Principles of Total Internal Reflection Microscopy (TIRFM)

INTRODUCTIONTotal internal reflection fluorescence microscopy (TIRFM) is a powerful technique for studying events that occur near a cell surface. The technique allows selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass. TIRFM has been used to study (1) exocytosis of single synaptic vesicles stained with FM1-43 in living goldfish retinal bipolar neurons and (2) exocytosis of single dense core granules stained with neuropeptide Y-enhanced green fluorescent protein (NPY-EGFP) in living bovine chromaffin cells. This article describes the basic theory behind TIRFM and provides an overview for setting up a TIRFM system.