Methods in Cellular Imaging
Fluorescence Resonance Energy Transfer and Lifetime Imaging Microscopy
The preceding chapters described various microscopy methodologies that primarily reveal the distribution of amounts of fluorescent stain in the cell, and the production of two- or three-dimensional images of intracellular structure from this information. From advances in molecular biology of the past decade, we know that protein-protein association underlies the specificity of signal transduction. X-ray diffraction, nuclear magnetic resonance, and electron microscopy methodology has been used for studying the structure and localization of proteins under non-physiological conditions. We now need new methods to study protein associations in living cells in 3D and in real-time. Fluorescence resonance energy transfer (FRET) provides such a method.
The visualization and quantitation of protein associations under physiological conditions are explained in the following four chapters using steady-state fluorescence, two-photon excitation, and time-resolved, or lifetime imaging microscopy, methods. The fluorescence lifetime is defined as the average time that a molecule remains in an excited state prior to returning to the ground state. Two lifetime methods, time-domain (Chapters 15, 17-18) and frequency domain (Chapter 16, 19), are described in this section.
Chapter 15 outlines the basics of FRET as well as lifetime and various biological applications of FRET (monitoring of protease activity of cellular proteins during apoptosis, calcium using calcium sensitive cameleons, plasma membrane potential), and FRET screening assay for quantification of gene expression using b-lactamase. The author also describes bioluminescence resonance energy transfer (BRET), which uses luciferase fusion protein as a donor and GFP as an acceptor. This Chapter introduces for the first time in the literature the correction for bleed-through (or cross-talk), which is an inherent problem in steady-state (or digitized video) FRET microscopy. The author also explains the importance of lifetime imaging for FRET imaging, which provides dynamic events of the proteins (spatial and temporal), since the lifetime method is not dependent on excitation intensity or fluorophore concentration targeted to the proteins. Theoretical and experimental techniques are described in Chapter 16 for frequency domain lifetime FRET imaging. Double-exponential decays are resolved using a single frequency FLIM instrument for protein phosphorylation between a fused green fluorescent protein and an inodcyanine dye (Cy3) bound to an antibody against a phosphoamino acid.
Chapter 17 compares the various FRET microscopic techniques, such as wide-field, confocal, two-photon excitation, and double exponential lifetime decay for FRET imaging. Moreover, this chapter describes the methodology of the correction for bleed-through in intensity based FRET imaging techniques. The author also points out that it is possible to quantitate one- or more protein associations in lifetime method is compared to the other three techniques. Lifetime FRET images are provided for the donor (CFP) in the presence and the absence of the acceptor (YFP) for enhancer binding protein alpha (C/EBPa) dimerization in pituitary GHFT1-5 living cells.
Chapter 18 describes the time-domain methodology of acquiring one- and two-photon excitation fluorescence lifetime imaging (FLIM) for calcium, pH, and oxygen concentration in biofilm. In the time-resolved, pump-probe microscopy (Chapter 19), two laser beams at different wavelengths (and modulation frequencies) are focused onto a diffraction limited spot and this allows recording of time-resolved images and lifetime measurements for cytoplasm and nuclear areas of the fibroblast cells.