Total Internal Reflection Fluorescence (TIRF) Microscopy

Abstract

Total internal reflection fluorescence (TIRF) microscopy (TIRFM) is an elegant optical technique that provides for the excitation of fluorophores in an extremely thin axial region ("optical section"). The method is based on the principle that when excitation light is completely internally reflected in a transparent solid (e.g., coverglass) at its interface with liquid, an electromagnetic field, called the evanescent wave, is generated in the liquid at the solid-liquid interface and is the same frequency as the excitation light. Since the intensity of the evanescent wave exponentially decays with distance from the surface of the solid, only fluorescent molecules within a few hundred nanometers of the solid are efficiently excited. This overview will review the history, optical theory, and hardware configurations used in TIRFM. In addition, it will provide experimental details and methodological considerations for studying receptors at the plasma membrane in neurons.

Single Particle Hopping as an Indicator for Evaluating Electrocatalysts

“… Notably, the rise of fluorescence signal was initiated (∼0 V) earlier than the onset potential in electrochemical measurement (∼0.60 V), indicating the existence of partial highly active catalytic sites that facilitate the heterogeneous oxygen bubble nucleation (Figure c). Furthermore, the size and lifetime distribution of gas bubbles could be estimated from the variations of R6G fluorescence trajectories in the evanescent field (Figure d). − The average lifetime of the gas bubbles was obtained from counting the blinking time of R6G fluorescence and calculated to be 3.85 and 5.77 s in anodic and subsequent cathodic polarization, respectively. The calculated bubble lifetimes were 2 orders of magnitude longer than the theory predicted values. , Because of the oversaturation region around gas bubbles, the mass transfer of molecules across the liquid–gas interface behaved as the limiting step in the gas bubble’s shrinkage and growth .…”

Multiscale fluorescence imaging of living samples

“…1 b). For studying biological dynamics in two dimensions within several hundred nanometers of the coverslip surface, total internal reflection fluorescence (TIRF) microscopy (Fish 2009 ) is an excellent choice. By restricting illumination to a thin zone of evanescently decaying energy, out-of-focus background is dramatically suppressed, leading to clear images with excellent SNR and contrast.…”

Fluorescence-based sensing of the bioenergetic and physicochemical status of the cell

“…Super resolution microscopy encompass a set of techniques that allow obtaining wide-field images of the analyzed probes at a resolution level beyond the diffraction limit via detection of single molecules (Khater, Nabi, & Hamarneh, 2020). Detection of single molecules can be achieved by using Total Internal Reflection Fluorescence (TIRF) microscopes (Fish, 2009), which use a source of light to illuminate the sample at a sufficiently oblique angle such that the light wave is totally reflected without refraction into the sample, allowing to image a very thin region of the cell, usually less than 200 nm. TIRF is an extremely powerful technique to image fluorescently labeled molecules that are in the vicinity of the glass slide onto which the cell or vesicle sample is loaded (Fish, 2009).…”

“…Detection of single molecules can be achieved by using Total Internal Reflection Fluorescence (TIRF) microscopes (Fish, 2009), which use a source of light to illuminate the sample at a sufficiently oblique angle such that the light wave is totally reflected without refraction into the sample, allowing to image a very thin region of the cell, usually less than 200 nm. TIRF is an extremely powerful technique to image fluorescently labeled molecules that are in the vicinity of the glass slide onto which the cell or vesicle sample is loaded (Fish, 2009). To measure fluorescence deeper inside the vesicles or cells, it is necessary for the light to pass through the sample and excite the fluorescent probes in a confined area.…”