Douglass, A.D. and Vale R.D. (2005). In: Cell Biology: A Laboratory Handbook, 3rd ed., Elsevier Sci (USA), pp. 129-136.
Traditional approaches to molecular and cell biology rely on techniques that infer the behavior of biomolecules based on the properties of a large ensemble. While using such methods to derive the average behavior of a system clearly has been valuable in developing our understanding of biology, bulk measurements often fail to provide information on heterogeneity within the population. The development of methods to measure the electrical activity of single ion channeels (Neher et al., 1978) first illustrated this point quite clearly, and the ensuing years have seen an explosion of interest in expanding our ability to study biological meterials on a single molecule level, especially using optical techniques. Early studies n the field focused primarily on in vitro systems containing a limited number pf purified components (Funatsu et al., 1995; Dickenson et al., 1997; Pierce et al, 1997) and greatly advanced our ability to detect variations not only within a population of molecules, but within the behavior of individual molecules over time. More recently, several groups have demonsrated the possibility of adapting single molecule techniques to living cells (Sako et al., 2000; Ueda et al, 2001).
These new abilities have been facilitated by several factors, including (1) the advent of green fluorescent protein (GFP) as a fluorescent tag for proteins in their natural environment, (2) the improved sensitivity of intensified charge-coupled devices (CCDs) and other detectors, and (3) the development and refinement of total internal reflectioin fluorescence (TIRF) microscopy (Axelrod, 2003). While other techniques, such as near-field scanning, optical microscopy (NSOM) and more traditional wide-field approaches, have been used for single molecule imaging in cells (Schutz et al., 1997; de Lange et al., 2001), TIRF is by far the most commonly used. In TIRF illumination, the excitation light is directed at the sample at a sufficently large angle that it is totally internally reflected when it strikes the interface between the aqueous buffer and the glass substrate upon which it lies. This creates an exponentially decaying evansecent wave that penetrates into the sample to a depth of only 100-200 nm, the exact distance depending on the refractive indices of the two materials. As a result, only fluorophores lying near the interface are excited efficently. This greatly decreases the background signal contributed by out-of-focus fluorescence and helps increase the signal-to-noise ratio in the output image to the level required for single molecule detection. Ready-built TIRF systems are available from several microscope vendors (Olympus, Nikon); however, it is relatively simple to add TIRF optics to an ordinary, inverted microscope, which is often preferable to commercial options due to the greater flexibility such an approach provides for later modifications to the system. This chapter provides an overview of how to construct, align, and use a TIRF system for single molecule imaging in living cells.