作　　者： ; ; ;

机构地区： 深圳大学光电工程学院光电子器件与系统教育部重点实验室

出　　处： 《深圳大学学报（理工版）》 2011年第1期1-16,共16页

摘　　要： 针对复杂生物学系统的研究和观测,指出对活体细胞内的分子及其相关事件,以高时空分辨率实现可视化、跟踪和定量处理,采用远场荧光成像是目前细胞内分子检测及成像的主要工具.在述评的基础上,介绍该课题组在活体细胞内分子检测及成像中的荧光显微成像和非标记检测技术的研究进展.围绕提高荧光显微成像分辨率,研究图像信息获取速率高的宽场成像方法,包括结构光照明和单分子定位显微.在结构光照明显微研究中,采用可编程的数字微镜器件代替需要精确移动的光栅对生物样品进行显微成像,获得了超分辨显微成像;在单分子定位显微成像研究中,搭建单分子定位显微成像系统,实测分辨率达到48nm,并获得了HeLa细胞突起中微丝束结构的纳米分辨图像;针对厚样品成像时存在的荧光串扰难题,提出利用双波长空间非相干光干涉照明,理论分析证明,其可实现厚度为35nm半高全宽的单一薄层的轴向选择性激发.在非标记检测成像技术方面,围绕相干反斯托克斯拉曼散射(coherentanti-Stokes Ramanscattering,CARS)方法中存在的问题展开研究.为获得完整的物质分子CARS光谱,利用飞秒激光脉冲泵浦PCF获得超连续谱激光输出同时作为泵浦光和斯托克斯光,实现超宽带时间分辨CARS光谱探测和显微成像技术;通过数值模拟获得优化超连续谱激光输出的时谱结构的实验条件,初步实现了满足实验所需的SC激光光源;通过调节探测光脉冲与SC激光脉冲之间的时间延迟实现时间分辨CARS,达到有效抑制非共振背景噪声,提高系统探测灵敏度的目的.利用超宽带时间分辨CARS光谱探测和显微成像技术,获得多种有机溶液在387～4092cm-1范围内,光谱分辨率达14cm-1的不同分子的CARS光谱信号;通过分析探讨实现超分辨CARS显微成像技术的途径,提出利用双探测光实现纳米分辨的方案.未来研究方向,在荧� Visualization,tracking and analysing molecules and their events in cells are critical in understanding a complex biological system.Far-field fluorescent microscopy is a common tool in detecting and imaging molecules in cells.In order to improve the resolution,we focused on two far-field imaging methods with high speed,structured illumination microscopy and single molecule localization microscopy.A home-built structured illumination microscope was set up,in which a programmable DMD instead of a piezo activated grating was used to produce stable and reliable structured illumination patterns.A single molecule localization microscope with the spatial resolution （FWHM） of 48nm was built on an IX71 inverted fluorescent microscope.Images of filopodias in HeLa cells were reconstructed with nano-resolution.Furthermore,a key question in imaging thick samples was taken into consideration on how to reduce the background brought by the fluorescence crosstalk.A background suppression method based on dual-wavelength incoherent interference illumination was presented.Simulation results demonstrated that a single layer with thickness of 35 nm （FWHM） could be selectively activated.In microscopic imaging techniques without fluorescent labels,we started and have carried out a great deal of research work based on the application requirements.In order to obtain the whole CARS spectral signals of the interested molecules in the sample,the optimal experimental conditions for improving the temporal-spectral structure of the supercontinuum （SC） are obtained by the computer simulations.Under the appropriately experimental conditions,a beam of femtosecond laser pulse is used as the seed pulse to pump a photonic crystal fiber （PCF） to generate the SC pulse that is simultaneously used as the pump and Stokes.By this method,an ultra-broadband time-resolved CARS spectroscopy and microscopy is established,in which the delay time between the probe and SC is adjusted to suppress the non-resonance background （NRB） for improving t

关 键 词： 信息光学 超分辨显微成像 远场荧光成像 结构光照明 单分子定位 相干反斯托克斯拉曼散射 纳米分辨 分子振动谱 超连续谱光源

领　　域： [生物学] [机械工程] [理学] [理学]