FLUORESCENCE LIFETIME IMAGING MICROSCOPY (FLIM) - SPATIAL-RESOLUTION OF MICROSTRUCTURES ON THE NANOSECOND TIME-SCALE
FLUORESCENCE LIFETIME IMAGING MICROSCOPY (FLIM) - SPATIAL-RESOLUTION OF MICROSTRUCTURES ON THE NANOSECOND TIME-SCALE
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DOI：
10.1016/0301-4622(93)85012-7
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发表时间：
1993-12-01
影响因子：
3.8
通讯作者：
CLEGG, RM
中科院分区：
文献类型：
--
作者：
GADELLA, TWJ;JOVIN, TM;CLEGG, RM
研制了一种频域荧光寿命成像显微镜（FLIM）。落射照明荧光显微镜的连续波激光激发源以高频f（A）调制。调制荧光发射的寿命由荧光信号相对于激发光的相位延迟和调制深度确定。相位检测通过在接近（外差法）或（零差法）f（A）的频率下调制微通道板图像增强器的高压放大级而在图像中的每个位置处同时完成。增强器的外差或零差图像输出聚焦到冷却的高分辨率电荷耦合器件相机上，用于数字记录和随后的相位和调制分析。该技术具有正常稳态显微镜的灵敏度，并且相对简单。我们提出了几个例子，说明FLIM的应用程序，用于确定提示荧光寿命在皮升均匀的解决方案，寿命成像的单细胞，并在荧光图像中的相位抑制特定的寿命成分。本文介绍并讨论了寿命分辨图像处理的几个独特方面，包括数据的分析、统计评估和显示。荧光图像的空间和时间方面的耦合大大扩展了定量荧光显微镜的可能性。
A frequency domain fluorescence lifetime imaging microscope (FLIM) has been developed. A continuous wave laser excitation source of an epi-illumination fluorescence microscope is modulated at a high frequency f(A). The lifetime of the modulated fluorescence emission is determined from the phase delay and modulation depth of the fluorescence signal relative to that of the excitation light. Phase detection is accomplished simultaneously at every location in the image by modulating the high voltage amplification stage of a microchannel plate image intensifier at a frequency near (heterodyne method) or at (homodyne method) f(A). The heterodyne or homodyne image output of the intensifier is focused onto a cooled high resolution charge-coupled-device camera for digital recording and subsequent analysis of phase and modulation. The technique has the sensitivity of normal steady state microscopy, and is relatively simple to employ. We present several examples illustrating the applications of FLIM for determining prompt fluorescence lifetimes in picoliter homogeneous solutions, for lifetime imaging of single cells, and for phase suppressing particular lifetime components in fluorescence images. Several unique aspects of lifetime resolved image processing are featured and discussed, including the analysis, statistical evaluation, and display of the data. Coupling of the spatial and temporal aspects of fluorescence images extends considerably the possibilities for quantitative fluorescence microscopy.