D L Taylor

Fluorescence ratio imaging microscopy

Publisher Summary Rapid advancements in low-light-level imaging technology have made it possible to apply the fundamental principles of fluorescence spectroscopic analyses to living cells. The quantitation of an optical signal from living cells attached to a substrate is very difficult. Living cells move, change shape, and vary the local number density of membrane-bound organelles. Fluorescence ratio imaging provides a means of overcoming many of the problems associated with quantifying the complex temporal and spatial dynamics of molecules and organelles in living cells. This chapter describes the issues of fluorescence ratio imaging microscopy, including basic requirements of the instrumentation, validation of the basic tenets of ratioing as applied to microscopy, and some methods and issues in the application of ratio imaging. With continued development and improvement in fluorescent probes for ion measurements as well as the development of probes for other important molecules, the potential applicability of ratio imaging is enormous. Fluorescence ratio imaging is a spectroscopic technique and is usable for any technique that can be expressed by the relationship of signals at two or more wavelengths.

A Photocross-linking Fluorescent Indicator of Mitochondrial Membrane Potential'

Ionic dyes that distribute across membranes according to electrical potential have proven valuable as fluorescent indicators of mitochondrial energetics in living cells. Applications have been limited, however, as potential-dependent staining is lost during cell fixation. We have produced a membrane potential indicator whose potential-dependent distribution can be made permanent, to enable correlation of membrane potential with cytochemical information from immunofluorescence. A carbocyanine dye was derivatized with a photoreactive nitrophenylazide moiety so that irradiation would induce nonspecific, covalent attachment to nearby molecules. Photo-induced cross-linking was observed in paper chromatography, when irradiation caused immobilization of the dye. The new dye, named PhoCy (photofixable cyanine), showed specific staining of mitochondria in living Swiss 3T3 fibroblasts. When living cells were stained, irradiated, and fixed with formaldehyde, mitochondrial staining was retained owing to cross-linking with mitochondrial components. Omission of irradiation eliminated mitochondrial staining in fixed cells. Labeling, irradiation, and fixation procedures were optimized to produce bright specific staining with minimal background. The indicators sensitivity to mitochondrial potential was demonstrated by treating cells with 2,4-dinitrophenol, an uncoupler of mitochondrial electron transport, which decreased mitochondrial staining in living cells and in the corresponding fixed specimens.