[1]牛憨笨,陈丹妮,尹君.细胞内分子检测及成像技术研究[J].深圳大学学报理工版,2011,28(No.1(001-095)):1-16.
 Han-ben NIU,CHEN Dan-ni,and YIN Jun.Advances in approaches of molecules detecting and imaging in cells[J].Journal of Shenzhen University Science and Engineering,2011,28(No.1(001-095)):1-16.
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细胞内分子检测及成像技术研究()
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《深圳大学学报理工版》[ISSN:1000-2618/CN:44-1401/N]

卷:
第28卷
期数:
2011年No.1(001-095)
页码:
1-16
栏目:
光电工程
出版日期:
2011-01-10

文章信息/Info

Title:
Advances in approaches of molecules detecting and imaging in cells
文章编号:
1000-2618(2011)01-0001-16
作者:
牛憨笨陈丹妮尹君
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,广东省光电子器件与系统重点实验室,深圳 518060
Author(s):
Han-ben NIUCHEN Dan-niand YIN Jun
College of Optoelectronics,Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education,Key Laboratory of Optoelectronic Devices and Systems of Guangdong Province,Shenzhen University,Shenzhen 518060
P.R.China
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
Keywords:
information opticssuper-resolution microscopyfluorescent microscopystructured illuminationsingle molecule localizationcoherent anti-stokes Raman scattering (CARS)nano-resolutionmolecular vibrationssupercontinuum
分类号:
Q 631; O 437
文献标志码:
A
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48 nm,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35 nm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(coherent anti-Stokes Raman scattering,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387~4 092 cm-1范围内,光谱分辨率达14 cm-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,在荧光显微方面,将结合双波长空间非相干光干涉照明和轴向纳米定位,实现完整细胞三维纳米分辨荧光成像,通过发展高量子效率的小分子荧光开关材料及高灵敏度高帧频的探测器,将单分子定位显微应用到活细胞三维纳米成像和分子追踪上;在非标记成像方面,将重点发展基于CARS原理的单分子检测和纳米成像方法,尤其是基于改进后的超连续谱的超宽带CARS光谱技术和纳米分辨的CARS显微技术.
Abstract:
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 48 nm 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 the sensitivity of the system.The ultra-broadband CARS spectral signals of some organic liquids in 387-4092 cm-1 and with spectral resolution of 14 cm-1 have been obtained.We have also theoretically investigated the way to break the diffraction limitation to achieve the CARS microscopic imaging with nanometer spatial resolution.An original method with two probe beams,one with Gaussian fashion and another with ring fashion,has been presented.In the future studies on fluorescent imaging,dual-wavelength incoherent interference illumination and axial localization methods will be combined to image a whole cell with three dimensional nano-resolution.Small molecules with high quantum fluorescence efficiency and detectors with high sensitivity and high framing rate will also be developed in our group.As for the label-free studies,we will focus on the single molecule detection and nano-imaging methods based on the CARS principle,especially on the development of ultra-broadband CARS spectroscopy and CARS microscopy with nanometer spatial resolution under basis of the improved SC.A simple,accurate,stable and effective research method and tool will be developed for the life and medical sciences through our further efforts.

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备注/Memo

备注/Memo:
收稿日期:2010-11-29;修回日期:2010-12-20
基金项目:国家自然科学基金资助项目(60878053);广东省高等学校科技创新团队资助项目(06CXTD009)
作者简介:牛憨笨(1940-),男(汉族),山西省壶关县人,中国工程院院士、深圳大学研究员.E-mail:hbniu@szu.edu.cn
更新日期/Last Update: 2011-01-12