Generating live cell data using total internal reflection fluorescence microscopy

Abstract

Live cell fluorescent microscopy is important in elucidating dynamic cellular processes such as cell signaling, membrane trafficking, and cytoskeleton remodeling. Often, transient intermediate states are revealed only when imaged and quantitated at the single-molecule, vesicle, or organelle level. Such insight depends on the spatiotemporal resolution and sensitivity of a given microscopy method. Confocal microscopes optically section the cell and improve image contrast and axial resolution (>600 nm) compared with conventional epifluorescence microscopes. Another approach, which can selectively excite fluorophores in an even thinner optical plane (<100 nm) is total internal reflection fluorescence microscopy (TIRFM). The key principle of TIRFM is that a thin, exponentially decaying, evanescent field of excitation can be generated at the interface of two mediums of different refractive index (RI) (e.g., the glass coverslip and the biological specimen); as such, TIRFM is ill-suited to deep imaging of cells or tissue. However, for processes near the lower cell cortex, the sensitivity of TIRFM is exquisite. The recent availability of a very high numerical-aperture (NA) objective lens (>1.45) and turnkey TIRFM systems by all the major microscopy manufacturers has made TIRFM increasingly accessible and attractive to biologists, especially when performed in a quantitative manner and complemented with orthogonal genetic and molecular manipulations. This article discusses sample preparation for TIRFM, acquisition of time-lapse movies, and quantitative analysis. It also gives examples of imaging cytoskeleton dynamics and exo- and endocytosis using TIRFM.