method was not used by many biologists but rather limited to use by physicists. The other method is known as the objective lens method which has increased the use of TIRFM in cellular microscopy and increased furthermore since a commercial solution became available. In this mechanism, one can easily switch between standard widefield fluorescence and TIRF by changing the off-axis position of the beam focus at the objective's back focal plane. There are several developed ways to change the positions of the beam such as using an actuator that can change the position in relation to the fluorescence illuminator that is attached to the microscope. Application In cell and molecular biology, a large number of molecular events in cellular surfaces such as cell adhesion, binding of cells by hormones, secretion of neurotransmitters, and membrane dynamics have been studied with conventional fluorescence microscopes. However, fluorophores that are bound to the specimen surface and those in the surrounding medium exist in an equilibrium state. When these molecules are excited and detected with a conventional fluorescence microscope, the resulting fluorescence from those fluorophores bound to the surface is often overwhelmed by the background fluorescence due to the much larger population of non-bound molecules. TIRFM allows for selective excitation of the surface-bound fluorophores, while non-bound molecules are not excited and do not fluoresce. Due to the fact of sub-micron surface selectivity, TIRFM has become a method of choice for single molecule detection. There are many applications of TIRFM in cellular microscopy. Some of these applications include: Measuring the kinetics of receptor endocytosis in response to ligand binding and receptor movement Observing exocytic events through the loading of vesicles undergoing exocytosis with fluorescent dyes Qualitatively and quantitatively describing the roles different proteins play in exocytosis/endocytosis Observing the size, movement, and distance apart of the regions of contact between a cell and a solid substrate With the ability to resolve individual vesicles optically and follow the dynamics of their interactions directly, TIRFM provides the capability to study the vast number of proteins involved in neurobiological processes in a manner that was not possible before. Benefits TIRFM provides several benefits over standard widefield and confocal fluorescence microscopy such as: The background is substantially decreased so structures can be seen clearly There is virtually no out-of-focus fluorescence collected which decrease blurring effects Cells are exposed to a significantly smaller amount of light which limits phototoxicity to cells Overview