Lakowicz J R, Szmacinski H, Nowaczyk K, Lederer W J, Kirby M S, Johnson M L
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore.
Cell Calcium. 1994 Jan;15(1):7-27. doi: 10.1016/0143-4160(94)90100-7.
We describe the first fluorescence lifetime images of cells. To demonstrate this new capability we measured intracellular images of Ca2+ in COS cells based on the Ca(2+)-dependent fluorescence lifetime of Quin-2. Apparent fluorescence lifetimes were measured by the phase-modulation method using a gain-modulated image intensifier and a slow-scan CCD camera. We describe methods to correct the images for photobleaching during acquisition of the data, and to correct for the position-dependent response of the image intensifier. The phase angle Quin-2 images were found to yield lower than expected Ca2+ concentrations, which appears to be the result of the formation of fluorescent photoproducts by Quin-2. Fluorescence lifetime imaging (FLIM) does not require wavelength-radiometric probes and appears to provide new opportunities for chemical imaging of cells.
我们描述了细胞的首张荧光寿命图像。为展示这一新技术，我们基于Quin-2的Ca(2+)依赖性荧光寿命，测量了COS细胞内Ca2+的图像。使用增益调制图像增强器和慢扫描电荷耦合器件相机，通过相位调制法测量表观荧光寿命。我们描述了在数据采集过程中对光漂白进行图像校正以及对图像增强器的位置依赖性响应进行校正的方法。发现相位角Quin-2图像产生的Ca2+浓度低于预期，这似乎是Quin-2形成荧光光产物的结果。荧光寿命成像（FLIM）不需要波长辐射探针，似乎为细胞化学成像提供了新的机会。
