Kusumi A, Tsuji A, Murata M, Sako Y, Yoshizawa A C, Kagiwada S, Hayakawa T, Ohnishi S
Department of Pure and Applied Sciences, College of Arts and Sciences, University of Tokyo, Japan.
Biochemistry. 1991 Jul 2;30(26):6517-27. doi: 10.1021/bi00240a024.
A time-resolved microscope fluorimeter based on a synchroscan streak camera and a fast pulsed laser system has been developed to measure the fluorescence lifetime decay under the fluorescence microscope. This system allows one to measure the nanosecond fluorescence lifetimes of fluorophores in a small spot (0.8-6.3 microns diameter) in single cultured cells under a fluorescence microscope, while the cells are being viewed under a high-power objective lens. A signal acquisition time between a second and a minute was usually sufficient to obtain fluorescence decay curves with good quality for 10(3)-10(5) fluorophores localized in 1 microns 2 domain. A signal-to-noise ratio better than 30 was obtained for approximately 30,000 fluorescein-labeled band 3 molecules in a 2 microns 2 region in a single human erythrocyte ghost after signal accumulation for 30 s. The measured lifetimes for a variety of fluorescent probes attached to proteins in solution and lipids in liposomes showed a good agreement with those measured in a cuvette under standard conditions by time-correlated single photon counting. With the development of this instrument, microscope fluorimetry has become a practical, straightforward, quantitative technique for investigation of molecular processes in single cells in culture. Time-resolved microscope fluorimetry has been applied to observe fusion of liposomes in vitro and that of endosomes in single cells by monitoring resonance energy transfer. Inspection of individual liposomes and endosomes revealed the extent of fusion for each vesicle. Since the use of time-resolved microscope fluorimetry eliminates the need for subcellular fractionation or the complex correction procedures in steady-state microfluorimetry, it greatly simplifies the assay for endosome fusion in vivo. The results showed that extensive fusion of sequentially formed endosomes takes place all over the cell matrix in cultured cells. This suggests that extensive fusion with incoming endosomes takes place in many endosomal compartments, possibly sorting organelles, or that the early endosomes fuse with the preexisting network of tubular cisternae of the endosomal compartment at many points in the network. It is concluded that time-resolved microscope fluorimetry is a powerful noninvasive technique for studies of in situ biochemistry and biophysics using cells and tissues.
基于同步扫描条纹相机和快速脉冲激光系统开发了一种时间分辨显微镜荧光计，用于测量荧光显微镜下的荧光寿命衰减。该系统能够在荧光显微镜下，当细胞在高倍物镜下观察时，测量单个培养细胞中一个小光斑（直径0.8 - 6.3微米）内荧光团的纳秒级荧光寿命。对于位于1平方微米区域内的10³ - 10⁵个荧光团，通常采集一秒到一分钟的信号时间就足以获得高质量的荧光衰减曲线。在单个人类红细胞血影的2平方微米区域内，对大约30,000个荧光素标记的带3分子进行30秒的信号积累后，获得了优于30的信噪比。对溶液中蛋白质和脂质体中脂质上连接的各种荧光探针测量的寿命，与在标准条件下通过时间相关单光子计数在比色皿中测量的结果显示出良好的一致性。随着该仪器的发展，显微镜荧光测定法已成为研究培养的单个细胞中分子过程的一种实用、直接的定量技术。时间分辨显微镜荧光测定法已被应用于通过监测共振能量转移来观察体外脂质体和单细胞内吞体的融合。对单个脂质体和内吞体的检查揭示了每个囊泡的融合程度。由于使用时间分辨显微镜荧光测定法无需进行亚细胞分级分离或稳态微荧光测定法中的复杂校正程序，它极大地简化了体内内吞体融合的检测。结果表明，在培养细胞的整个细胞基质中发生了依次形成的内吞体的广泛融合。这表明在许多内吞体区室（可能是分选细胞器）中与进入的内吞体发生了广泛融合，或者早期内吞体在网络中的许多点与内吞体区室预先存在的管状池网络融合。结论是，时间分辨显微镜荧光测定法是一种用于使用细胞和组织进行原位生物化学和生物物理学研究的强大非侵入性技术。
