Fluorescence lifetime imaging by asynchronous pump-probe microscopy
Fluorescence lifetime imaging by asynchronous pump-probe microscopy
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DOI：
10.1016/s0006-3495(95)80148-7
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发表时间：
1995-12-01
影响因子：
3.4
通讯作者：
Gratton, E
中科院分区：
文献类型：
--
作者：
Dong, CY;So, PTC;Gratton, E
我们报告的发展扫描寿命荧光显微镜使用异步，泵浦探测（受激发射）的方法。这种技术有两个显著的优点。首先，由重叠的泵浦和探测激光器产生的互相关信号导致i）类似于共焦和双光子激发显微镜中的轴向切片效果，以及ii）与传统的单光子荧光显微镜相比提高的空间分辨率。第二，低频，互相关信号生成允许寿命分辨成像，而不使用快速光电探测器。本文给出的数据包括：1）确定泵浦-探测信号线性的激光源阈值功率; 2）使用0.28 μ m荧光乳胶球表征泵浦-探测强度分布; 3）高频水中罗丹明B的寿命测量（最高6.7 GHz）和4）荧光乳胶球、用罗丹明DHPE染色的人红细胞和小鼠成纤维细胞以及用溴化乙锭和罗丹明DHPE标记的小鼠成纤维细胞的寿命分辨图像。
We report the development of a scanning lifetime fluorescence microscope using the asynchronous, pump-probe (stimulated emission) approach. There are two significant advantages of this technique. First, the cross-correlation signal produced by overlapping the pump and probe lasers results in i) an axial sectioning effect similar to that in confocal and two-photon excitation microscopy, and ii) improved spatial resolution compared to conventional one-photon fluorescence microscopy. Second, the low-frequency, cross-correlation signal generated allows lifetime-resolved imaging without using fast photodetectors. The data presented here include 1) determination of laser sources' threshold powers for linearity in the pump-probe signal; 2) characterization of the pump-probe intensity profile using 0.28 mu m fluorescent latex spheres; 3) high frequency (up to 6.7 GHz) lifetime measurement of rhodamine B in water; and 4) lifetime-resolved images of fluorescent latex spheres, human erythrocytes and a mouse fibroblast cell stained by rhodamine DHPE, and a mouse fibroblast labeled with ethidium bromide and rhodamine DHPE.