current of the four quadrants, which is used as a characteristic parameter of the focused spot of the monitoring beam to characterize the shape of the focused spot.
同样，将待测样品放在三维纳米扫描平台8上，通过调整三维纳米扫描平台8的轴向位置，并实时记录QD上四象限上的输出电流，并计算四象限输出电流差分值Δr，从而得到四象限输出电流差分值Δr与待测样品的轴向漂移量的关系，曲线如图4所示，把这个关系式作为系统标定函数输入计算机12，完成系统的标定。Similarly, the sample to be tested is placed on the three-dimensional nano-
系统标定好后，用于样品轴向漂移补偿：当待测样品发生轴向漂移时，QD将计算得到的四象限输出电流差分值Δr传送给计算机12，计算机12在标定好的系统内跟踪查询到相应的待测样品的轴向位置漂移量，并据此发出指令调整三维纳米扫描平台8的轴向位置，实现待测样品轴向漂移补偿。当四象限输出电流差分值Δr与样品无轴向漂移时的标定值相等时，即完成了对待测样品轴向位置漂移补偿。After the system is calibrated, it is used for sample axial drift compensation: when the sample to be tested has axial drift, QD will transmit the calculated four-quadrant output current differential value Δr to the
整个过程实时监控并不断循环进行，从而完成对待测样品轴向漂移的实时补偿。The whole process is monitored in real time and continuously circulated, so as to complete the real-time compensation for the axial drift of the sample to be tested.
图1中黑框内结构可以构成一个独立的样品轴向漂移补偿模块应用于高精度超分辨显微镜的测量光路中，实时地对待测样品的轴向漂移进行补偿。具体工作光路如图2所示。The structure inside the black frame in Figure 1 can constitute an independent sample axial drift compensation module, which can be applied to the measurement optical path of a high-precision super-resolution microscope to compensate the axial drift of the sample to be measured in real time. The specific working optical path is shown in Figure 2.
即，采用了本发明样品轴向漂移补偿模块的高精度超分辨显微镜系统，包括：显微物镜7，三维纳米扫描平台8，计算机12，二向色镜13、14，基于象散的样品轴向漂移补偿模块15，显微镜聚焦透镜16和显微镜探测器17。That is, the high-precision super-resolution microscope system using the sample axial drift compensation module of the present invention includes: a microscope
其中基于象散的样品轴向漂移补偿模块15的内部具体结构如图1中黑框内部结构所示，包括：激光器1、单模光纤2、准直透镜3、分光棱镜4、第一柱面镜5、第二柱面镜6、聚焦透镜9、光衰减器10和光电感应器件11。The internal specific structure of the sample axial
将本发明基于象散的样品轴向漂移补偿模块15应用于高精度超分辨显微镜系统中对样品轴向漂移进行补偿的方法如下：The method of applying the astigmatism-based sample axial
由基于象散的样品轴向漂移补偿模块15发出的第一象散光束R1，经过二向色镜14后形成反射，反射光束和显微镜工作光束R3经显微物镜7聚焦后投射到待测样品上。经待测样品反射的象散光束和显微镜工作光束在经过二向色镜14时形成分离，其中经待测样品反射的象散光束被二向色镜14反射，由基于象散的样品轴向漂移补偿模块15接收并转变为监控光束R2；而经待测样品反射的显微镜工作光束则透过二向色镜14，之后再透过二向色镜13形成显微镜成像光束R4并与入射的工作光束分离，显微镜成像光束R4经过显微镜聚焦透镜16聚焦后被显微镜探测器17接收，用于显微成像。The first astigmatic beam R1 emitted by the sample axial
基于象散的样品轴向漂移补偿模块15根据监控光束R2所成的聚焦光斑信息输出相应的监控信号并传送给计算机12，经过计算机12处理后转变为相应的控制信号用于调整三维纳米扫描平台8的轴向位置，从而实现对待测样品轴向漂移的补偿。The sample axial
在实际操作中，整个过程实时监控并不断循环进行，从而完成对待测样品轴向漂移的实时补偿。In actual operation, the whole process is monitored in real time and continuously circulated, so as to complete the real-time compensation for the axial drift of the sample to be tested.
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