Single-molecule fluorescence imaging by total internal reflection fluorescence microscopy (IUPAC Technical Report)

Abstract

Total internal reflection fluorescence (TIRF) is a popular illumination technique in microscopy, with many applications in cell and molecular biology and biophysics. The chief advantage of the technique is the high contrast that can be achieved by restricting fluorescent excitation to a thin layer. We summarise the optical theory needed to understand the technique and various aspects required for a practical implementation of it, including the merits of different TIRF geometries. Finally, we discuss a variety of applications including super-resolution microscopy and high-throughput DNA sequencing technologies.

High Axial and Lateral Resolutions on Self-Assembled Gold Nanoparticle Metasurfaces for Live-Cell Imaging

“…The nanoscale structural changes shown in Figure b were not detectable by regular optical microscopy. The red-colored brightest spots were composed of a few pixels in the shortest direction, which are comparable to or slightly smaller than the theoretical diffraction limit (<200 nm in the lateral plane), even though the image was obtained during movement under a widefield microscope …”

“…The red-colored brightest spots were composed of a few pixels in the shortest direction, which are comparable to or slightly smaller than the theoretical diffraction limit (<200 nm in the lateral plane), even though the image was obtained during movement under a widefield microscope. 40 As mentioned in Section 1, several methods have been developed to improve the spatial resolution of fluorescence microscopy systems, not only conventional SIM, STED, and PALM/STORM but also point accumulation in nanoscale topography (PAINT), repetitive optical selective exposure (ROSE), and many other newly developed systems. 41−43 All of these microscopes use sophisticated and expensive superresolution microscopic techniques to localize the emitting points beyond the diffraction limit.…”

Total Internal Reflection Fluorescence Microscopy To Investigate the Distribution of Residual Bitumen in Oil Sands Tailings

“…Because bitumen is dispersed throughout the bulk of the sample, fluorescence from deeper planes creates significant background noise, resulting in the loss of information from the focused region of the sample. For that reason, to image bitumen distribution in MFT, we adapted a Total Internal Reflection Fluorescence (TIRF), which until now was used only in cell-biology research, − to image bitumen distribution in MFT. In TIRF, the sample is illuminated by an evanescent wave near the surface, in contrast to illumination by a propagating wave in a conventional confocal fluorescence microscope.…”