Wang Jun, Bao Ning, Paris Leela L, Geahlen Robert L, Lu Chang
Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, Indiana 47907, USA.
Anal Chem. 2008 Dec 15;80(24):9840-4. doi: 10.1021/ac801940w.
Total internal reflection fluorescence microscopy (TIRFM) has been widely used to explore biological events that are close to the cell membrane by illuminating fluorescent molecules using the evanescent wave. However, TIRFM is typically limited to the examination of a low number of cells, and the results do not reveal potential heterogeneity in the cell population. In this report, we develop an analytical tool referred to as total internal reflection fluorescence flow cytometry (TIRF-FC) to examine the region of the cell membrane with a throughput of approximately 100-150 cells/s and single cell resolution. We use an elastomeric valve that is partially closed to force flowing cells in contact with the glass surface where the evanescent field resides. We demonstrate that TIRF-FC is able to detect the differences in the subcellular location of an intracellular fluorescent protein. Proper data processing and analysis allows TIRF-FC to be quantitative. With the high throughput, TIRF-FC will be a very useful tool for generating information on cell populations with events and dynamics close to the cell surface.
全内反射荧光显微镜（TIRFM）已被广泛用于通过利用倏逝波激发荧光分子来探索靠近细胞膜的生物事件。然而，TIRFM通常限于对少量细胞的检测，并且结果并未揭示细胞群体中潜在的异质性。在本报告中，我们开发了一种称为全内反射荧光流式细胞术（TIRF-FC）的分析工具，以大约100-150个细胞/秒的通量和单细胞分辨率检测细胞膜区域。我们使用一个部分关闭的弹性阀，迫使流动的细胞与存在倏逝场的玻璃表面接触。我们证明TIRF-FC能够检测细胞内荧光蛋白亚细胞定位的差异。适当的数据处理和分析使TIRF-FC具有定量性。凭借高通量，TIRF-FC将成为生成有关细胞群体信息的非常有用的工具，这些信息涉及靠近细胞表面的事件和动态。
