Dynamic, real time imaging of ion activities in single living cells using fluorescence video microscopy and image analysis

Abstract

Quantitative Fluorescence Microscopy is a powerful analytical tool for the in vivo determination of molecules and ions concentrations in cell populations. Until recently the results obtained represent the average value from a large number of cells under the microscope. The recent development of ratiometric ion-sensitive fluorochromes has provided us with a molecular stopwatch, with which we can monitor microscopic and submicroscopic cellular events in individual cells. These probes can be introduced into the cytoplasm of living cells and as the ionic concentration of, i.e., calcium and hydrogen in the cell interior changes, the dyes undergo changes in fluorescence consisting of wavelength shifts and quantum efficiency. Combining this powerful new technique with ultra-sensitive low-light-level video cameras and digital image processing to the fluorescence microscope permits the study of both spatial and temporal distribution of ions localized on or within intact single cells. Details of the procedures and the equipment required for such a fluorescence ratio imaging system are described. A review of some of the fluorescent probes used for measuring intracellular free Ca²⁺ and pH will be illustrated with examples of data obtained with a Digital Image Processing System in our laboratory. The use of quantitative video fluorescence microscopy is proposed as a means of studying intracellular chemical pathology both for the dynamic investigations of intracellular processes and for diagnosis of diseases.

Key Words:

• BCECF
• cytosolic free Ca²⁺
• Fura-2
• digital image processing
• Indo-1
• pH
• low-light-level video camera