Chapter 12 - Confocal and Nonlinear Optical Imaging of Potentiometric Dyes

This laboratory has been engaged in the design and synthesis of voltage-sensitive dyes for over 20 years (Loew et al., 1978, Loew et al., 1979a, Loew et al., 1979b). Several important general-purpose dyes have emerged from this effort including di-5-ASP (Loew et al., 1979a), di-4-ANEPPS (Fluhler et al., 1985, Loew et al., 1992), di-8-ANEPPS (Bedlack et al., 1992, Loew, 1994), and TMRM and TMRE (Ehrenberg et al., 1988). Potentiometric dyes are designed either to measure membrane potentials in cell populations with a macroscopic system such as a spectrofluorometer or to be used in conjunction with a microscope to measure voltages associated with individual cells or organelles. The latter kinds of applications are the primary interest of this laboratory and have directed the dye chemistry development. Such experiments have been of great utility to neuroscientists interested in mapping patterns of electrical activity in complex neuronal preparations with numerous examples spanning the past 15 years (recent examples: Wu et al., 1994, Prechtl et al., 1997, Obaid et al., 1999, Tsodyks et al., 1999). In addition, the dyes have been used to map the spatial (Gross et al., 1985, Bedlack et al., 1992, Bedlack et al., 1994) and temporal (Shrager and Rubinstein, 1990, Zecevic, 1996) patterns of electical activity along single cell membranes and have measured potentials in fine processes and at synapses (Salzberg, 1989). We have even been able to measure the membrane potentials across the inner membranes of individual mitochondria within a single living cell (Loew et al., 1993, Fink et al., 1998). All of these experiments could not be accomplished with conventional electrical measurements using microelectrode or patch-clamp techniques.

The application of confocal microscopy to such measurements can significantly extend our ability to map the electrical properties of cell and organelle membranes with high spatial resolution. In addition to the obvious enhancement of resolution, especially along the optical axis, confocal microscopy offers opportunities for quantitative measurements of fluorescence intensities at each point in an image. Methods for calibrating such measurements and transforming them into spatial profiles of membrane potential will be described in this chapter. We will also review some new techniques for using nonlinear optical microscopy to probe for membrane potential in living cells. Specifically, we have developed a new nonlinear optical modality, second harmonic imaging microscopy (SHIM), that can deliver signals that are highly sensitive to membrane potential.