Total Internal Reflection Fluorescence Microscopy

The goal in fluorescence microscopy is to detect the signal of fluorescently labeled molecules with great sensitivity and minimal background noise. In epifluorescence microscopy, it is difficult to observe weak signals along the optical axis, owing to the overpowering signal from the out-of-focus particles. Confocal microscopy uses a small pinhole to produce thin optical sections (∼500 nm), but the pinhole rejects some of the in-focus photons as well. Total internal reflection fluorescence microscopy (TIRFM) is a wide-field illumination technique that illuminates only the molecules near the glass coverslip. It has become widely used in biological imaging because it has a significantly reduced background and high temporal resolution capability. TIRFM has been used to study proteins in vitro as well as signaling cascades by hormones and neurotransmitters, intracellular cargo transport, actin dynamics near the plasma membrane, and focal adhesions in living cells. Because TIRF illumination is restricted to the glass-water interface and does not penetrate the specimen, it is well suited for studying the interaction of molecules within or near the cell membrane in living cells.