Simultaneous Analysis of Multiple Fluorescent Probes in Single Cells by Microspectroscopic Imaging

摘要

A spectrograph based imaging system attached to a microscope is described for use in monitoring the fluorescence of multiple probes loaded into single cells. Movements of ions, important in regulating cell function, are inter-related under many conditions. Since we are interested in the mechanisms underlying coupling between ion movements and metabolism, we required a microscope based system to analyze these parameters at the level of a single cell. The spectral imaging system was implemented for two purposes. (1) To resolve heterogeneities which exist in the response of individual cells in a population, and within any given cell due to loading of dye into more than one subcellular compartment, and (2) to study the temporal relations between several physiological parameters from a single cell. Since spectral imaging resolves signal from multiple probes simultaneously, the concentrations of several ions or metabolic factors can be followed in a single cell. Moreover, spatial information is acquired along one axis, providing information from probes located in individual compartments. The spectral imaging microscope and some specific applications are described.