CN108801863A - The femtosecond optical optical tweezers system of colloidal particle dynamics and image-forming information in solution can be obtained - Google Patents
The femtosecond optical optical tweezers system of colloidal particle dynamics and image-forming information in solution can be obtained Download PDFInfo
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Abstract
本发明涉及一种可获取溶液中胶体粒子动力学及成像信息的飞秒光镊系统,其特征在于该系统包括:用于作为飞秒捕获激光为待测样品提供稳定捕获光阱的飞秒光镊捕获激光单元;用于探测被捕获待测样品在相对于光阱中心做受限布朗运动时位置分布的位置探测单元;用于作为激发光照射待测样品进行荧光激发的激发光源单元;用于将飞秒捕获激光收集到位置探测单元,且将激发光源单元出射的激发光引入到位于捕获光阱中的待测样品,使得待测样品的荧光染料分子受激发产生荧光信号的光学显微单元;用于收集待测样品产生的荧光信号,完成对荧光染料包被的单个胶体粒子的实时荧光成像,获得待测样品在不同粘弹性特征体系中的单个胶体粒子的动力学信息的单分子荧光成像单元。
The invention relates to a femtosecond optical tweezers system capable of obtaining dynamics and imaging information of colloidal particles in a solution, which is characterized in that the system includes: a femtosecond light used as a femtosecond capture laser to provide a stable capture optical trap for a sample to be tested A tweezers capture laser unit; a position detection unit used to detect the position distribution of the captured sample to be measured when doing limited Brownian motion relative to the center of the optical trap; an excitation light source unit used to irradiate the sample to be measured with excitation light for fluorescence excitation; The optical microscope is to collect the femtosecond capture laser light into the position detection unit, and introduce the excitation light emitted by the excitation light source unit into the sample to be measured in the capture light trap, so that the fluorescent dye molecules of the sample to be measured are excited to generate fluorescence signals Unit; used to collect the fluorescent signal generated by the sample to be tested, complete the real-time fluorescence imaging of a single colloidal particle coated with a fluorescent dye, and obtain the single molecule of the kinetic information of a single colloidal particle of the sample to be tested in different viscoelastic characteristic systems Fluorescence Imaging Unit.
Description
技术领域technical field
本发明是关于一种可获取溶液中胶体粒子动力学及成像信息的飞秒光镊系统,涉及胶体科学以及生物物理基础研究领域。The invention relates to a femtosecond optical tweezers system capable of acquiring dynamics and imaging information of colloidal particles in a solution, and relates to the fields of colloid science and basic biophysics research.
背景技术Background technique
光镊技术是一种可以捕获和操控微小粒子以及进行力学测量的重要单分子技术,自美国贝尔实验室的阿什金于1986年发明,经过三十多年的发展已经日益成熟,在理论以及应用方面都做了大量的研究工作,由单光镊逐步发展为双光镊、多光镊、飞秒光镊以及涡旋光镊等各类光镊,这一技术被广泛应用于生物物理、纳米加工、胶体科学以及物理学等多个领域,并且光镊由于易与其他一些技术相结合,例如单分子荧光成像,荧光共振能量转移等,使其成为相对于单一技术或测量手段的一大优势。飞秒光镊,是人们将光镊的捕获光源用飞秒激光代替连续波激光,飞秒激光具有飞秒的脉宽以及高重复频率,使其在满足连续波激光捕获要求的同时,还具备连续波激光不具备的性质,在较低的平均功率下就可实现捕获。毛方林等人2004年在理论上首次提出并计算了飞秒光镊的横向光学力,同年B.Agate等人用飞秒光镊实现了双光子荧光激发以及原位控制,发现其捕获所需的激光平均光强明显低于连续波激光;降雨强等在2010年首次发现由于强聚焦飞秒光镊的非线性捕获形成的光阱劈裂的现象;Goswami等人基于新的飞秒光镊的微流变方法在一个微小体积内直接原位测量了固-液界面处的温度等。相较于传统光镊,飞秒激光自身的复杂特性使得飞秒光镊的相关实验研究都还很有限,因此,将飞秒光镊与全内反射荧光成像相结合,一种可获取溶液中胶体粒子的动力学信息测量的高灵敏度高精度的成像测量方法的建立是非常有必要的。Optical tweezers technology is an important single-molecule technology that can capture and manipulate tiny particles and perform mechanical measurements. It was invented by Ashkin of Bell Laboratories in the United States in 1986. After more than 30 years of development, it has become increasingly mature. In theory and A lot of research work has been done in terms of application, and the single optical tweezers have gradually developed into various types of optical tweezers such as dual optical tweezers, multi-optical tweezers, femtosecond optical tweezers, and vortex optical tweezers. This technology is widely used in biophysics, nanoscale Processing, colloid science and physics and other fields, and optical tweezers are easy to combine with other technologies, such as single-molecule fluorescence imaging, fluorescence resonance energy transfer, etc., making it a great advantage over a single technology or measurement method . Femtosecond optical tweezers is the use of femtosecond laser instead of continuous wave laser as the capture light source of optical tweezers. Femtosecond laser has femtosecond pulse width and high repetition rate, which makes it meet the requirements of continuous wave laser capture and also has CW lasers do not have the property that trapping can be achieved at lower average power. Mao Fanglin and others theoretically proposed and calculated the transverse optical force of femtosecond optical tweezers for the first time in 2004. In the same year, B.Agate et al. used femtosecond optical tweezers to realize two-photon fluorescence excitation and in-situ control, and found that the capture required The average light intensity of the laser is significantly lower than that of the continuous wave laser; Rain Qiang and others first discovered the phenomenon of optical trap splitting due to the nonlinear capture of strongly focused femtosecond optical tweezers in 2010; Goswami et al. based on the new femtosecond optical tweezers The microrheological method directly measures the temperature at the solid-liquid interface in a small volume in situ. Compared with the traditional optical tweezers, the complex characteristics of the femtosecond laser itself make the relevant experimental research of the femtosecond optical tweezers very limited. It is very necessary to establish a high-sensitivity and high-precision imaging measurement method for the measurement of the kinetic information of colloidal particles.
发明内容Contents of the invention
针对上述问题,本发明的目的是提供一种可获取溶液中胶体粒子动力学及成像信息的飞秒光镊系统,能够对胶体体系、细胞进行精确主动操控的同时还能够进行高分辨率、高灵敏度的单分子测量与成像,进而得到胶体粒子或细胞在溶液中的动力学信息。In view of the above problems, the purpose of the present invention is to provide a femtosecond optical tweezers system that can obtain the dynamics and imaging information of colloidal particles in solution, which can perform precise and active manipulation of colloidal systems and cells, and can also perform high-resolution, high-resolution imaging. Sensitive single-molecule measurement and imaging, and then obtain kinetic information of colloidal particles or cells in solution.
为实现上述目的,本发明采取以下技术方案:一种可获取溶液中胶体粒子动力学及成像信息的飞秒光镊系统,该系统包括:用于作为飞秒捕获激光为待测样品提供稳定捕获光阱的飞秒光镊捕获激光单元;用于探测被捕获待测样品在相对于光阱中心做受限布朗运动时位置分布的位置探测单元;用于作为激发光照射待测样品进行荧光激发的激发光源单元;用于将飞秒捕获激光收集到所述位置探测单元,且将所述激发光源单元出射的激发光引入到位于捕获光阱中的待测样品,使得待测样品的荧光染料分子受激发产生荧光信号的光学显微单元;用于收集待测样品产生的荧光信号,完成对荧光染料包被的单个胶体粒子的实时荧光成像,获得待测样品在不同粘弹性特征体系中的单个胶体粒子的动力学信息的单分子荧光成像单元。In order to achieve the above object, the present invention adopts the following technical solutions: a femtosecond optical tweezers system that can obtain colloidal particle dynamics and imaging information in solution, the system includes: used as a femtosecond capture laser to provide stable capture for the sample to be tested The femtosecond optical tweezers capture laser unit of the optical trap; the position detection unit used to detect the position distribution of the trapped sample to be measured when it is doing limited Brownian motion relative to the center of the optical trap; used to irradiate the sample to be measured with excitation light for fluorescence excitation An excitation light source unit; used to collect the femtosecond capture laser light to the position detection unit, and introduce the excitation light emitted by the excitation light source unit into the sample to be measured in the capture optical trap, so that the fluorescent dye of the sample to be measured An optical microscopic unit that generates fluorescent signals when molecules are excited; it is used to collect the fluorescent signals generated by the samples to be tested, to complete real-time fluorescence imaging of single colloidal particles coated with fluorescent dyes, and to obtain the characteristics of the samples to be tested in different viscoelastic characteristic systems. Single-molecule fluorescence imaging unit for kinetic information of individual colloidal particles.
进一步地,所述光学显微单元采用倒置荧光显微镜结构,包括显微物镜、第一~第三二向色镜和聚光镜;激发光经所述显微物镜聚焦到位于捕获光阱中的待测样品,经激发光激发待测样品产生的荧光经所述显微物镜收集后发射到第一二向色镜,经所述第一二向色镜出射的荧光信号发射到所述单分子荧光成像测量单元。Further, the optical microscope unit adopts an inverted fluorescence microscope structure, including a microscope objective lens, first to third dichroic mirrors, and a condenser lens; For the sample, the fluorescence generated by the sample to be tested is collected by the microscope objective lens and emitted to the first dichroic mirror, and the fluorescence signal emitted by the first dichroic mirror is emitted to the single-molecule fluorescence imaging unit of measurement.
进一步地,所述飞秒光镊捕获激光单元包括飞秒脉冲激光器、扩束和准直镜以及偏转镜,所述飞秒脉冲激光器出射的飞秒脉冲激光经所述扩束和准直镜进行扩束和准直,扩束和准直后的飞秒脉冲激光经所述偏转镜进行偏转后发射到第二二向色镜,经所述第二二向色镜出射的光发射到所述显微物镜。Further, the femtosecond optical tweezers capture laser unit includes a femtosecond pulse laser, a beam expander and collimator mirror, and a deflection mirror, and the femtosecond pulse laser emitted by the femtosecond pulse laser passes through the beam expander and collimator mirror. Beam expansion and collimation, the femtosecond pulse laser after beam expansion and collimation is deflected by the deflection mirror and then sent to the second dichroic mirror, and the light emitted by the second dichroic mirror is sent to the microscope objective.
进一步地,所述位置探测单元包括会聚透镜、位置探测装置、数据采集卡和计算机;经所述显微物镜出射的前向散射光形成的干涉图样经所述聚光镜收集后发射到第三二向色镜,经所述第三二向色镜出射的光经所述会聚透镜聚焦所述位置探测装置,所述位置探测装置通过所述数据采集卡连接所述计算机获得待测样品所处光阱的刚度系数以及待测样品在不同粘弹性的体系中的粘度。Further, the position detection unit includes a converging lens, a position detection device, a data acquisition card and a computer; the interference pattern formed by the forward scattered light emitted by the microscope objective lens is collected by the condenser lens and then emitted to the third dichotomous A color mirror, the light emitted by the third dichroic mirror is focused on the position detection device through the converging lens, and the position detection device is connected to the computer through the data acquisition card to obtain the optical trap where the sample to be measured is located The coefficient of stiffness and the viscosity of the sample to be tested in different viscoelastic systems.
进一步地,所述位置探测装置采用位置敏感探测器或数字相机。Further, the position detecting device adopts a position sensitive detector or a digital camera.
进一步地,所述激发光源单元包括固体激光器、格兰泰勒棱镜、四分之一波片、扩束和准直镜、透镜以及滤光片;所述固体激光器发出的连续波依次经所述格兰-泰勒棱镜和四分之一波片发射到所述扩束和准直镜,经所述扩束和准直镜出射的圆偏振光经依次经上所述透镜和滤光片聚焦所述显微物镜的后焦面上。Further, the excitation light source unit includes a solid-state laser, a Glan-Taylor prism, a quarter-wave plate, a beam expander and collimator mirror, a lens, and an optical filter; the continuous wave emitted by the solid-state laser passes through the lattice The blue-Taylor prism and the quarter-wave plate emit to the beam expander and collimator mirror, and the circularly polarized light emitted by the beam expander and collimator mirror is focused by the above lens and filter in turn. back focal plane of the microscope objective.
进一步地,所述固体激光器出口处设置中性密度滤波片。Further, a neutral density filter is arranged at the exit of the solid-state laser.
进一步地,所述单分子荧光成像单元采用EMCCD相机和PCI数据采集卡,所述EMCCD相机经所述PCI数据采集卡连接所述计算机。Further, the single-molecule fluorescence imaging unit adopts an EMCCD camera and a PCI data acquisition card, and the EMCCD camera is connected to the computer via the PCI data acquisition card.
进一步地,所述待测样品为聚苯乙烯荧光小球、金纳米颗粒、无荧光的聚苯乙烯小球或二氧化硅小球、人体活性细胞。Further, the sample to be tested is polystyrene fluorescent beads, gold nanoparticles, non-fluorescent polystyrene beads or silicon dioxide beads, human active cells.
本发明由于采取以上技术方案,其具有以下优点:1、本发明将高重复频率的飞秒脉冲光镊与单分子全内反射荧光成像相结合,能够获取溶液中被测量体系的动力学信息。2、本发明在能够实现捕获操控的同时,采用高灵敏度的EMCCD相机对待测样品进行激发光激发以及荧光信号的收集,进而实现包括双光子荧光信号以及普通多荧光信号的探测。3、本发明的待测样品为不同粘弹性体系中的胶体小球或金纳米颗粒等,因此可以实现不同粘弹性体系、不同尺寸的胶体粒子进行测量。本发明可以广泛地应用于纳米颗粒、胶体粒子、凝胶、细胞等多种体系的相关动力学研究,探索其各自特殊性质的基础物理根源。Due to the adoption of the above technical solutions, the present invention has the following advantages: 1. The present invention combines femtosecond pulsed optical tweezers with high repetition rate and single-molecule total internal reflection fluorescence imaging to obtain dynamic information of the measured system in solution. 2. While the present invention can achieve capture control, it uses a high-sensitivity EMCCD camera to excite the sample to be tested with excitation light and collect fluorescence signals, thereby realizing detection including two-photon fluorescence signals and ordinary multi-fluorescence signals. 3. The samples to be tested in the present invention are colloid balls or gold nanoparticles in different viscoelastic systems, so colloid particles of different viscoelastic systems and different sizes can be measured. The present invention can be widely applied to related dynamic research of various systems such as nanoparticles, colloidal particles, gels, cells, etc., and explores the basic physical roots of their respective special properties.
附图说明Description of drawings
图1是本发明的飞秒光镊系统测量原理示意图;Fig. 1 is a schematic diagram of the measurement principle of the femtosecond optical tweezers system of the present invention;
图2是本发明的飞秒激光捕获光源单元与激发光源单元光路示意图;Fig. 2 is a schematic diagram of the light path of the femtosecond laser capture light source unit and the excitation light source unit of the present invention;
图3是本发明实施例的胶体粒子在光阱中运动的位移分布示意图;3 is a schematic diagram of the displacement distribution of colloidal particles moving in the optical trap according to an embodiment of the present invention;
图4是本发明实施例中被捕获的半径100nm的荧光PS小球的双光子激发成像图。Fig. 4 is a two-photon excitation imaging diagram of trapped fluorescent PS beads with a radius of 100 nm in an embodiment of the present invention.
具体实施方式Detailed ways
以下结合附图来对本发明进行详细的描绘。然而应当理解,附图的提供仅为了更好地理解本发明,它们不应该理解成对本发明的限制。在本发明的描述中,需要理解的是,术语“第一”、“第二”等仅仅是用于描述的目的,而不能理解为指示或暗示相对重要性。The present invention will be described in detail below in conjunction with the accompanying drawings. However, it should be understood that the accompanying drawings are provided only for better understanding of the present invention, and they should not be construed as limiting the present invention. In the description of the present invention, it should be understood that the terms "first", "second" and so on are only used for the purpose of description, and should not be understood as indicating or implying relative importance.
如图1所示,本发明提供的可获取溶液中胶体粒子动力学及成像信息的飞秒光镊系统,包括飞秒光镊捕获激光单元1、位置探测单元2、激发光源单元3、光学显微单元4和单分子荧光成像单元5,其中:As shown in Figure 1, the femtosecond optical tweezers system provided by the present invention, which can obtain the dynamics and imaging information of colloidal particles in the solution, includes a femtosecond optical tweezers capture laser unit 1, a position detection unit 2, an excitation light source unit 3, an optical display Micro unit 4 and single molecule fluorescence imaging unit 5, wherein:
飞秒光镊捕获激光单元1用于作为飞秒捕获激光为待测样品提供稳定的捕获光阱;The femtosecond optical tweezers capture laser unit 1 is used as a femtosecond capture laser to provide a stable capture optical trap for the sample to be tested;
位置探测单元2用于探测被捕获待测样品在相对于光阱中心做受限布朗运动时的位置分布;The position detection unit 2 is used to detect the position distribution of the captured sample to be measured when performing limited Brownian motion relative to the center of the optical trap;
激发光源单元3用于作为激发光照射待测样品进行荧光激发;The excitation light source unit 3 is used as excitation light to irradiate the sample to be tested for fluorescence excitation;
光学显微单元4用于将飞秒捕获激光收集到位置探测单元2,以及将激发光源单元3出射的激发光引入到位于捕获光阱中的待测样品,使得待测样品的荧光染料分子受激发产生荧光信号;The optical microscope unit 4 is used to collect the femtosecond capture laser light into the position detection unit 2, and introduce the excitation light emitted by the excitation light source unit 3 into the sample to be measured in the capture light trap, so that the fluorescent dye molecules of the sample to be measured are subjected to Excited to generate a fluorescent signal;
单分子荧光成像单元5用于收集待测样品产生的荧光信号,完成对荧光染料包被的单个胶体粒子的实时荧光成像,获得待测样品在不同粘弹性特征体系中的单个胶体粒子的动力学信息;另外,单分子荧光成像单元5也可用于满足柯勒照明条件的普通的明场成像。The single-molecule fluorescence imaging unit 5 is used to collect the fluorescent signal generated by the sample to be tested, complete the real-time fluorescence imaging of the single colloidal particle coated with the fluorescent dye, and obtain the dynamics of the single colloidal particle of the sample to be tested in different viscoelastic characteristic systems Information; In addition, the single-molecule fluorescence imaging unit 5 can also be used for ordinary bright-field imaging that meets Koehler illumination conditions.
在一个优选的实施例中,如图1所示,光学显微单元4可以采用倒置荧光显微镜结构,包括显微物镜40、三个二向色镜41、聚光镜42、反射镜43和光阑44;激发光经显微物镜40聚焦到位于捕获光阱中的待测样品,经激发光激发待测样品产生的荧光经显微物镜40收集后发射到二向色镜41c,经二向色镜41c出射的荧光信号经发射光高通滤波片发射到反射镜43,经反射镜43反射的荧光经光阑44发射到单分子荧光成像测量单元5。In a preferred embodiment, as shown in FIG. 1 , the optical microscope unit 4 can adopt an inverted fluorescence microscope structure, including a microscope objective lens 40, three dichroic mirrors 41, a condenser lens 42, a mirror 43 and an aperture 44; The excitation light is focused by the microscope objective lens 40 to the sample to be measured in the capture light trap, and the fluorescence generated by the excitation light to excite the sample to be measured is collected by the microscope objective lens 40 and then emitted to the dichroic mirror 41c, and passed through the dichroic mirror 41c The emitted fluorescence signal is transmitted to the reflector 43 through the high-pass filter of the emitted light, and the fluorescence reflected by the reflector 43 is transmitted to the single-molecule fluorescence imaging measurement unit 5 through the aperture 44 .
在一个优选的实施例中,如图2所示,飞秒光镊捕获激光单元1包括提供飞秒捕获激光的飞秒脉冲激光器10、若干反射镜11、扩束和准直镜12、光阑13、偏转镜14和两带通滤光片15。飞秒脉冲激光器10可以采用TEM00模式的钛蓝宝石光纤激光器,飞秒脉冲激光器10出射的飞秒脉冲激光经反射镜11a反射到扩束和准直镜12进行扩束和准直,扩束和准直后的飞秒脉冲激光依次经光阑13和反射镜11b和11c发射到偏转镜14,经偏转镜14偏转出射的光经带通滤光片15a发射到二向色镜41a,经二向色镜41a出射的光经带通滤光片15b发射到显微物镜40,扩束后束径大小应等于或略大于显微物镜40后瞳大小,不同数值孔径的显微物镜40会略有差别,约5~6.3mm,这样能够保证获得尽可能大的梯度力,从而有利于实现并保持稳定的捕获,其中,偏转镜14可以采用压电驱动的高精度偏转镜,可以通过计算机控制驱动偏转镜14发生微弧度偏转,实现飞秒光镊的主动操控。In a preferred embodiment, as shown in Figure 2, the femtosecond optical tweezers capture laser unit 1 includes a femtosecond pulsed laser 10 that provides femtosecond capture laser light, several mirrors 11, a beam expander and collimator mirror 12, an aperture 13. A deflection mirror 14 and two bandpass filters 15. Femtosecond pulsed laser 10 can adopt the titanium sapphire fiber laser of TEM00 mode, the femtosecond pulsed laser light that femtosecond pulsed laser 10 emerges is reflected to beam expander and collimating mirror 12 by reflector 11a and carries out beam expansion and collimation, beam expander and The collimated femtosecond pulsed laser light is transmitted to the deflection mirror 14 through the diaphragm 13 and the mirrors 11b and 11c in turn, and the light deflected by the deflection mirror 14 is transmitted to the dichroic mirror 41a through the band-pass filter 15a, and passed through the dichroic mirror 41a. The light emitted by the chromatic mirror 41a is emitted to the microscopic objective lens 40 through the band-pass filter 15b. There is a difference, about 5-6.3mm, which can ensure the gradient force as large as possible, which is conducive to realizing and maintaining stable capture. Among them, the deflection mirror 14 can be a high-precision deflection mirror driven by piezoelectricity, which can be controlled by a computer The deflection mirror 14 is driven to deflect in micro-radians, so as to realize the active control of the femtosecond optical tweezers.
在一个优选的实施例中,如图1所示,位置探测单元2包括会聚透镜20、高通低阻滤光片21、位置敏感探测器22、NI数据采集卡23和计算机24;经显微物镜40出射的前向散射光形成的干涉图样经聚光镜42(condenser,使光束更好地照到样品上的部件,目的是尽可能的收集前向散射光)收集后发射到二向色镜41b,经二向色镜41b出射的光依次经会聚透镜20和高通低阻滤光片21聚焦位置敏感探测器22,位置敏感探测器22通过NI数据采集卡23连接计算机24。需要说明的是,位置敏感探测器22可以替换为数字相机,数字相机将探测接收的光强信号转换为电信号经NI数据采集卡23发送到计算机24也可以进行分析获得位置与电压的关系,进而获得待测样品所处光阱的刚度系数以及获得待测样品在不同粘弹性的体系中的粘度,其中,高通低阻滤光片21可以采用可见光和红外干涉滤光片。In a preferred embodiment, as shown in Figure 1, the position detection unit 2 includes a converging lens 20, a high-pass low-resistance filter 21, a position-sensitive detector 22, a NI data acquisition card 23 and a computer 24; The interference pattern formed by the forward scattered light emitted by 40 is collected by the condenser lens 42 (condenser, the part that makes the light beam better shine on the sample, the purpose is to collect the forward scattered light as much as possible) and emits to the dichroic mirror 41b, The light emitted by the dichroic mirror 41b is focused on the position-sensitive detector 22 through the converging lens 20 and the high-pass low-resistance filter 21 in turn, and the position-sensitive detector 22 is connected to the computer 24 through the NI data acquisition card 23 . It should be noted that the position-sensitive detector 22 can be replaced by a digital camera, and the digital camera converts the detected light intensity signal into an electrical signal and sends it to the computer 24 through the NI data acquisition card 23, which can also be analyzed to obtain the relationship between position and voltage. Further, the stiffness coefficient of the optical trap where the sample to be measured is located and the viscosity of the sample to be measured in different viscoelastic systems are obtained, wherein, the high-pass low-resistance filter 21 can use visible light and infrared interference filters.
在一个优选的实施例中,高重复频率的飞秒脉冲激光由于自身的非线性效应可以实现双光子激发,在捕获的同时又可以获得双光子激发,因此所选择荧光染料的激发波长要适当,本发明的激发光源单元3采用高稳定性TEM00模式连续波激光,连续波激光的波长需要与所激发的染料分子相匹配,激发光源单元3包括固体激光器30(本实施例采用532nm波长,以此为例,不限于此)、中性密度滤波片31(中性密度滤波片31可以采用激发光带通滤波片)、若干反射镜32、格兰泰勒棱镜33、四分之一波片34、扩束和准直镜35、透镜36以及两滤光片37;固体激光器30发出的连续波激光经中性密度滤波片31发射到反射镜32a,经反射镜32a出射的激光依次经格兰-泰勒棱镜33、四分之一波片34和反射镜32b发射到扩束和准直镜35,经扩束和准直镜出射的圆偏振光经依次经透镜36、滤光片37a、反射镜32c和滤光片37b聚焦到显微物镜40的后焦面上,由于高倍显微物镜40具有很高的数值孔径可以实现全内反射,能够实现比宽场荧光成像信噪比更高的全内反射荧光成像。其中,此处扩束的目的是用于将激发光束直径扩大以确保其尺寸大于显微物镜40的进光孔以确保激发光是高质量的圆偏振光。In a preferred embodiment, the high repetition rate femtosecond pulsed laser can realize two-photon excitation due to its own nonlinear effect, and can obtain two-photon excitation while capturing, so the excitation wavelength of the selected fluorescent dye should be appropriate, The excitation light source unit 3 of the present invention adopts a high-stability TEM 00 mode continuous wave laser, the wavelength of the continuous wave laser needs to match the excited dye molecules, and the excitation light source unit 3 includes a solid-state laser 30 (the present embodiment adopts a 532nm wavelength, with This is an example, not limited thereto), neutral density filter 31 (neutral density filter 31 can adopt excitation light band-pass filter), some reflection mirrors 32, Glan Taylor prism 33, quarter wave plate 34 , beam expander and collimating mirror 35, lens 36 and two optical filters 37; The continuous wave laser that solid-state laser 30 sends is transmitted to reflecting mirror 32a through neutral density filter 31, and the laser light that exits through reflecting mirror 32a passes successively through gran -Taylor prism 33, quarter-wave plate 34 and reflector 32b emit to beam expander and collimator mirror 35, and the circularly polarized light through beam expander and collimator mirror exits through lens 36, optical filter 37a, reflection successively The mirror 32c and the optical filter 37b are focused on the back focal plane of the microscopic objective lens 40. Since the high-magnification microscopic objective lens 40 has a very high numerical aperture, total internal reflection can be realized, and a higher signal-to-noise ratio than wide-field fluorescence imaging can be achieved. Total internal reflection fluorescence imaging. Wherein, the purpose of beam expansion here is to expand the diameter of the excitation beam to ensure that its size is larger than the light entrance hole of the microscope objective lens 40 to ensure that the excitation light is high-quality circularly polarized light.
在一个优选的实施例中,单分子荧光成像单元5可以采用EMCCD相机和PCI数据采集卡,EMCCD相机通过PCI数据卡连接计算机24,EMCCD相机具备单光子的极高灵敏度,可以获取单个聚合物分子的散焦图像。In a preferred embodiment, single-molecule fluorescence imaging unit 5 can adopt EMCCD camera and PCI data acquisition card, and EMCCD camera is connected computer 24 by PCI data card, and EMCCD camera possesses the extremely high sensitivity of single photon, can obtain single polymer molecule defocused image.
在一个优选的实施例中,待测样品可以是聚苯乙烯荧光小球、金纳米颗粒、量子点、无荧光的聚苯乙烯小球或二氧化硅小球、人体活性细胞;其中,聚苯乙烯荧光小球半径约为100nm,金纳米颗粒和量子点的半径为25nm、30nm等,远小于飞秒脉冲激光器10的束腰半径,无荧光聚苯乙烯小球或二氧化硅小球半径为0.80μm、1.08μm、2.02μm等,接近或大于飞秒脉冲激光器10的束腰半径,人体活性细胞的半径为5μm,远大于飞秒脉冲激光器10的束腰半径,因此,本发明可以捕获不同尺寸的米氏粒子,获得直观的图像。In a preferred embodiment, the sample to be tested can be polystyrene fluorescent beads, gold nanoparticles, quantum dots, non-fluorescent polystyrene beads or silica beads, human active cells; wherein, polystyrene The radius of the ethylene fluorescent ball is about 100nm, the radius of the gold nanoparticles and quantum dots is 25nm, 30nm, etc., which are far smaller than the beam waist radius of the femtosecond pulsed laser 10, and the radius of the non-fluorescent polystyrene ball or silicon dioxide ball is 0.80 μm, 1.08 μm, 2.02 μm, etc., close to or greater than the beam waist radius of femtosecond pulsed laser 10, the radius of human active cells is 5 μm, much larger than the beam waist radius of femtosecond pulsed laser 10, therefore, the present invention can capture different Mie particles of different sizes to obtain intuitive images.
下面通过具体实施例详细说明采用本发明的可获取溶液中胶体粒子动力学及成像信息的飞秒光镊系统的使用过程。The process of using the femtosecond optical tweezers system of the present invention capable of obtaining dynamics and imaging information of colloidal particles in solution will be described in detail below through specific examples.
实施例1:采用本发明获取不同粘度下溶液中单个胶体粒子的动力学信息,具体过程为:Embodiment 1: adopt the present invention to obtain the kinetic information of a single colloidal particle in the solution under different viscosities, the specific process is:
1、将本发明的飞秒光镊系统放置在光学平台上,调整飞秒光镊系统中的各光学器件的位置使得符合本发明飞秒光镊系统的光路传播条件;1. Place the femtosecond optical tweezers system of the present invention on an optical platform, adjust the position of each optical device in the femtosecond optical tweezers system so as to meet the optical path propagation conditions of the femtosecond optical tweezers system of the present invention;
2、将半径为1.08μm聚苯乙烯微球作为待测样品放置于样品池中,样品池底部为0.17mm的盖玻片;2. Place polystyrene microspheres with a radius of 1.08 μm as the sample to be tested in the sample pool, and the bottom of the sample pool is a 0.17 mm cover glass;
3、打开飞秒捕获激光光源单元1的飞秒脉冲激光器10,使得飞秒脉冲激光器10发出的激光经扩束和准直镜12扩大到束径满足显微物镜40的孔径要求,扩束后的激光经准直使之成为平行光;3. Turn on the femtosecond pulse laser 10 of the femtosecond capture laser light source unit 1, so that the laser beam emitted by the femtosecond pulse laser 10 is expanded to a beam diameter that meets the aperture requirements of the microscopic objective lens 40 through beam expansion and collimator mirror 12. After beam expansion The laser beam is collimated to make it parallel light;
4、将显微物镜40切换到油浸显微物镜,在显微物镜40上加入20~100μL镜油,并对其进行调节使之达到合适的聚焦位置;4. Switch the microscopic objective lens 40 to an oil-immersed microscopic objective lens, add 20-100 μL of lens oil to the microscopic objective lens 40, and adjust it so that it reaches a suitable focus position;
5、打开显微镜40的卤素灯,并调节到合适的亮度,通过数字相机或EMCCD相机,初步找到在溶液中悬浮的胶体小球;5. Turn on the halogen lamp of the microscope 40 and adjust it to a suitable brightness, and use a digital camera or EMCCD camera to initially find the colloidal beads suspended in the solution;
6、打开NI数据采集卡23采集数据;6. Open the NI data acquisition card 23 to collect data;
7、结束数据集后将数据导出,做出位置的统计概率分布图(如图3所示),通过Boltzmann统计法校准得到光阱刚度为0.800pN/μm,粘度为0.0013泊(室内温度22℃)。7. After finishing the data set, export the data, make a statistical probability distribution map of the position (as shown in Figure 3), and calibrate through the Boltzmann statistical method to obtain an optical trap with a stiffness of 0.800pN/μm and a viscosity of 0.0013 poise (indoor temperature 22°C ).
8、根据斯托克斯-爱因斯坦公式计算得到待测样品的扩散系数D为0.016μm2/s。8. According to the Stokes-Einstein formula, the diffusion coefficient D of the sample to be tested is calculated to be 0.016 μm 2 /s.
实施例2:采用本发明完成光镊捕获下纳米荧光小球荧光成像,具体过程为:Embodiment 2: Using the present invention to complete fluorescence imaging of nano-fluorescence beads captured by optical tweezers, the specific process is as follows:
1、将本发明的飞秒光镊系统放置在光学平台上,将荧光染料包被的聚苯乙烯小球置于显微物镜4正上方,调整各光学器件使得符合本发明的光路传播条件,1. Place the femtosecond optical tweezers system of the present invention on an optical platform, place the polystyrene pellets coated with fluorescent dye directly above the microscope objective lens 4, adjust each optical device so as to meet the optical path propagation conditions of the present invention,
2、将待测样品放置于样品池中,样品池底部使用0.17mm的盖玻片;2. Place the sample to be tested in the sample cell, and use a 0.17mm cover glass at the bottom of the sample cell;
3、打开激发光源单元3的固体激光器30,使固体激光器30出射的连续波激光经扩束调整后使激发光在显微物镜40的后焦平面处聚焦,从而使得通过显微物镜40后出射为平行光;3. Turn on the solid-state laser 30 of the excitation light source unit 3, so that the continuous-wave laser emitted by the solid-state laser 30 is adjusted by beam expansion to focus the excitation light at the back focal plane of the microscopic objective lens 40, so that it is emitted after passing through the microscopic objective lens 40 for parallel light;
4、将显微物镜40切换到油浸显微物镜,在显微物镜40上加入20-100μL镜油,并对其进行调节使之达到最佳聚焦位置;4. Switch the microscopic objective 40 to an oil-immersed microscopic objective, add 20-100 μL of lens oil to the microscopic objective 40, and adjust it to achieve the best focus position;
5、打开飞秒捕获激光光源单元中1的飞秒脉冲激光器10并对其光强进行调节;5. Turn on the femtosecond pulse laser 10 in the femtosecond capture laser light source unit 1 and adjust its light intensity;
6、打开单分子荧光成像单元5对单个荧光小球进行实时荧光成像(如图4所示),通过荧光信号强度随时间阶梯式的变化从而获取被捕获胶体粒子变化个数的信息。6. Turn on the single-molecule fluorescence imaging unit 5 to perform real-time fluorescence imaging of a single fluorescent bead (as shown in FIG. 4 ), and obtain information on the number of captured colloidal particles through the stepwise change of fluorescence signal intensity with time.
上述各实施例仅用于说明本发明,其中各部件的结构、连接方式和制作工艺等都是可以有所变化的,凡是在本发明技术方案的基础上进行的等同变换和改进,均不应排除在本发明的保护范围之外。The above-mentioned embodiments are only used to illustrate the present invention, wherein the structure, connection mode and manufacturing process of each component can be changed to some extent, and any equivalent transformation and improvement carried out on the basis of the technical solution of the present invention should not excluded from the protection scope of the present invention.
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