Imaging with total internal reflection fluorescence microscopy for the cell biologist

Abstract

SummaryTotal internal reflection fluorescence (TIRF) microscopy can be used in a wide range of cell biological applications, and is particularly well suited to analysis of the localization and dynamics of molecules and events near the plasma membrane. The TIRF excitation field decreases exponentially with distance from the cover slip on which cells are grown. This means that fluorophores close to the cover slip (e.g. within ~100 nm) are selectively illuminated, highlighting events that occur within this region. The advantages of using TIRF include the ability to obtain high-contrast images of fluorophores near the plasma membrane, very low background from the bulk of the cell, reduced cellular photodamage and rapid exposure times. In this Commentary, we discuss the applications of TIRF to the study of cell biology, the physical basis of TIRF, experimental setup and troubleshooting.

Microscopic Visualization of Metabotropic Glutamate Receptors on the Surface of Living Cells Using Bifunctional Magnetic Resonance Imaging Probes

“…Due to its experimental design, TIRF microscopy facilitates very shallow depth penetration and primarily illuminates the fluorophores near to the coverslip adhered cell surface; hence, the signal from intracellular regions is reduced to a minimum. 21 A signal from the imaging probes at a maximum distance of 150 nm from the plasma membrane could be obtained employing this microscopy technique. Using the same cell preparations, live-cell laser scanning confocal microscopy (LSCM) studies were also performed.…”

The Nicotine Metabolite, Cotinine, Alters the Assembly and Trafficking of a Subset of Nicotinic Acetylcholine Receptors

“…TIRFM allows the excitation of fluorophores within ϳ100 -200 nm from the cell-glass bottom dish interface, visualizing receptors localized to the plasma membrane or peripheral endoplasmic reticulum (38). SEP was excited with a 488-nm diode-pumped solid-state laser (ϳ1.00 watt/cm 2 ) through the objective (Olympus, 1.49NA, 60ϫ oil immersion) and detected by an electron-multiplying charge coupled device (Andor iXon Ultra 897).…”

“…After images were collected at pH 7.4, the solution was exchanged with an otherwise identical solution adjusted to pH 5.4. When the pH of the extracellular solution is Ͻ6, nAChRs located on the PM transition into the off state, so the observed fluorescence is solely from nAChRs in the peripheral ER (7,38,41). The integrated density of TIRF images, showing the relative number of fluorescent receptors, are collected at both pH 7.4 and pH 5.4.…”

DNA-Endonuclease Complex Dynamics by Simultaneous FRET and Fluorophore Intensity in Evanescent Field

“…In the TFM scheme, fluorophores of surface-immobilized biomolecules are excited by the evanescent electromagnetic field created by the total internal reflection (TIR) of the laser light off the glass-water interface (19). Because the intensity of this field decays exponentially with the distance from the glass surface with the exponent factor being of the order of a few hundreds of nanometers, only a shallow near-surface layer is excited, thus ensuring a better signal-to-noise ratio compared with a regular wide-field excitation (20). At the same time, this exponential field strength distance-dependence implies that the rate of excitation of a fluorophore should depend on its distance from the surface.…”