CN106970055B - A three-dimensional fluorescent differential super-resolution microscopy method and device - Google Patents
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Abstract
本发明公开一种三维荧光差分超分辨显微装置，包括激光器、承载待测样品的电动样品台和将光线投射到所述电动样品台的显微物镜；所述的激光器和显微物镜之间依次设有：用于将所述激光器发出的光束改变为线偏振光的起偏器；用于调制所述线偏振光偏振方向的第一二分之一波片；用于依次调制光束水平分量和垂直分量的空间光调制模块；用于对圆偏振光进行光路偏转的扫描振镜系统，由所述扫描振镜系统出射的圆偏振光经显微物镜投射到待测样品上；还包括采集待测样品发出的信号光的探测系统，以及控制所述空间光调制模块和扫描振镜系统的计算机。本发明还公开基于上述三维荧光差分超分辨显微装置实现的显微方法。
The invention discloses a three-dimensional fluorescent differential super-resolution microscopic device, which includes a laser, an electric sample stage carrying a sample to be tested, and a microscopic objective lens for projecting light onto the electric sample stage; It is provided in sequence: a polarizer for changing the beam emitted by the laser into linearly polarized light; a first half-wave plate for modulating the polarization direction of the linearly polarized light; and for sequentially modulating the horizontal component of the beam and a vertical component spatial light modulation module; a scanning galvanometer system for deflecting the optical path of circularly polarized light, and the circularly polarized light emitted by the scanning galvanometer system is projected onto the sample to be measured through a microscope objective lens; it also includes an acquisition A detection system for the signal light emitted by the sample to be tested, and a computer for controlling the spatial light modulation module and the scanning galvanometer system. The invention also discloses a microscopic method realized based on the above-mentioned three-dimensional fluorescent difference super-resolution microscopic device.
Description
技术领域technical field
本发明属于光学超分辨显微领域，特别涉及一种三维荧光差分超分辨显微方法及装置。The invention belongs to the field of optical super-resolution microscopy, in particular to a three-dimensional fluorescent differential super-resolution microscopy method and device.
背景技术Background technique
1873年，德国科学家Abbe提出光学成像系统的“衍射极限”，任何光学显微镜都存在一个分辨率极限，由光波长以及透镜的数值孔径决定。光学显微成像系统由于“衍射极限”的存在，在可见光波段无法实现200纳米以下的高分辨成像。为此人们不断努力，研究发展超分辨成像技术，希望突破衍射极限，获得更高的分辨率。2014年从事荧光超分辨光学显微术的三位科学家获得了诺贝尔化学奖，他们开启了人类利用荧光标记方法实现超分辨显微的大门。从此，人类的光学显微已进入超分辨时代。In 1873, the German scientist Abbe proposed the "diffraction limit" of the optical imaging system. Any optical microscope has a resolution limit, which is determined by the wavelength of light and the numerical aperture of the lens. Due to the existence of the "diffraction limit", optical microscopy imaging systems cannot achieve high-resolution imaging below 200 nanometers in the visible light band. For this reason, people continue to work hard to research and develop super-resolution imaging technology, hoping to break through the diffraction limit and obtain higher resolution. In 2014, three scientists engaged in fluorescence super-resolution optical microscopy won the Nobel Prize in Chemistry. They opened the door for humans to use fluorescent labeling methods to achieve super-resolution microscopy. Since then, human optical microscopy has entered the era of super-resolution.
现在主流超分辨显微成像技术可以大致分为两类：一类是基于经典共焦系统，如受激辐射淬灭显微术(STED)，荧光发射微分显微术(FED)；另一类是基于宽场成像系统，如随机光学重组显微术(STORM)、结构光照明显微术(SIM)、光子活化定位显微术(PALM)等等。近年来，超分辨技术得到了迅猛发展，其发展方向已经不在单单局限于横向分辨率的提高，而是向着提高三维分辨率、提高成像速度、系统集成化紧凑化发展。以上提到的超分辨显微术中，有的已经向三维方向发展，为医学或生物学研究人员提供更多的微观信息。Now the mainstream super-resolution microscopy imaging technology can be roughly divided into two categories: one is based on the classical confocal system, such as stimulated radiation quenching microscopy (STED), fluorescence emission differential microscopy (FED); It is based on wide-field imaging systems, such as Stochastic Optical Recombination Microscopy (STORM), Structured Illumination Microscopy (SIM), Photon Activation Localization Microscopy (PALM), etc. In recent years, super-resolution technology has developed rapidly, and its development direction is no longer limited to the improvement of lateral resolution, but toward the improvement of three-dimensional resolution, the improvement of imaging speed, and the development of system integration and compactness. Some of the super-resolution microscopy mentioned above have been developed in the three-dimensional direction to provide more microscopic information for medical or biological researchers.
随着分辨率的提升，光学超分辨显微方法和装置越来越受到医学和生物学领域研究人员的青睐。其快速直观无损的特定，使其得到了更多的应用，因此，高集成化、易使用，高分辨率的光学超分辨显微装置也成为了研究者们关注的重点。With the improvement of resolution, optical super-resolution microscopy methods and devices are increasingly favored by researchers in the fields of medicine and biology. Its fast, intuitive and non-destructive specificity makes it more widely used. Therefore, highly integrated, easy-to-use, and high-resolution optical super-resolution microscopy devices have also become the focus of researchers.
发明内容Contents of the invention
本发明提供了一种三维荧光差分超分辨显微方法及装置，可以实现超越衍射极限的三维分辨率。系统结构紧凑，单激发光路，调校方便，该方法装置简单，成像速度快，对样品没有特殊荧光染料要求。可应用于生物、医学研究中对衍射极限一下微观结构细节的三维成像。The invention provides a three-dimensional fluorescence differential super-resolution microscopy method and device, which can realize three-dimensional resolution beyond the diffraction limit. The system has a compact structure, a single excitation optical path, and is easy to adjust. This method has simple equipment, fast imaging speed, and no special fluorescent dye requirements for samples. It can be applied to three-dimensional imaging of microstructure details below the diffraction limit in biological and medical research.
本发明的具体技术方案如下：一种三维荧光差分超分辨显微装置，包括激光器、承载待测样品的电动样品台和将光线投射到所述电动样品台的显微物镜，所述激光器与显微物镜之间依次设有：The specific technical scheme of the present invention is as follows: a three-dimensional fluorescent differential super-resolution microscopic device, including a laser, a motorized sample stage carrying a sample to be tested, and a microscope objective lens that projects light onto the motorized sample stage, the laser and the microscope Between the micro-objective lenses, there are:
用于将所述激光器发出的激光转换为平行光的准直器；a collimator for converting the laser light emitted by the laser into parallel light;
用于将所述激光器发出的光束改变为线偏振光的起偏器；a polarizer for changing the light beam emitted by the laser into linearly polarized light;
用于调制所述光束偏振方向的第一二分之一波片；a first half-wave plate for modulating the polarization direction of the light beam;
用于依次调制光束水平分量和垂直分量的空间光调制模块；A spatial light modulation module for sequentially modulating the horizontal component and the vertical component of the light beam;
用于对所述相位调制后的光束进行光路偏转的扫描振镜系统；所述圆偏振光通过所述显微物镜投射到所述待测样品上；A scanning galvanometer system for deflecting the optical path of the phase-modulated light beam; the circularly polarized light is projected onto the sample to be measured through the microscope objective lens;
依次布置的分别用于对所述扫描振镜系统出射的光束进行聚焦和准直的扫描透镜和场镜；A scanning lens and a field lens respectively arranged in sequence for focusing and collimating the light beam emitted by the scanning galvanometer system;
并设有用于控制所述空间光调制器和扫描振镜系统的控制器及收集所述待测样品发出的信号光的探测系统。A controller for controlling the spatial light modulator and scanning galvanometer system and a detection system for collecting signal light emitted by the sample to be tested are also provided.
优选的，所述的空间光调制模块包括：Preferably, the spatial light modulation module includes:
空间光调制器，由所述计算机控制加载黑色背景或同时加载0～π相位调制图案和0～2π涡旋相位调图案；The spatial light modulator is controlled by the computer to load a black background or simultaneously load a 0-π phase modulation pattern and a 0-2π vortex phase modulation pattern;
反射镜，用于将空间光调制器反射的光束再次反射进入空间光调制器内；a reflector, configured to reflect the light beam reflected by the spatial light modulator into the spatial light modulator again;
位于所述空间光调制器和反射镜之间的第一四分之一波片，用于将两次经过的光束的偏振方向转过90度。The first quarter-wave plate located between the spatial light modulator and the reflector is used to rotate the polarization directions of the light beams passing twice through 90 degrees.
进一步优选的，所述的空间光调制模块和扫描振镜系统间设有用于将偏振光转换为圆偏振光的第二二分之一波片和第二四分之一波片。Further preferably, a second half-wave plate and a second quarter-wave plate for converting polarized light into circularly polarized light are provided between the spatial light modulation module and the scanning galvanometer system.
本发明中，探测系统包括：In the present invention, the detection system includes:
布置在第二四分之一波片和扫描振镜系统之间的分束镜。所述分束镜在待测样品为荧光样品时应选用二色镜。A beamsplitter mirror arranged between the second quarter-wave plate and the galvo scanning system. The beam splitter should be a dichromatic mirror when the sample to be tested is a fluorescent sample.
用于滤去分束镜出射的信号光中的杂散光的带通滤波片，所述带通滤波片在待测样品为非荧光样品时可以省略；A band-pass filter for filtering stray light in the signal light emitted by the beam splitter, the band-pass filter can be omitted when the sample to be measured is a non-fluorescent sample;
用于探测信号光束的光强信号的探测器，所述探测器选用光电倍增管(PMT)或雪崩光电二极管(APD)；A detector for detecting the light intensity signal of the signal beam, the detector is a photomultiplier tube (PMT) or an avalanche photodiode (APD);
用于将滤光后的信号光束聚焦到探测器上的聚焦透镜；用于对所述信号光束进行空间滤波的空间滤波器，其位于所述聚焦透镜的焦平面处，所述空间滤波器可以采用针孔或多模光纤，若采用针孔，所用针孔的直径应小于一个艾里斑直径。A focusing lens for focusing the filtered signal beam onto the detector; a spatial filter for spatially filtering the signal beam, which is located at the focal plane of the focusing lens, and the spatial filter can Use pinholes or multimode optical fibers, and if pinholes are used, the diameter of the pinholes used should be smaller than the diameter of an Airy disk.
所述激光器与起偏器之间依次设有用于对所述激光光束进行滤波的单模光纤。A single-mode optical fiber for filtering the laser beam is sequentially arranged between the laser and the polarizer.
所述空间光调制器液晶屏幕在左右两侧同时加载0～π相位调制图案和0～2π涡旋相位调图案；The liquid crystal screen of the spatial light modulator is simultaneously loaded with a 0-π phase modulation pattern and a 0-2π vortex phase modulation pattern on the left and right sides;
空间光调制器中加载调制图案和黑色背景的切换频率与扫描振镜系统的空间扫描频率相同，从而实现扫描振镜系统配合电动样品太每扫描一次三维空间，空间光调制器的调制函数切换一次。The switching frequency of the modulation pattern loaded in the spatial light modulator and the black background is the same as the spatial scanning frequency of the scanning galvanometer system, so that the modulation function of the spatial light modulator is switched once every time the scanning galvanometer system cooperates with the electric sample to scan the three-dimensional space. .
优选的，所述显微物镜的数值孔径NA＝1.49。Preferably, the numerical aperture of the microscope objective lens is NA=1.49.
根据上述的三维荧光差分超分辨显微装置，本发明的显微方法包括以下步骤：According to the above-mentioned three-dimensional fluorescent differential super-resolution microscopic device, the microscopic method of the present invention comprises the following steps:
1)激光器发出的激光光束在准直后转换为线偏振光；1) The laser beam emitted by the laser is converted into linearly polarized light after collimation;
2)调节第一二分之一波片，使光束的偏振方向与空间光调制器可调节偏振方向成α角；2) adjusting the first half-wave plate so that the polarization direction of the light beam and the adjustable polarization direction of the spatial light modulator form an angle α;
3)将偏振光入射至空间光调制器的屏幕一侧，利用该侧加载的0～π相位调制图案对偏振光进行相位调制；3) The polarized light is incident on one side of the screen of the spatial light modulator, and the phase modulation of the polarized light is performed by using the 0-π phase modulation pattern loaded on the side;
4)控制空间光调制器反射后的光束重新折返入射至空间光调制器的屏幕另一侧，利用该侧加载的0～2π涡旋相位调图案进行相位调制；4) controlling the light beam reflected by the spatial light modulator to return to the other side of the screen of the spatial light modulator, and use the 0-2π vortex phase modulation pattern loaded on this side to perform phase modulation;
5)两次调制后的激光光束在转化为圆偏后经扫描振镜系统和显微物镜聚焦到样品上并进行扫描；5) After the laser beam modulated twice is converted into a circular polarization, it is focused on the sample by the scanning galvanometer system and the microscope objective lens and scanned;
6)在扫描过程中实时收集被测样品各被激发点发出的信号光，得到一次扫描信号光强I1(x,y,z)；6) During the scanning process, the signal light emitted by each excited point of the sample to be tested is collected in real time, and the signal light intensity I 1 (x, y, z) of one scan is obtained;
7)将步骤3)和步骤4)内的空间光调制器上仅加载黑色背景，重复步骤3)～6)，对相同的三维空间进行第二次扫描，得到二次扫描信号光强I2(x,y,z)；7) Load only the black background on the spatial light modulator in step 3) and step 4), repeat steps 3) to 6), scan the same three-dimensional space for the second time, and obtain the light intensity of the second scanning signal I 2 (x,y,z);
8)根据公式I(x,y,z)＝I2(x,y,z)-r×I1(x,y,z)计算最终信号光强I(x,y,z)，并利用I(x,y,z)得到超分辨图像；其中r＝I2 max/2×I1 max，I2 max为I2(x,y,z)的最大值，I1 max为I1(x,y,z)中的最大值。8) Calculate the final signal light intensity I(x,y,z) according to the formula I(x,y,z)=I 2 (x,y,z)-r×I 1 (x,y,z), and use I(x,y,z) to obtain super-resolution images; where r=I 2 max /2×I 1 max , I 2 max is the maximum value of I 2 (x,y,z), and I 1 max is I 1 ( x, y, z).
本发明中，当待测样品为荧光样品时，所述信号光为所述圆偏振光经显微物镜投射后在样品上激发出的荧光；当待测样品为非荧光样品时，所述信号光为所述圆偏振光经显微物镜投射后经样品表面的反射光束。In the present invention, when the sample to be tested is a fluorescent sample, the signal light is the fluorescence excited on the sample after the circularly polarized light is projected by the microscope objective lens; when the sample to be tested is a non-fluorescent sample, the signal light The light is the reflected light beam from the surface of the sample after the circularly polarized light is projected by the microscope objective lens.
其中，被测样品上x，y，z轴方向由三维扫描方式决定。Among them, the directions of x, y, and z axes on the measured sample are determined by the three-dimensional scanning method.
作为优选的，最终信号光强I(x,y,z)为负值时，令I(x,y,z)＝0。Preferably, when the final signal light intensity I(x, y, z) is a negative value, set I(x, y, z)=0.
本发明原理如下：Principle of the present invention is as follows:
根据经典衍射理论，实际光学系统对平行光的聚焦效果，并非理想的点，而是一个可计算其空间尺寸的梭形空间分布，长轴延光轴方向，在焦面上即衍射斑或艾里斑。艾里斑范围内的样品都会被激发从而发出信号光，使得在艾里斑范围内的样品细节无法被分辨。因此，显微系统的分辨率受到衍射极限的限制。所以，突破衍射极限的限制，提高显微系统的分辨率，减小艾里斑面积是关键。理论上艾里斑是无法通过光学器件减小的，但是可以通过其他手段减小系统最终的等效激发面积，从而达到提高分辨率的目的。同理对于三维超分辨而言，提高显微镜分辨率，减小空间聚焦光斑的体积是关键。According to the classical diffraction theory, the focusing effect of an actual optical system on parallel light is not an ideal point, but a fusiform spatial distribution whose spatial size can be calculated. Spots. The samples within the range of the Airy disk will be excited to emit signal light, so that the details of the samples within the range of the Airy disk cannot be resolved. Therefore, the resolution of the microscopy system is limited by the diffraction limit. Therefore, it is the key to break through the limitation of the diffraction limit, improve the resolution of the microscopic system, and reduce the area of the Airy disk. Theoretically, the Airy disk cannot be reduced by optical devices, but other means can be used to reduce the final equivalent excitation area of the system, so as to achieve the purpose of improving resolution. Similarly, for three-dimensional super-resolution, improving the resolution of the microscope and reducing the volume of the spatially focused spot is the key.
在本发明方法中，空间光调制器左侧加载0～π相位调制图案右侧加载0～2π涡旋相位调制图案。当光束被0～π相位调制后，根据矢量光场衍射理论，由狄拜积分计算可知，此时光束经显微物镜聚焦后的光场，在焦面空间附近为一强度极弱的空心圆柱，圆柱两端为强度较强的细实心圆柱。。当光束被0～2π涡旋相位调制后，同理可计算得到，此时光束经显微镜物镜聚焦后在焦面空间附近为一个空心圆柱分布，在焦面上为一个甜甜圈形空心光斑In the method of the present invention, a 0-π phase modulation pattern is loaded on the left side of the spatial light modulator and a 0-2π vortex phase modulation pattern is loaded on the right side. When the beam is modulated by 0～π phase, according to the theory of vector light field diffraction, it can be known from the calculation of the Dibye integral that the light field after the beam is focused by the microscopic objective lens is a hollow cylinder with extremely weak intensity near the focal plane space. , the two ends of the cylinder are thin solid cylinders with strong strength. . When the beam is modulated by 0-2π vortex phase, it can be calculated similarly. At this time, after the beam is focused by the microscope objective lens, it is a hollow cylinder distribution near the focal plane space, and a donut-shaped hollow spot on the focal plane.
光束首先入射到空间光调制器的左侧，此时空间光调制器左侧加载的0～π相位图案只对光束水平方向的分量进行调制，垂直分量未被调制。当光束经偏振旋转90度后，再次入射到空间光调制器上左侧时，之前的水平分量变为垂直分量，其不会被再次调制，之前的垂直分量变为水平分量，被空间光调制器右侧加载的0～2π涡旋相位图案调制。这样，两个方向的分量被不同的调制图案进行调制，此时光束经显微物镜聚焦到焦面时，上述两种空间光场分布叠加，在焦面附近得到近似的空心椭球体光场分布，长轴延光轴方向。该空心椭球激发范围激发样品所得信号光强为I1(x,y,z)。The light beam is first incident on the left side of the spatial light modulator. At this time, the 0-π phase pattern loaded on the left side of the spatial light modulator only modulates the horizontal component of the beam, and the vertical component is not modulated. When the beam is polarized and rotated by 90 degrees, when it is incident on the left side of the spatial light modulator again, the previous horizontal component becomes a vertical component, which will not be modulated again, and the previous vertical component becomes a horizontal component, which is modulated by the spatial light The 0-2π vortex phase pattern modulation loaded on the right side of the detector. In this way, the components in the two directions are modulated by different modulation patterns. At this time, when the beam is focused to the focal plane by the microscope objective lens, the above two spatial light field distributions are superimposed, and an approximate hollow ellipsoid light field distribution is obtained near the focal plane. , the long axis extends along the optical axis direction. The signal light intensity obtained by exciting the sample in the hollow ellipsoid excitation range is I 1 (x, y, z).
当空间光调制器加载黑色背景是，理论上不对激发光做任何调制，我们可以认为空间光调制器只起到平面反射镜的作用。此时，由狄拜积分计算可知，光束经显微成像物镜聚焦后在焦面附近为一个实心光斑。该实心光斑激发范围内激发样品所得信号光强为I2(x,y,z)。根据公式I(x,y,z)＝I2(x,y,z)-r×I1(x,y,z)计算最终信号光强I(x,y,z)。显然I(x,y,z)所对应的各扫描点处的有效信号光发光体积将小于I2(x,y,z)所对应的各扫描点处发光体积。因此，与常规光学显微方法相比，本发明减小了有效信号光的发光体积，从而可以实现超衍射极限的分辨率。When the spatial light modulator is loaded with a black background, theoretically no modulation is performed on the excitation light, and we can think that the spatial light modulator only acts as a plane reflector. At this time, it can be seen from the calculation of the Debye integral that the light beam is a solid spot near the focal plane after being focused by the microscope imaging objective lens. The signal intensity obtained by exciting the sample within the excitation range of the solid spot is I 2 (x, y, z). The final signal light intensity I(x,y,z) is calculated according to the formula I(x,y,z)=I 2 (x,y,z)−r×I 1 (x,y,z). Obviously, the effective signal light luminous volume at each scanning point corresponding to I(x, y, z) will be smaller than the luminous volume at each scanning point corresponding to I 2 (x, y, z). Therefore, compared with conventional optical microscopy methods, the present invention reduces the luminous volume of effective signal light, so that super-diffraction-limited resolution can be achieved.
相对于现有的技术，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)可以实现在较低激发光功率的前提下，提供三维超衍射极限的分辨率；(1) Under the premise of low excitation light power, it can provide three-dimensional super-diffraction-limited resolution;
(2)由于只需要两次扫描，相比于以往类似方法3次扫描的系统，成像速度提高三分之一。(2) Since only two scans are required, the imaging speed is increased by one-third compared with the previous similar method with three scans.
(3)单激发光路，使得系统紧凑，省去多路调重合的步骤，易于调校。(3) The single excitation optical path makes the system compact, saves the step of multi-channel overlapping, and is easy to adjust.
(4)单空间光调制器加载两幅调制图案，简约成本。(4) A single spatial light modulator is loaded with two modulation patterns, reducing the cost.
附图说明Description of drawings
图1为本发明中基于荧光受激发射微分的三维超分辨装置示意图；Fig. 1 is a schematic diagram of a three-dimensional super-resolution device based on fluorescence stimulated emission differentiation in the present invention;
图2为本发明中0～π相位调制图案；Fig. 2 is 0～π phase modulation pattern among the present invention;
图3为本发明中被0～π相位调制后，光束聚焦得到的焦面xy方向及xz方向的光场分布；Fig. 3 is the light field distribution in the xy direction and xz direction of the focal plane obtained by focusing the beam after being modulated by 0～π phase in the present invention;
图4为本发明中0～2π涡旋相位调制图案；Fig. 4 is the 0～2π vortex phase modulation pattern in the present invention;
图5为本发明中被0～2π相位调制后，光束聚焦得到的焦面xy方向及xz方向的光场分布；Fig. 5 is the light field distribution in the xy direction and xz direction of the focal plane obtained by focusing the beam after being modulated by 0-2π phase in the present invention;
图6本发明中光束经两次调制后聚焦后的焦面xy方向及xz方向的光场分布；Fig. 6 is the light field distribution in the xy direction and xz direction of the focal plane after the light beam is focused twice after modulation in the present invention;
图7为普通共聚焦显微镜焦面处xy方向光场分布与本发明方法焦面处xy方向光场分布，即普通共焦显微镜横向有效发光面积和本发明系统横向有效发光面积；Fig. 7 is the light field distribution in the xy direction at the focal plane of the common confocal microscope and the light field distribution in the xy direction at the focal plane of the method of the present invention, that is, the transverse effective light-emitting area of the common confocal microscope and the transverse effective light-emitting area of the system of the present invention;
图8为普通共聚焦显微镜焦面处xz方向光场分布与本发明方法焦面处xz方向光场分布，即普通共焦显微镜纵向有效发光面积和本发明系统纵向有效发光面积。Figure 8 shows the light field distribution in the xz direction at the focal plane of a common confocal microscope and the light field distribution in the xz direction at the focal plane of the method of the present invention, that is, the longitudinal effective light-emitting area of the common confocal microscope and the longitudinal effective light-emitting area of the system of 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.
如图1所示三维荧光差分超分辨显微，包括：激光器1，单模光纤2a，准直器3，起偏器4，反射镜5a，1/2波片6a，D形反射镜7，空间光调制器8，1/4波片9a，透镜10，反射镜5b，反射镜5c，1/2波片6b，1/4波片9b，四带通二色镜11，振镜扫描系统12，扫描镜13，场镜14，显微物镜15，样品太16，四带通滤波片17，电动小孔18，单模光纤2b，探测器19，控制系统及PC机20。As shown in Figure 1, the three-dimensional fluorescent differential super-resolution microscope includes: a laser 1, a single-mode fiber 2a, a collimator 3, a polarizer 4, a mirror 5a, a 1/2 wave plate 6a, a D-shaped mirror 7, Spatial light modulator 8, 1/4 wave plate 9a, lens 10, reflector 5b, reflector 5c, 1/2 wave plate 6b, 1/4 wave plate 9b, four-bandpass dichromatic mirror 11, vibrating mirror scanning system 12. Scanning mirror 13, field lens 14, microscope objective lens 15, sample mirror 16, four-bandpass filter 17, motorized pinhole 18, single-mode optical fiber 2b, detector 19, control system and PC 20.
其中，单薄光纤2a、准直器3、起偏器4和反射镜5a依次位于激光器1出射的光轴之上，起偏器4的透光轴方向应使得透射后的光强最大。Wherein, the thin optical fiber 2a, collimator 3, polarizer 4 and reflector 5a are sequentially located on the optical axis emitted by the laser 1, and the direction of the transmission axis of the polarizer 4 should maximize the light intensity after transmission.
其中，D形反射镜位于折转后的激光器1光轴之上，并将光束第一次折转入射至空间光调制器8左侧。Wherein, the D-shaped reflector is located on the optical axis of the laser 1 after deflection, and deflects the light beam for the first time to enter the left side of the spatial light modulator 8 .
其中，1/4波片9a、透镜10、反射镜5b位于空间光调制器8折转后的光束光轴之上，反射镜5b也位于透镜10的焦面上。Wherein, the 1/4 wave plate 9a, the lens 10, and the mirror 5b are located on the optical axis of the light beam folded by the spatial light modulator 8, and the mirror 5b is also located on the focal plane of the lens 10.
光束被反射镜5b反射，并再次经过1/4波片9a、透镜10入射到空间光调制器右侧，第二次经空间光调制器反射至反射镜5c，由5c对光束进行转折。其中1/2波片6b，1/4波片9b及四带通二色镜11位于经反射镜5c折转后的光轴上。The light beam is reflected by the mirror 5b, and enters the right side of the spatial light modulator through the 1/4 wave plate 9a and the lens 10 again, and is reflected by the spatial light modulator to the mirror 5c for the second time, and the light beam is turned by 5c. Wherein the 1/2 wave plate 6b, the 1/4 wave plate 9b and the four-bandpass dichroic mirror 11 are located on the optical axis after being deflected by the mirror 5c.
光束被二色镜11反射进入振镜扫描系统12，其中扫描镜13、场镜14、显微物镜15和电动样品台16依次位于扫描振镜系统出射光束的光轴上。电动样品台16位于物镜15焦面处。The light beam is reflected by the dichroic mirror 11 and enters the galvanometer scanning system 12, wherein the scanning mirror 13, the field lens 14, the microscope objective lens 15 and the motorized sample stage 16 are sequentially located on the optical axis of the outgoing beam of the scanning galvanometer system. The motorized sample stage 16 is located at the focal plane of the objective lens 15 .
四带通滤波片17、电动小孔18和探测器19位于信号光光轴上。Four bandpass filters 17, motorized pinholes 18 and detectors 19 are located on the optical axis of the signal light.
控制系统及PC机20与空间光调制器8、探测器19及扫描振镜系统相连接，用于控制空间光调制器8上图案的切换。空间光调制器在上位PC机及控制系统20的控制下，在相位调制图案和黑色背景间切换。The control system and the PC 20 are connected with the spatial light modulator 8 , the detector 19 and the scanning galvanometer system for controlling the pattern switching on the spatial light modulator 8 . Under the control of the host PC and the control system 20, the spatial light modulator switches between the phase modulation pattern and the black background.
上述装置中，显微物镜15的数值孔径NA＝1.49；所用小孔为一电动小孔装置，其自带聚焦透镜和可切换直径的一系列小孔，在本发明中装置中使用0.7个艾里斑的小孔；探测器19为光电倍增管(PMT)。In the above-mentioned device, the numerical aperture NA=1.49 of microscopic objective lens 15; Used aperture is an electric aperture device, and it carries a series of apertures of focusing lens and switchable diameter, uses 0.7 Å in device among the present invention A small hole in the spot; the detector 19 is a photomultiplier tube (PMT).
采用图1所示装置实现三维超分辨的过程如下：The process of using the device shown in Figure 1 to realize three-dimensional super-resolution is as follows:
激光器1发出的光束耦合进单模光纤2a中，通过单模光纤2a导入准直器3，光束从准直器3出射后为平行光，经偏振片4转换为线偏振光。经过反射镜5a，1/2波片6a和D形反射镜7入射到空间光调制器8左侧。其中，调节1/2波片快轴，使得光束的偏振方向与水平方向夹角为54.5度。此时空间光调制器8左侧加载0～π相位调制图案，如图2所示。0～π相位调制的调制函数可以用极坐标表示为，The light beam emitted by the laser 1 is coupled into the single-mode fiber 2a, and then guided into the collimator 3 through the single-mode fiber 2a. After passing through the mirror 5a, the 1/2 wave plate 6a and the D-shaped mirror 7 are incident on the left side of the spatial light modulator 8. Wherein, the fast axis of the 1/2 wave plate is adjusted so that the angle between the polarization direction of the light beam and the horizontal direction is 54.5 degrees. At this time, the left side of the spatial light modulator 8 is loaded with a 0-π phase modulation pattern, as shown in FIG. 2 . The modulation function of 0～π phase modulation can use polar coordinates Expressed as,
其中，θmax为入射光半径的最大值；Among them, θ max is the maximum value of the incident light radius;
此时，偏振方向与水平方向成54.5度的光束的水平分量被上述0～π相位调制函数调制。其在转化成圆偏光后，经物镜聚焦后的焦面附近处光场分布如图3所示。光束被空间光反射，经过1/4波片9a和透镜10，被反射镜5b反射后，再次经过透镜10和1/4波片9a，入射到空间光调制器右侧。反射镜5b位于透镜10的焦点上，使得反射镜的面形对光束波前的影响降到最低。调节1/4波片9a的快轴，使得入射偏振光束两次经过1/4波片9后，偏振方向转过90度入射到空间光调制器8右侧。空间光调制器右侧加载0～2π涡旋相位调制图案，如图4所示，其相位调制函数可以写成：At this time, the horizontal component of the light beam whose polarization direction is 54.5 degrees from the horizontal direction is modulated by the above-mentioned 0-π phase modulation function. After it is converted into circularly polarized light, the light field distribution near the focal plane after being focused by the objective lens is shown in Figure 3. The light beam is reflected by the spatial light, passes through the 1/4 wave plate 9a and the lens 10, is reflected by the mirror 5b, passes through the lens 10 and the 1/4 wave plate 9a again, and enters the right side of the spatial light modulator. The mirror 5b is located at the focal point of the lens 10, so that the influence of the shape of the mirror on the wavefront of the beam is minimized. The fast axis of the 1/4 wave plate 9 a is adjusted so that the incident polarized light beam passes through the 1/4 wave plate 9 twice, and the polarization direction is rotated by 90 degrees to enter the right side of the spatial light modulator 8 . The right side of the spatial light modulator is loaded with a 0-2π vortex phase modulation pattern, as shown in Figure 4, and its phase modulation function can be written as:
此时，之前被空间光调制器8左侧图案调制的水平分量变为垂直分量，无法被调制。之前未被调制的垂直分量变为水平分量，即被空间光调制器8右侧加载的0～2π涡旋相位调制图案调制。该分量转化为圆偏光后，经物镜聚焦后的焦面附近处光场分布如图5所示。At this time, the horizontal component previously modulated by the pattern on the left side of the spatial light modulator 8 becomes a vertical component and cannot be modulated. The vertical component that was not modulated before becomes the horizontal component, that is, modulated by the 0-2π vortex phase modulation pattern loaded on the right side of the spatial light modulator 8 . After this component is converted into circularly polarized light, the light field distribution near the focal plane after being focused by the objective lens is shown in Figure 5.
光束经空间光调制器右侧反射后，再被反射镜5c反射，经过1/2波片6b和1/4波片9b转化为圆偏光。圆偏振光束经四带通二色镜11反射，进入振镜扫描系统12，再经扫描镜13和场镜14进入物镜15，聚焦到样品面上，其光场分布如图6所示，即为图3和图5所示光场的叠加。样品上被圆偏光束激发的区域，发出信号光，经场镜14、扫描镜13、振镜系统12、四带通二色镜11、四带通滤光片17进入电动小孔18，由电动小孔18自带的透镜聚焦至小孔，再由单模光纤2b将信号光导入探测器19，进而进入PC机20内存。After the light beam is reflected by the right side of the spatial light modulator, it is reflected by the mirror 5c, and converted into circularly polarized light by the 1/2 wave plate 6b and the 1/4 wave plate 9b. The circularly polarized light beam is reflected by the four-bandpass dichroic mirror 11, enters the galvanometer scanning system 12, and then enters the objective lens 15 through the scanning mirror 13 and the field lens 14, and focuses on the sample surface. The light field distribution is shown in Figure 6, namely is the superposition of the light fields shown in Figure 3 and Figure 5. The region excited by the circularly polarized light beam on the sample sends signal light, and enters the motorized small hole 18 through the field mirror 14, the scanning mirror 13, the galvanometer system 12, the four-band-pass dichromatic mirror 11, and the four-band-pass filter 17. The lens attached to the motorized pinhole 18 focuses on the pinhole, and then the signal light is guided into the detector 19 by the single-mode optical fiber 2b, and then enters the memory of the PC 20 .
控制器及PC机20和空间光调制器8、探测器19、振镜扫描系统12及电动样品台相连。由控制器及PC机20控制振镜扫描系统12及电动样品台16完成三维空间的逐点扫描，并记录各点信号，从而得到信号光强I1(x,y,z)。The controller and the PC 20 are connected with the spatial light modulator 8, the detector 19, the galvanometer scanning system 12 and the motorized sample stage. The controller and PC 20 control the galvanometer scanning system 12 and the motorized sample stage 16 to complete point-by-point scanning in three-dimensional space, and record the signals of each point, so as to obtain the signal light intensity I 1 (x, y, z).
通过控制器调整空间光调制器8上的图案，使其全屏加载黑色背景，重复上述步骤，得到信号光强I2(x,y,z)。利用公式I(x,y,z)＝I2(x,y,z)-r×I1(x,y,z)得到最终有效信号光强I(x,y,z)。普通共聚焦显微镜焦面处xy方向光场分布与本发明所述方法焦面处xy方向光场分布如图7所示。普通共聚焦显微镜焦面处xz方向光场分布与本发明所述方法焦面处xz方向光场分布如图8所示。The pattern on the spatial light modulator 8 is adjusted by the controller so that the full screen is loaded with a black background, and the above steps are repeated to obtain the signal light intensity I 2 (x, y, z). The final effective signal light intensity I(x,y,z) is obtained by using the formula I(x,y,z)=I 2 (x,y,z)-r×I 1 (x,y,z). The light field distribution in the xy direction at the focal plane of a common confocal microscope and the light field distribution in the xy direction at the focal plane of the method according to the present invention are shown in FIG. 7 . The light field distribution in the xz direction at the focal plane of a common confocal microscope and the light field distribution in the xz direction at the focal plane of the method according to the present invention are shown in FIG. 8 .
以上所述仅为本发明的较佳实施举例，并不用于限制本发明，凡在本发明精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The above descriptions are only examples of the preferred implementation of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention within.
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