3 - An Introduction to Filters and Mirrors for FRET

Abstract

This chapter discusses some of the filters used for protein imaging of several Förster resonance energy transfer (FRET) pairs such as fluorphores or chromophores with high-resolution FRET techniques. FRET is a process that involves the direct transfer of excitation energy between two fluorochromes (molecules that have the potential to fluoresce) with overlapping emission and excitation spectra. FRET is a fairly simple optical arrangement: an excitation filter and dichroic mirror matches the wavelength of the donor molecule and an emission filter matches the fluorescence emitted by the acceptor. The excitation filter is designed to block all light from the source except those wavelengths that will be used to “excite” the fluorochrome of interest. This optic, traditionally made of float glass, is used at 0° angle of incidence (AOI), and is not normally ground and polished. The second optic, the dichromatic beamsplitter, is also called the dichroic or simply the mirror. This optic is not glass but is made from quartz or fused silica, and it must be made to very exacting tolerances of both physical flatness and optical characteristics. The emission/barrier filter in the set has the primary function of blocking the excitation wavelengths transmitted by the excitation filter of the set to an optical density of 6.