Background

Total internal reflection (TIR) occurs when the angle of incidence of a beam of light upon a refractive surface exceeds a certain threshold, called the critical angle, resulting in reflection of the incident beam of light. While this implies an absence of transmission of light across the interface, there is an escape of electromagnetic energy in the form of an evanescent wave. Total internal reflection fluorescence microscopy (TIRFM) relies on this exponentially decaying evanescent wave that penetrates to a depth of ~100 nm into the sample area. Given that TIR occurs at the interface of two materials with distinct refractive indices, in cell biology experiments the result is that the evanescent wave will only excite fluorescence capable moieties in the evanescent field at or near the cell surface. Thus, TIRFM illuminates fluorescent particles at the cell membrane below the Rayleigh limit for resolution, making the technique well suited for the study of membrane proteins and membrane-delimited signaling events, in fixed or live cell samples.

Several types of microscopy are utilized in the literature to achieve optical sectioning with the intent to localize fluorescent biological moieties to various cellular fractions. Confocal microscopy is a common and versatile technique that has the potential to target any plane of the sample rather than only the glass/liquid interface, as in TIRFM. As with any microscopy, the resolution is determined by the lateral (X-Y) and axial dimensions of the imaging voxel (the Z-plane of a 3-dimensional pixel), each of which is dependent upon numerous factors. Thus, the resolution of the image is determined by the hardware (lens and detector), software, and sample preparation, as well as the fluorophore (protein or dye) and the light source in use. Commonplace confocal microscopes typically operate with a lateral dimension above the Abbe limit of ~250 nm. However, adaptations such as Airy disc scanners and stimulated emission dyes that form the basis of many super resolution techniques can reduce the lateral dimension to below 100 nm. Technologies such as Airy disc confocal, stimulated emission depletion, photoactivated localization microscopy, and stochastic optical reconstruction microscopy have yielded unprecedented information about subcellular architecture and protein localization (Vangindertael et al., 2018). However, they typically require a fixed sample (particularly where specific emission dyes must be employed) and have lower axial resolution (equivalent to the depth of the slice of up to ~500 nm) (Axelrod, 2001) and imaging speeds that are below the biological timescale. The issue of low axial resolution can be improved with two-photon localization microscopy, which, in some cases, can be combined with super resolution in the X-Y plane (Zong et al., 2017). However, this suite of tools is typically expensive and requires specialized hardware, software, and sample preparation. In contrast, TIRFM provides precise localization of fluorescence moieties, at or close to the cell membrane (axial resolution below ~100 nm). In addition to being commercially available, TIRFM systems can be homemade and constructed to image a range of user-preferred fluorophores, including genetically encoded fluorescent proteins, in live cells at relatively high speed in a cost-effective manner. Therefore, TIRFM is the imaging modality of choice to study membrane-delimited proteins with a high signal-to-noise ratio in fixed and live samples.