ABSTRACT
Most optical probes in biology are based on a fluorescent readout and are designed to report biological or even physiological parameters in living samples. Effectively, this enables researchers to gain information on a molecular scale, well below the resolution limit of light microscopy. Förster resonance energy transfer (FRET) is one method for gaining information on a molecular level, and it is frequently utilized in fluorescent biosensor design. FRET involves the nonradiative transfer of energy between two fluorophores, and it can occur only if a fluorescent molecule-the donor-is in very close proximity to another aromatic system such as a dye or another chromophore. The maximal distance is usually below 10 nm, which is conveniently close to the size of an average protein. The efficiency with which energy transfer occurs depends very strongly on the orientation and distance between the donor and the acceptor. The efficiency of energy transfer is inversely proportional to the sixth power of the donor and acceptor distance, rendering it very useful for readouts of, for example, protein conformational changes or protein cleavage (Schultz 2007). In addition, FRET can be exploited to determine interactions between molecules (Jares-Erijman and Jovin 2003) and as a molecular ruler on a single-molecule level to measure distances in molecules or complexes (Stryer 1978). This chapter provides an overview of FRET and complementary techniques that are useful in combination with optical probes or FRET experiments. Chapter 1 introduced fluorescent proteins (FPs) and their properties, which are widely exploited in the design of fluorescent biosensors. The use of genetically encoded constructs based on FPs is often advantageous, because they are easily expressed in living cells and investigated in vivo. The design and application of probes for biomolecules are described in subsequent chapters in Part II. A key prerequisite to gaining physiological readouts with these probes is the use of the appropriate imaging technique while keeping the environment of the specimen at physiological conditions. Because elevated light intensity can influence the physiological state of cells, it is important to use an imaging setup with high sensitivity (Gräf et al. 2005; Brown 2007) to minimize bleaching and phototoxicity. These imaging requirements are discussed in the first part of this chapter. The
2.3.2.6 FLIM-Reduced Fluorescence Lifetime of the Donor 58 2.3.2.7 Polarization Anisotropy Imaging 60
2.3.3 FRET Method Selection 60 2.4 Fluorescence Recovery after Photobleaching 60 2.5 Fluorescence Correlation Spectroscopy 62 2.6 Bimolecular Fluorescence Complementation 64 2.7 Dimerization-Dependent Fluorescence 65 2.8 Conclusion and Outlook 65 References 65
second part focuses on advanced microscopy methods, which are interesting in the context of biosensors. The main topic in this respect is FRET, as a large number of fluorescent biosensors depend on FRET, which is used to report the biosensor status due to conformational changes on binding of a ligand (e.g., calcium in case of cameleon sensors). In addition to biosensor readout, the mobility of molecules is another important parameter to measure. Fluorescence recovery after photobleaching (FRAP) and fluorescence cross-correlation spectroscopy (FCCS) are techniques used to determine protein dynamics, including binding and diffusion, and are complementary methods to FRET.
