CN113533294B - Time domain, space domain and spectrum domain single molecule characterization device based on nanometer gap electrode pair - Google Patents
Time domain, space domain and spectrum domain single molecule characterization device based on nanometer gap electrode pair Download PDFInfo
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Abstract
Description
技术领域technical field
本发明涉及光电技术领域,尤其是涉及一种基于纳米间隙电极对下的时域、空域和谱域单分子表征装置。The invention relates to the field of optoelectronic technology, in particular to a single-molecule characterization device in time domain, space domain and spectrum domain based on a pair of nano-gap electrodes.
背景技术Background technique
单分子尺度的高时空分辨率表征是发展分子光电子学、解决生物、化学以及医疗挑战的关键,也被认为发展精准医学以及解决主要医疗挑战的关键之一。High temporal and spatial resolution characterization at the single-molecule scale is the key to the development of molecular optoelectronics, solving biological, chemical and medical challenges, and is also considered to be one of the keys to developing precision medicine and solving major medical challenges.
当前主要依赖于高能探针(如透射电子显微镜、X射线衍射以及扫描电子显微镜)、扫描探针(如原子力显微镜、扫描隧道显微镜、光谱指纹探针等)、荧光探针以及纳米孔技术来实现。高能探针技术已成熟运用于表面特性的特征,一般具有很好空间分辨率,但对材料特性的分析能力较弱,特别需要真空导电等特殊条件,在实际应用中存在很大的局限性,尤其对动态生物体与单分子变化过程的表征分析目前为止还是无法实现。At present, it mainly relies on high-energy probes (such as transmission electron microscope, X-ray diffraction and scanning electron microscope), scanning probes (such as atomic force microscope, scanning tunneling microscope, spectral fingerprint probe, etc.), fluorescent probes and nanopore technology to achieve . High-energy probe technology has been maturely applied to the characteristics of surface properties, generally has a good spatial resolution, but the ability to analyze material properties is weak, especially requires special conditions such as vacuum conduction, and has great limitations in practical applications. In particular, the characterization and analysis of dynamic organisms and single-molecule change processes has not been realized so far.
光学成像技术今年来飞速发展,对于荧光标记的样品空间分辨率可达几十纳米,无需真空、超低温条件,样品观测条件友好,但在更小空间尺度(如5纳米以下)的单分子实时、纳米到亚纳米尺度的动态追踪和非荧光标记原位检测仍然存在很大困难。Optical imaging technology has developed rapidly in recent years. The spatial resolution of fluorescently labeled samples can reach tens of nanometers. It does not require vacuum and ultra-low temperature conditions, and the sample observation conditions are friendly. However, single-molecule real-time, There are still great difficulties in dynamic tracking and in situ detection with non-fluorescent labels at the nanometer to subnanometer scale.
纳米孔单分子检测技术利用纳米限域通道为单元组件,外部施加激励信号(如恒电位)来测量目标物通过纳米孔时的信号变化(如离子电流),实现对单分子的分析,具有高灵敏、无标记、单分子检测等性能,但这种技术一般只能提供准稳态单一信息,时间分辨率多为秒级,极大限制分子体系动力学信息的认识。The nanopore single-molecule detection technology uses the nano-confined channel as a unit component, and externally applies an excitation signal (such as a constant potential) to measure the signal change (such as ion current) when the target passes through the nanopore, so as to realize the analysis of single molecules, with high Sensitive, label-free, single-molecule detection and other performances, but this technology generally can only provide quasi-steady-state single information, and the time resolution is mostly at the second level, which greatly limits the understanding of molecular system dynamics information.
为了同时获取时间和空间分辨信息,一种可行的方案是将泵浦-探测的方法引入具有空间分辨的表征技术,目前已经有结合电子显微镜或扫描探针的泵浦-探测技术显示其功能。对于结合电子显微镜的时间分辨透射电子显微镜技术,其特殊的环境要求限制了其在生物与化学工程领域的应用的问题依然存在。而与扫描探针结合的时间分辨扫描隧道显微镜(STM)技术,利用超快激光脉冲诱导样品中电子处于非平衡态,结合隧穿电子的高空间分辨成像可获取电子激发态变化的超快动力学特性。然而这个技术同样拥有明显的缺陷,一是缺乏稳定可靠的电学探针,光激发造成STM探针的不稳定;其二是激发隧穿电流的成分占总体隧穿电流的比重非常小,采用传统的STM常导致信号淹没于噪声,因此提高光激发引起隧穿电流变化是实验中非常关键的问题。In order to obtain temporal and spatial resolution information at the same time, a feasible solution is to introduce the pump-probe method into a spatially resolved characterization technique. At present, the pump-probe technology combined with electron microscope or scanning probe has shown its function. For time-resolved transmission electron microscopy combined with electron microscopy, the special environmental requirements limit its application in the fields of biological and chemical engineering. The time-resolved scanning tunneling microscope (STM) technology combined with the scanning probe uses ultrafast laser pulses to induce electrons in the sample to be in a non-equilibrium state, and combined with high spatial resolution imaging of tunneling electrons, the ultrafast dynamics of electronic excited state changes can be obtained. academic characteristics. However, this technology also has obvious defects. One is the lack of stable and reliable electrical probes, and optical excitation causes the instability of STM probes; The STM often causes the signal to be submerged in noise, so improving the tunneling current change caused by photoexcitation is a very critical issue in the experiment.
另外纳米传感器件近年来也受到了人们的充分重视,包括基于纳米孔、隧穿传感、场效应晶体管等单分子检测技术获得了广泛的关注和发展。其中隧穿传感的测量模式,以亚5纳米间隙电极器件为分析元件,测量施加恒电位测量目标物带来的隧穿电流的变化而应用于DNA测序、单分子检测及单分子化学反应的机制研究,是当前世界发展的重要前沿领域之一。然而,恒电位模式下的隧穿传感测量方法也有着不可避免的技术局限性,其信号单一、灵敏度差、选择性低,也无法提供分析物动力学信息,极大限制了量子隧穿传感的应用。In addition, nanosensor devices have also received sufficient attention in recent years, including single-molecule detection technologies based on nanopores, tunneling sensing, and field-effect transistors, which have gained widespread attention and development. Among them, the measurement mode of tunneling sensing uses the sub-5nm gap electrode device as the analysis element to measure the change of tunneling current caused by applying a constant potential to measure the target, and is applied to DNA sequencing, single-molecule detection and single-molecule chemical reaction. Mechanism research is one of the important frontier fields of current world development. However, the tunneling sensing measurement method in the constant potential mode also has inevitable technical limitations, such as single signal, poor sensitivity, low selectivity, and inability to provide analyte dynamics information, which greatly limits quantum tunneling sensing. sense application.
发明内容SUMMARY OF THE INVENTION
本发明提供了一种基于纳米间隙电极对下的时域、空域和谱域单分子表征方法和装置,利用纳米间隙的多功能集成纳米隧道探针替代传统STM的金属针尖,引入飞秒激励的单分子隧穿技术,同时利用独特的探针结构实现等离子激元局域增强拉曼探针与荧光探针的结合,进而实现对单分子的精准多功能纳米尺度时空综合表征。The present invention provides a single molecule characterization method and device in the time domain, space domain and spectral domain based on the nano-gap electrode pair, using the multi-functional integrated nano-tunnel probe of the nano-gap to replace the metal tip of the traditional STM, and introducing femtosecond excitation The single-molecule tunneling technology uses a unique probe structure to realize the combination of plasmonic localized enhanced Raman probes and fluorescent probes, and then realizes the precise and multifunctional nanoscale spatiotemporal comprehensive characterization of single molecules.
本发明所公开的基于纳米间隙电极对下的时域、空域和谱域单分子表征装置系统,主要由探针与隧穿电学测试系统(子系统一)、超快光调制隧穿探测系统(子系统二)、单分子拉曼探测系统(子系统三)三个探测子系统组成,三个探测子系统之间还由相应的控制处理器,所有的控制处理器与计算机共同组成了系统控制系统(子系统四)。The time domain, space domain and spectrum domain single molecule characterization device system based on the nano-gap electrode pair disclosed in the present invention mainly consists of a probe and tunneling electrical test system (subsystem one), an ultrafast light modulation tunneling detection system ( Subsystem 2) and single-molecule Raman detection system (subsystem 3) are composed of three detection subsystems. There are corresponding control processors between the three detection subsystems. All the control processors and computers together form the system control system. system (subsystem four).
1)探针与隧穿电学测试系统:该系统由纳米间隙探针、三维纳米精密移动台和隧道信号探测模块组成。1) Probe and tunneling electrical test system: The system consists of a nano-gap probe, a three-dimensional nano-precision mobile stage and a tunnel signal detection module.
作为优选的,纳米间隙探针可以实现对更小纳米尺度的单分子进行分析,通过施加恒定电位观测单分子在经过探针电极间隙过程中的隧穿电流变化,具备实现DNA测序、单分子检测及单分子化学反应的机制研究的能力,同时与超快光调制相结合能进一步实现时间分辨率的提升;三维纳米精密移动台可以将纳米探针精密移动到要求的位置,进行探测样品的纳米扫描和高精度亚纳米定位;高精度的隧穿信号探测模块可以实现与控制系统关联并且飞秒激光器的脉冲激光的时延与精确同步。Preferably, the nanogap probe can realize the analysis of single molecules at a smaller nanometer scale, and observe the change of tunneling current of single molecules passing through the probe electrode gap by applying a constant potential, which is capable of realizing DNA sequencing and single molecule detection. and the ability to study the mechanism of single-molecule chemical reactions. At the same time, combining with ultrafast light modulation can further improve the time resolution; the three-dimensional nano-precision mobile stage can precisely move the nano-probe to the required position to detect the nano-scale of the sample. Scanning and high-precision sub-nanometer positioning; the high-precision tunneling signal detection module can realize the time delay and precise synchronization of the pulsed laser associated with the control system and the femtosecond laser.
2)超快光调制隧穿探测系统:主要包括激光光源与光束偏振调制模块,共聚焦扫描模块以及飞秒激光泵浦-探测模块。2) Ultrafast optical modulation tunneling detection system: mainly includes laser light source and beam polarization modulation module, confocal scanning module and femtosecond laser pumping-detecting module.
本子系统可以将隧穿电学检测平台与飞秒激光泵浦-探测系统结合,可为测量过程提供多参数的信号输出(如隧穿电流、荧光光谱、荧光成像等),实现较高时间和空间分辨率的单分子分析。This subsystem can combine the tunneling electrical detection platform with the femtosecond laser pump-detection system, which can provide multi-parameter signal output (such as tunneling current, fluorescence spectrum, fluorescence imaging, etc.) Single-molecule analysis at high resolution.
作为优选的,光源调控模块包括钛宝石激光器飞秒脉冲高重频可调激光器,准直与扩束单元,波前调控单元、二维高精度扫描单元以及共焦聚焦单元。飞秒激光光束经过扩束准直之后,利用脉冲选择器选择脉冲数目,空间光调制器或偏振器进行光束调控,以便产生不同的泵浦光子状态,形成更强的激发信号编码。同时脉冲激光经过共聚焦扫描模块,经过高数值孔径物镜聚焦到样品的针尖上,可以诱发局域荧光效应与隧穿结势垒变化。每个脉冲的变化,是表明系统能够探测的载流子激发与跃迁的能力,而脉冲的频次则反映这种状态的持续变化。将输出飞秒激光脉冲同步信号,控制隧穿传感的单分子光电一体检测系统,实现纳米探针-隧穿检测与采用飞秒脉冲波的时间门控。Preferably, the light source control module includes a titanium sapphire laser femtosecond pulse high repetition frequency tunable laser, a collimation and beam expansion unit, a wavefront control unit, a two-dimensional high-precision scanning unit and a confocal focusing unit. After the femtosecond laser beam is expanded and collimated, a pulse selector is used to select the number of pulses, and a spatial light modulator or polarizer is used to regulate the beam, so as to generate different pump photon states and form a stronger excitation signal code. At the same time, the pulsed laser passes through the confocal scanning module and focuses on the needle tip of the sample through the high numerical aperture objective lens, which can induce the local fluorescence effect and the change of the tunneling junction barrier. The change of each pulse indicates the ability of the system to detect the excitation and transition of carriers, and the frequency of the pulse reflects the continuous change of this state. The femtosecond laser pulse synchronization signal will be output to control the single-molecule photoelectric integrated detection system for tunneling sensing to realize nanoprobe-tunneling detection and time gating using femtosecond pulse waves.
3)单分子拉曼探测模块:本发明利用隧道结的金属纳米结构,构建特殊的针尖表面等离子波局域场增强特性,可结合荧光和拉曼光谱探测技术,获取隧道结内分子的指纹光谱信息。3) Single-molecule Raman detection module: This invention uses the metal nanostructure of the tunnel junction to construct a special needle-tip surface plasmon wave local field enhancement characteristic, which can be combined with fluorescence and Raman spectrum detection technology to obtain the fingerprint spectrum of molecules in the tunnel junction information.
作为优选的,本发明采用直流激光器,直流激光器出射的激光经过扩束准直后,经过高数值孔径物镜,汇聚照射于样品与电极探针,产生局域等离子激元波,诱发产生样品的拉曼辐射。将拉曼辐射收集后经过拉曼单色系统形成光谱图像,利用面阵EMCCD相机检测光谱信号,实现实时高分辨率成像和光谱分析利用。Preferably, the present invention adopts a DC laser. After the laser beam emitted by the DC laser is expanded and collimated, it passes through a high numerical aperture objective lens, converges and irradiates the sample and the electrode probe to generate localized plasmon waves, and induces the pull of the sample. Mann radiation. After the Raman radiation is collected, the Raman monochromatic system forms a spectral image, and the area array EMCCD camera is used to detect the spectral signal to realize real-time high-resolution imaging and spectral analysis.
基于等离激元增强效应,其电磁场场强分布与拉曼增强指数衰减的特性,具有近场增强效应,可极大地提高拉曼分子信号,并降低了背景光噪声的干扰。当分子通过隧道结时,具拉曼活性的分子探针,可实时定位与跟踪待测分子。利用表面增加拉曼或荧光等方法,通过分子的拉曼指纹信息、光强、波长、寿命等变化可反映穿孔分子与隧道结相互作用,达到光谱监测分子通过隧道结的目的,并与直流隧穿传感分析技术互为补充。Based on the plasmon enhancement effect, its electromagnetic field intensity distribution and Raman enhancement exponential attenuation characteristics have near-field enhancement effect, which can greatly improve the Raman molecular signal and reduce the interference of background light noise. When the molecule passes through the tunnel junction, the molecular probe with Raman activity can locate and track the molecule to be detected in real time. Using methods such as adding Raman or fluorescence on the surface, the interaction between the perforated molecule and the tunnel junction can be reflected through the changes in the Raman fingerprint information, light intensity, wavelength, and lifetime of the molecule, so as to achieve the purpose of spectroscopic monitoring of the molecule passing through the tunnel junction, and the interaction with the DC tunnel junction. Wear sensing analysis techniques complement each other.
4)系统控制系统:主要包括光源控制器、光斑调制控制器、扫描控制器、信号探测控制器四个部分,均与计算机相连。4) System control system: It mainly includes four parts: light source controller, light spot modulation controller, scanning controller, and signal detection controller, all of which are connected to the computer.
作为优选的,控制系统的在时间上的高精度同步与时间延迟,控制移动与扫描系统亚纳米级的运动与对准,控制光谱的探测,图像探测,隧穿电流信号的探测。Preferably, the high-precision synchronization and time delay of the control system in time, control the sub-nanometer movement and alignment of the moving and scanning system, control the detection of spectrum, image detection, and tunneling current signal detection.
本发明公开的基于纳米间隙电极对下的光-电综合表征单分子方法,利用纳米探针检测隧穿电流实现对单分子的高精度检测,并利用超快光调制隧穿探测系统将隧穿电学检测平台与飞秒激光泵浦-探测系统结合,可为测量过程提供多参数的信号输出(如隧穿电流、荧光光谱、荧光成像等),实现较高时间和光分辨率的单分子分析;利用探针针尖表面等离子波局域场增强特性,可结合荧光和拉曼光谱探测技术,获取隧道结内分子的指纹光谱信息,从而实现对单分子在时域、空域和谱域的表征。The opto-electrical comprehensive characterization method for single molecules based on nano-gap electrode pairs disclosed in the present invention uses nanoprobes to detect tunneling currents to achieve high-precision detection of single molecules, and utilizes ultrafast optical modulation tunneling detection systems to detect tunneling currents. The combination of the electrical detection platform and the femtosecond laser pump-detection system can provide multi-parameter signal output (such as tunneling current, fluorescence spectrum, fluorescence imaging, etc.) for the measurement process, and realize single-molecule analysis with higher temporal and optical resolution; Utilizing the surface plasmon wave local field enhancement characteristics of the probe tip, combined with fluorescence and Raman spectroscopy detection technology, the fingerprint spectrum information of molecules in the tunnel junction can be obtained, so as to realize the characterization of single molecules in the time domain, space domain and spectral domain.
附图说明Description of drawings
图1为本发明的装置系统图;Fig. 1 is a device system diagram of the present invention;
图2为纳米间隙电极对的单分子检测装置的结构的示意图;2 is a schematic diagram of the structure of a single-molecule detection device with a pair of nano-gap electrodes;
图3为单链DNA分子存在下隧穿电极的电导-时间变化图。Fig. 3 is a conductance-time variation graph of the tunneling electrode in the presence of single-stranded DNA molecules.
具体实施方式Detailed ways
在下面的描述中阐述了很多具体细节以便于充分理解本发明,但是,本发明还可以采用其他不同于在此描述的其他方式来实施,因此,本发明并不限于下面公开的具体实施例的限制。In the following description, many specific details are set forth in order to fully understand the present invention, but the present invention can also be implemented in other ways different from those described here, therefore, the present invention is not limited to the specific embodiments disclosed below limit.
下面详细描述本发明的实施例,所述实施例的示例在附图中示出,其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的,旨在用于解释本发明,而不能理解为对本发明的限制。The following describes in detail the embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals refer to the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the figures are exemplary and are intended to explain the present invention and should not be construed as limiting the present invention.
如图1所示,本实施例的基于亚5纳米间隙电极对的多功能单分子表征装置系统装置,包括:波长为632nm的激光器1,单模保偏光纤2a和单模保偏光纤2b,准直扩束镜3a和准直扩束镜3b,反射镜4a、反射镜4b和反射镜4c,飞秒激光器5,脉冲选择器6,二色镜7a和二色镜7b,二分之一波片8,四分之一波片9,分束镜10,扫描振镜系统11,透镜12a、透镜12b和透镜12c,场镜13,显微物镜14,样品台15,20-60倍收集物镜16,CCD17,纳米间隙电极隧穿探针18,滤光片19,多模光纤20a和多模光纤20b,探测器雪崩光电二极管APD21,拉曼光谱仪22,三维纳米精密移动台23,隧道结单分子分析仪24,同步控制FPGA(Field ProgrammableGate Array)板25,信号采集FPGA板26,计算机27。As shown in Figure 1, the multifunctional single molecule characterization device system device based on the sub-5 nanometer gap electrode pair in this embodiment includes: a laser 1 with a wavelength of 632nm, a single-mode polarization-maintaining fiber 2a and a single-mode polarization-maintaining
其中,脉冲选择器6用于得到不同延迟时间的飞秒激光脉冲,可得到飞秒到微秒范围内的连续时间延迟,从而可以得到样品激发态的动态信息,提高时间分辨率到飞秒。Among them, the
其中,显微物镜14为高数值孔径物镜,数值孔径NA为1.4;收集物镜16和CCD17用来得到样品的宽场成像信息。Wherein, the
其中,纳米间隙一般指形成或以其他方式提供在材料中的孔眼、通道或通路,然后与电极相连可接入传感区,并可用于测量单分子引起的隧穿电流变化(如图2)。同时纳米金属间隙探针能够产生局域等离基元激元震荡,诱发产生增强拉曼辐射;三维纳米精密移动台23用以将纳米探针精密移动到要求的位置,进行探测样品的纳米扫描和高精度亚纳米定位。Among them, nanogap generally refers to the holes, channels or passages formed or provided in other ways in the material, and then connected to the electrode to access the sensing area, and can be used to measure the change of tunneling current caused by single molecule (as shown in Figure 2) . At the same time, the nano-metal gap probe can generate localized plasmon excitations and induce enhanced Raman radiation; the three-dimensional nano-precision
其中,拉曼光谱仪用以接收探针局域等离子激元波增强的拉曼辐射信号,实现拉曼光谱检测。Among them, the Raman spectrometer is used to receive the Raman radiation signal enhanced by the localized plasmon wave of the probe to realize Raman spectrum detection.
其中,通过计算机控制同步控制FPGA板25实现对激光器1,飞秒激光器5,脉冲选择器6,扫描振镜系统11,三维纳米精密移动台23和信号采集FPGA板26的同步控制,而信号采集FPGA板26可以同步采集CCD17的宽场信号,隧道结单分子分析仪24的电信号,探测器雪崩光电二极管APD21的共聚焦激发探测的荧光信号,拉曼光谱仪22的光谱信号,并将相关信息传递到计算机27中传达给使用者。Wherein, realize the synchronous control to laser 1, femtosecond laser 5,
采用图1所示的装置获得单分子表征时域信息过程如下:Using the device shown in Figure 1 to obtain time-domain information for single-molecule characterization is as follows:
本装置包含隧穿电流探测模块可以采用传统的直流恒电位模式来记录隧穿电流,通过采用离子流检测技术,隧穿单分子可提供分子本征物性信息,包括分子亚结构和能级结构信息。图1中简化表达了隧穿探测模块,由纳米探针18和隧道结单分子分析仪24组成,而在图2中表达的单分子检测实施例详细表现了本发明装置获得单分子表针时域信息、探测隧穿电流的过程。The device includes a tunneling current detection module that can use the traditional DC constant potential mode to record the tunneling current. By using the ion current detection technology, the tunneling single molecule can provide molecular intrinsic property information, including molecular substructure and energy level structure information . The tunneling detection module is simplified and expressed in Fig. 1, which is composed of a
图2为纳米间隙电极对的单分子检测装置的结构的示意图,探针由亚纳米间隙电极对28a~28b、电介质29a~29b构成,A端为近样品端,B端为电流检测端。单分子分析仪由测量电源30、电泳电极对31a~31b、电泳电源32、电流放大模块35、安培计36和控制单元33组成。控制单元33可控制测量电源30、电泳电源32和电流放大模块35,负责控制隧道探针的偏置电压以及电流信号的放大和探测,实现隧穿电学信息探测,并将放大后的电流传递到信号采集FPGA板26中。2 is a schematic diagram of the structure of a single-molecule detection device with nano-gap electrode pairs. The probe is composed of sub-nano-gap electrode pairs 28a-28b and
在DNA单链检测的实施例中,A端如图2(b)所示,通过电泳电源32设置合适的电泳电极对31a~31b的电压,从而控制单分子的运动经过隧道结。测量电源30可以对纳米间隙电极对28a~28b的电极施加电压,当单分子34经过隧道结时,纳米间隙电极对28a~28b产生隧穿电流,电流在纳米间隙电极对28a~28b中从A端传导至B端,从而方便在B端对电流进行测量。B端连接电流放大模块35,安培计36测量经过放大后的隧穿电流将电流送至信号采集FPGA板26中。在待分析物在驱动力作用下通过隧穿结(纳米电极间隙)时,隧穿电流发生瞬时变化,从而引起电导发生变化,其大小和持续时间反映了分析物信息,通过对大量脉冲电流的统计分析可实现对分析物的检测。图3为单链DNA分子持续经过间隙约为1.1纳米的隧穿电极时产生的电导-时间图,当DNA分子位于电极间隙中间时,可以检测到一个尖刺装的脉冲信号(隧穿电流),因此电流(电导)的变化反映了每个单分子经过电极对所对应的信息。In the embodiment of DNA single-strand detection, as shown in FIG. 2( b ), the
为了提高隧道电流检测的时间分辨率和空间分辨率,装置还可以通过飞秒激光泵浦-探测来实现光耦合隧穿电流探测。该装置采用共聚焦系统进行激发,实现空间分辨率的提升。飞秒激光器5通过单模保偏光纤2b输出飞秒激光在经过准直扩束镜3b后进入到脉冲选择器6,脉冲选择器可以通过不同的延时设置来控制飞秒激光的脉冲延迟。在经过脉冲选择器调制后,激发光通过二色镜7a和二色镜7b的反射后进入到扫描振镜系统11实现激发光在样品面上进行扫描,并且被二分之一波片8和四分之一波片9调制成圆偏振光(或其他所需要的偏振光)。在经过透镜12a和场镜13组成的4f系统后,飞秒激发光被显微物镜14聚焦到纳米探针18的针尖上。飞秒激光的激发刺激产生隧穿结的势垒变化,形成快速变化,再经过隧道结单分子分析仪24的控制下对隧穿电极的快速准确的电信号探测,实现隧道电流检测的时间分辨率的显著提升。In order to improve the time resolution and spatial resolution of tunnel current detection, the device can also realize optically coupled tunnel current detection through femtosecond laser pump-probe. The device uses a confocal system for excitation to achieve improved spatial resolution. The femtosecond laser 5 outputs the femtosecond laser through the single-mode polarization-maintaining
采用图1所示的装置获得单分子表征空域信息的方法如下:Using the device shown in Figure 1 to obtain single-molecule characterization spatial information is as follows:
本发明可以基于装置本身的共聚焦系统获取单分子表征的荧光信息,在激发光经过上文所述的共聚焦系统后,可以被显微物镜14聚焦到固定在样品台15上的样品,样品产生的荧光首先可以通过收集物镜16收集然后被CCD17接收并采集至信号采集FPGA板26中,同时可被显微物镜14收集,再次经过场镜13和透镜12a的4f系统后,再次通过扫描振镜系统解扫描。然后荧光信号透过二色镜7b,经过滤光片19滤去杂散光后被多模光纤20a收集并送到探测器APD21中,最终被信号采集FPGA板采集得到荧光信息,在共聚焦系统的激发探测下,实现空间域的分辨率提高。The present invention can obtain fluorescence information of single molecule characterization based on the confocal system of the device itself. After the excitation light passes through the above-mentioned confocal system, it can be focused by the
除此之外,本发明同样可以利用纳米探针的等离子激发获得单分子的空间信息。在合适的电压控制或者飞秒激发光激发下,纳米探针18与样品在近场实现局域等离子体激发,激发出来的光信号在经过显微物镜14收集后再经过场镜13,反射镜4b,透镜12a,扫描振镜系统11,二色镜7b和滤光片19,经过透镜12b的会聚后被多模光纤20a和探测器APD21收集,通过同步控制FPGA板控制三维纳米精密移动台23实现对探针的高精度移动,从而实现对样品的扫描,得到样品的超高分辨率空间信息。In addition, the present invention can also use the plasma excitation of the nanoprobe to obtain the spatial information of the single molecule. Under appropriate voltage control or femtosecond excitation light excitation, the
采用图1所示的装置获得单分子表征谱域信息的方法如下:The method for obtaining single-molecule characterization spectral domain information using the device shown in Figure 1 is as follows:
波长为632nm的激光器1通过单模保偏光纤2a输出激发光,在经过准直扩束镜3a准直扩束、反射镜4a反射后透过二色镜7a与飞秒激发光光路合并,进入到共聚焦系统。在经过前面所述光路后,经显微物镜聚焦到纳米探针和样品之间,产生局域等离子体激元波,诱发产生样品的拉曼辐射,拉曼辐射信号再次经过显微物镜14收集,通过场镜13,反射镜4b,透镜12a,扫描振镜系统11,经过分束镜10的反射后进入拉曼光谱探测光路,经反射镜4c反射,透镜12c聚焦到多模光纤20b中传递到拉曼光谱仪22中收集分析,最终光谱信息被信号采集FPGA板26收集。The laser 1 with a wavelength of 632nm outputs the excitation light through the single-mode polarization-maintaining fiber 2a. After being collimated and expanded by the
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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