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FRET Imaging in the Wide-Field Microscope

Fred Schaufele, Ignacio Demarco, and Richard N. Day

1. Introduction

We have witnessed remarkable advances over the past decade in the application of light microscopy to visualize dynamic processes inside the living cell. The de-velopment of new fluorescent probes, coupled with advances in digital image ac-quisition and analysis, has dramatically improved our ability to obtain quantitative measurements from living cells. For example, the cloning of the jellyfish green fluo-rescent protein (GFP) afforded the enormous benefit of fluorescence labeling that is genetically encoded by transferable DNA sequences (reviewed by van Roessel and Brand, 2001; Zhang et al., 2002). This has transformed studies in cell biology by allowing the behavior of proteins to be tracked by fluorescence microscopy in their natural environment within the living cell. Strategies of mutagenesis of GFP, and the discovery of new GFP-like proteins are yielding an assortment of fluores- cent proteins (FPs) that emit from the blue to the red range of the visible spectrum (discussed below; Patterson et al., 2001; Matz et al., 2002; Zhang et al., 2002; Verkhusha and Lukyanov, 2004).

The FPs are widely used as noninvasive markers in living cells because their

fluorescence does not require the addition of cofactors, and they are very stable and well tolerated by most cell-types. The minimal toxicity of these probes is best illustrated by the many examples of healthy transgenic mice that express the FP markers (reviewed by Hadjantonakis and Nagy, 2001; Feng et al., 2000; Walsh and Lichtman, 2003). For instance, Brewer et al. (2002) generated “knockin” mice that express the GFP-glucocorticoid receptor under endogenous regulatory control. The expression and function of the chimeric receptor were indistinguishable from the endogenous counterpart it replaced, demonstrating that these imaging probes can be truly non-invasive. Further, since the FPs have no intrinsic intracellular target- ing, they're most useful for monitoring the subcellular localization and trafficking properties of proteins.

Importantly, the spectral properties of the FPs allow them to be used as probes in Förster resonance energy transfer (FRET) microscopy. FRET microscopy can detect the result of the direct transfer of energy from a donor to an acceptor FP that labels proteins of interest in the living cell. Since energy transfer can only occur over distances of less than about 8 nm, the detection of FRET provides information about the spatial relationships of those proteins on the scale of angstroms (Stryer, 1978; Selvin, 1995; Periasamy and Day, 1999). In this chapter, we describe the char-acteristics and discuss the limitations of the FPs as labels for proteins expressed in living cells. We review the principals of acquiring FRET measurements using wide-field microscopy (WFM) of cells expressing the FPs. We then demonstrate three different methods for detecting FRET signals from living cells, and consider the limi-tations of each of these approaches. Finally, we will discuss potential problems associated with the expression of proteins fused to the FPs for FRET-based mea-surements from living cells.