New Microscopic Techniques to Investigate Intracellular Localization and Reactions of Photosensitizers: Laser Scanning Fluorescence Lifetime Imaging (LS-FLIM)
New Microscopic Techniques to Investigate Intracellular Localization and Reactions of Photosensitizers: Laser Scanning Fluorescence Lifetime Imaging (LS-FLIM)
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DOI：
10.1078/1615-1615-00045
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发表时间：
2002
期刊：
影响因子：
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通讯作者：
A. Rück;Matthias Kress;F. Dolp;N. Akgün;T. Meier;R. Steiner
中科院分区：
文献类型：
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作者：
A. Rück;Matthias Kress;F. Dolp;N. Akgün;T. Meier;R. Steiner
本工作描述了活细胞中5-ALA诱导PPIX的时间分辨荧光特性。细胞内荧光寿命由时间门控光谱和单光子计数确定，使用ps脉冲二极管激光器进行荧光激发。将波长为398nm、持续时间为70ps的二极管激光器耦合到共聚焦激光扫描显微镜上。时间分辨光谱采用了由切尔尼·特纳光谱仪和mcp门控加强型CCD相机组成的装置。通过将激光束置于“点扫描”模式，获得细胞内的时间门控光谱。此外，开发了一种新型的时间相关单光子计数模块，用于从单个点确定荧光寿命并记录寿命图像。PPIX的荧光寿命为7.4 ns，较短的3.6 ns可能是由于PPIX的光产物和聚集体。与单独的荧光强度图像相比，不能区分不同的荧光种类。然而，在寿命图像中，细胞质中的结构化荧光分布与较长的寿命相关，并且可能与线粒体一致。短脉冲二极管激光器耦合到激光扫描显微镜，配备适当的检测单元，因此可以实现高空间分辨率的时间分辨光谱和寿命成像，并为细胞和药物研究提供了许多可能性。
Summary This work describes the time-resolved fluorescence characteristics of 5-ALA induced PPIX in living cells. The intracellular fluorescence lifetime was determined from time-gated spectra as well as single photon counting, using a ps pulsed diode laser for fluorescence excitation. The diode laser which emits pulses at 398 nm with 70 ps duration was coupled to a confocal laser scanning microscope. For time resolved spectroscopy a setup consisting of 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 novel developed time-correlated single photon counting module was used to determine the fluorescence lifetime from single spots and to record lifetime images. A fluorescence lifetime of 7.4 ns was found for PPIX, a shorter lifetime at 3.6 ns was probably attributed to photoproducts and aggregates of PPIX. In contrast from fluorescence intensity images alone, different fluorescence species could not be distinguished. However, in the lifetime image a structured fluorescence distribution in the cytoplasm was correlated with the longer lifetime and probably coincides with mitochondria. Short pulsed diode lasers coupled to a laser scanning microscope, equipped with appropriate detection units allows therefore time-resolved spectroscopy and lifetime imaging with high spatial resolution and provides numerous possibilities in cellular and pharmaceutical research.