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首页> 外文会议>Conference on Manipulation and Analysis of Biomolecules, Cells, and Tissues Jan 28-29, 2003 San Jose, California, USA >Fluorescence lifetime imaging (FLIM) using ps pulsed diode lasers in laser scanning microscopes
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Fluorescence lifetime imaging (FLIM) using ps pulsed diode lasers in laser scanning microscopes

机译：在激光扫描显微镜中使用ps脉冲二极管激光器的荧光寿命成像（FLIM）

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A setup consisting on a laser scanning microscope equipped with appropriate detection units was developed for time-resolved intracellular fluorescence spectroscopy and fluorescence lifetime imaging (FLIM) for online detection of structural changes of various biomolecules. Short-pulsed excitation was performed with a diode laser which emits pulses at 398 nm with 70 ps duration. The laser was coupled to the laser scanning microscope. For time resolved spectroscopy a setup consisting on a Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Time-gated spectra within the cells were acquired by placing the laser beam in "spot scan" mode. In addition, a time-correlated single photon counting module was used to determine the fluorescence lifetime from single spots and to record lifetime images (τ-mapping). The time-resolved fluorescence characteristics of 5-ALA (5-aminolevulinic-acid), as well as 5-ALAhe (5-aminolevulinic-acid-hexylester)- induced protoporphyrine IX (PPIX) were investigated before and during PDT with subcellular resolution. For cells which were incubated with 5-ALA, a component with a fluorescence lifetime of about 7 ns was correlated with a structured fluorescence, which probably coincides with mitochondria, whereas a shorter lifetime was found in the cytoplasm. In the case of 5-ALAhe the lifetime of PPIX was longer, which could be due to different localization. During PDT the component with the longer lifetime completely vanished, whereas the shorter lifetime was retained. It seems that FLIM is a valuable method to selectively identify and localize the photodynamically active photosensitizer.
机译：开发了一种由配备有适当检测单元的激光扫描显微镜组成的设备，用于时间分辨细胞内荧光光谱和荧光寿命成像（FLIM），用于在线检测各种生物分子的结构变化。用二极管激光器进行短脉冲激发，该二极管激光器在398 nm处发射脉冲，持续时间为70 ps。激光耦合到激光扫描显微镜。对于时间分辨光谱，使用了由Czerny Turner光谱仪和MCP门控和增强CCD相机组成的装置。通过将激光束置于“点扫描”模式来获取细胞内的时间门控光谱。另外，使用时间相关的单光子计数模块确定单个斑点的荧光寿命并记录寿命图像（τ映射）。在PDT之前和期间，以亚细胞分辨率研究了5-ALA（5-氨基乙酰丙酸）和5-ALAhe（5-氨基乙酰丙酸-己酸酯）诱导的原卟啉IX（PPIX）的时间分辨荧光特性。对于与5-ALA孵育的细胞，荧光寿命约为7 ns的成分与结构化荧光相关，这可能与线粒体相符，而在细胞质中发现寿命较短。在5-ALAhe情况下，PPIX的寿命更长，这可能是由于不同的定位所致。在PDT期间，使用寿命更长的组件完全消失，而使用寿命较短。似乎FLIM是一种有价值的方法，可以有选择地识别和定位光动力活性光敏剂。

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