Total internal reflection fluorescence (TIRF) microscopy illuminator for improved imaging of cell surface events

Total internal reflection fluorescence (TIRF) microscopy is a high-contrast imaging technique suitable for observing biological events that occur on or near the cell membrane. The improved contrast is accomplished by restricting the thickness of the excitation field to over an order of a magnitude narrower than the z-resolution of an epi-fluorescence microscope. This technique also increases signal-to-noise, making it a valuable tool for imaging cellular events such as vesicles undergoing exocytosis or endocytosis, viral particle formation, cell signaling, and dynamics of membrane proteins. This protocol describes the basic procedures for setting up a through-the-objective TIRF illuminator and a prism-based TIRF illuminator. In addition, an alternate protocol for incorporating an automated deflection system into through-the-objective TIRF is given. This system can be used to decrease aberrations in the illumination field, to quickly switch between epi- and TIRF illumination, and to adjust the penetration depth during multicolor TIRF applications. In the commentary, a description of the total internal reflection phenomenon is given, critical parameters of a TIRF microscope are discussed, and technical challenges and considerations are reviewed.