Monitoring Integrated Activity of Individual Neurons Using FRET-Based Voltage-Sensitive Dyes

Abstract

Fluorescence resonance energy transfer (FRET) is a physical dipole-dipole coupling between the excited state of a donor fluorophore and an acceptor chromophore that causes relaxation of the donor to a non-fluorescent ground state, which excites fluorescence in the acceptor. Initially described by Förster (1948), FRET has been extensively reviewed (Stryer 1978; Clegg 1995; Selvin 2000; Lakowicz 2006). In practical terms, the efficiency of FRET depends on the properties of the chromophores and the distance between them, measured as the Förster radius (R o): the distance between the donor and the acceptor at which half the energy is transferred. The magnitude of R o depends on the donor quantum yield and the spectral overlap between the donor emission and the acceptor absorbance spectra. For commonly used synthetic FRET pairs, R o values range from 2 to 8 nm (Wu and Brand 1994). FRET efficiency is inversely proportional to the sixth power of the donor and acceptor distance, providing a sensitive readout of intermolecular distances near R o. Experimentally, FRET is measured either as the decrease in the lifetime or intensity of donor fluorescence after the addition of acceptor, or as the increase in acceptor fluorescence after the addition of donor. Because typical R o values are similar to protein and membrane dimensions, FRET has proven useful in a wide variety of biochemical and cellular applications to investigate protein-protein interactions , protein and DNA conformational analysis, and membrane topography (Stryer 1978; Clegg 1995; Selvin 2000; Lakowicz 2006). Furthermore, with the advent of a large variety of fluorescent protein color variants, FRET has become a natural method to probe cellular biochemistry (Piston and Kremers 2007). Here, we review how FRET probes have been used to measure cellular membrane potentials. Voltage-sensitive dyes (VSDs) based upon FRET are composed of two molecules, with either the donor or acceptor being a hydrophobic anion introduced into the plasma membrane acting as the voltage sensor by translocating between the energy minima at the intracellular and extracellular membrane-water interfaces (Fig. 6.1A) (Gonzalez and Tsien 1995). When the transmembrane potential changes, the hydrophobic anion redistributes as an exponential function of the potential according to the Nernst equation. A second impermeant fluorophore is attached to one face of the membrane where it can undergo FRET with the mobile molecule, in proportion to the distance between the two molecules. When the impermeant fluorophore is bound to the extracellular membrane surface and the cell is at its normal (negative) resting potential, the anions are predominately near the extracellular face of the memb rane, so that the two molecules produce efficient FRET. When the membrane depolarizes, for whatever reason, the anions equilibrate at a higher density at the intracellular membrane surface, thereby decreasing FRET. When both molecules are fluorescent , all increases in the acceptor emission are at the expense of the donor emission, and vice versa (Fig. 6.2A, B), thus providing a ratiometric signal of the membrane potential change. FRET-based dyes are sometimes called "slow" dyes, which is true relative to electrochromic dyes that have typical response times of a few microseconds (Ebner and Chen 1995; Baker et al. 2005), but FRET dye time constants actually span a large range, from 400 ms to 500 ms, depending on the properties of the mobile anion. While not the fastest, FRET dyes have yielded some of the largest observed fractional fluorescence changes, ranging from 10-20% per 100 mV in intact tissue (Cacciatore et al. 1999) to 100-300% per 100 mV in isolated cells (Gonzalez and Maher 2002). Following a brief account of the development of FRET VSDs, we discuss their temporal resolution, sensitivity, and phototoxicity. We then present examples of how these dyes have been used to image neurons. Finally, we provide detailed staining protocols as a reference and starting points for use in other systems. 6.2 development of fret dye paIrs Gonzalez and Tsien (1995) were first to image voltage-dependent responses from a FRET-based VSD. They used negatively charged hydrophobic oxonol derivatives, bis-(1,3-dialkyl-2-thiobarbiturate)-trimethineoxonol, DiSBAC x (3), where x refers to the number of 6