Center for Fluorescence Spectroscopy, Department of Biological Chemistry, University of Maryland at Baltimore, 660 West Redwood Strect, 21201, Baltimore, Maryland.
J Fluoresc. 1992 Mar;2(1):47-62. doi: 10.1007/BF00866388.
We describe imaging of calcium concentrations using the long-wavelength Ca(2+) indicators, Calcium Green, Orange, and Crimson. The lifetimes of these probes were measured using the frequency-domain method and were found to increase from 50% to severalfold in response to calcium. The two-dimensional images of the calcium concentration were obtained using a new apparatus for fluorescence lifetime imaging (FLIM). We also describe procedures to correct for the position-dependent frequency response of the gain-modulated image intensifier used in the FLIM apparatus. Importantly, the FLIM method does not require the probe to display shifts in the excitation or emission spectra. Using the FLIM method, calcium imaging is possible using probes which display changes in lifetime in response to calcium. Consequently, calcium imaging is possible with excitation wavelengths ranging from 488 to as long as 620 nm, where autofluorescence and/or photochemical damage is minimal. These probes are also suitable for calcium measurements of single cells using lifetime-based flow cytometry.
我们描述了使用长波长 Ca(2+)指示剂 Calcium Green、Orange 和 Crimson 来测量钙离子浓度的成像技术。使用频域方法测量了这些探针的寿命，发现它们的寿命在响应钙离子时从 50%增加到数倍。使用荧光寿命成像 (FLIM) 的新设备获得了二维钙离子浓度图像。我们还描述了校正 FLIM 设备中使用的增益调制像增强器的位置相关频率响应的程序。重要的是，FLIM 方法不需要探针显示激发或发射光谱的位移。使用 FLIM 方法，可以使用在响应钙离子时显示寿命变化的探针进行钙离子成像。因此，可以使用从 488nm 到最长 620nm 的激发波长进行钙离子成像，在此范围内，自发荧光和/或光化学损伤最小。这些探针也适用于基于寿命的流式细胞术对单细胞进行钙离子测量。
