A STED-FLIM microscope applied to imaging the natural killer cell immune synapse
A STED-FLIM microscope applied to imaging the natural killer cell immune synapse
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用于自然杀伤细胞免疫突触成像的 STED-FLIM 显微镜
DOI：
10.1117/12.875018
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Lenz M
中科院分区：
文献类型：
--
作者：
Lenz M
我们提出了一种受激发射耗尽（STED）荧光寿命成像（FLIM）显微镜，激发的微结构光纤超连续光源，泵浦飞秒钛宝石激光器，这也是用于耗尽。在激光扫描共聚焦荧光显微镜系统上使用压电扫描台实现，FLIM使用时间相关单光子计数（TCSPC）实现，这提供了共聚焦和STED-FLIM之间的方便切换，空间分辨率低至60 nm以下。我们将提出我们的设计考虑，使一个强大的仪器，生物应用，包括固定相位板和空间光调制器（SLM）的方法来塑造STED光束和STED和共焦FLIM显微镜的相关性之间的比较。继我们以前的应用程序FLIM-FRET研究细胞间信号在免疫突触（IS），我们采用STED显微镜表征的空间分布的细胞分子与subdiffraction分辨率在IS。特别是，我们正在成像自然杀伤细胞激活免疫突触的细胞骨架结构。我们还将介绍我们对多标记STED显微镜的进展，以确定如何相对空间的分子组织，以前无法检测到的传统显微镜技术，是重要的NK细胞的细胞毒性功能。关键词：STED，受激发射耗尽显微镜，自然杀伤细胞，荧光寿命成像，FLIM，超分辨显微镜。
We present a stimulated emission depletion (STED) fluorescence lifetime imaging (FLIM) microscope, excited by a microstructured optical fibre supercontinuum source that is pumped by a femtosecond Ti:Sapphire-laser, which is also used for depletion. Implemented using a piezo-scanning stage on a laser scanning confocal fluorescence microscope system with FLIM realised using time correlated single photon counting (TCSPC), this provides convenient switching between confocal and STED-FLIM with spatial resolution down to below 60 nm. We will present our design considerations to make a robust instrument for biological applications including a comparison between fixed phase plate and spatial light modulator (SLM) approaches to shape the STED beam and the correlation of STED and confocal FLIM microscopy. Following our previous application of FLIM-FRET to study intercellular signalling at the immunological synapse (IS), we are employing STED microscopy to characterize the spatial distribution of cellular molecules with subdiffraction resolution at the IS. In particular, we are imaging cytoskeletal structure at the Natural Killer cell activated immune synapse. We will also present our progress towards multilabel STED microscopy to determine how relative spatial molecular organization, previously undetectable by conventional microscopy techniques, is important for NK cell cytotoxic function. Keywords: STED, Stimulated Emission Depletion Microscopy, Natural Killer (NK) cell, Fluorescence lifetime imaging, FLIM, Super resolution microscopy.
DOI：
10.1073/pnas.96.26.15062
发表时间：
1999-12-21
影响因子：
11.1
作者：
Davis, DM;Chiu, I;Strominger, JL
通讯作者：
Strominger, JL