Imaging Membrane Proteins Using Total Internal Reflection Fluorescence Microscopy (TIRFM) in Mammalian Cells

Abstract

The cell surfaceome is of vital importance across physiology, developmental biology, and disease states alike. The precise identification of proteins and their regulatory mechanisms at the cell membrane has been challenging and is typically determined using confocal microscopy, two-photon microscopy, or total internal reflection fluorescence microscopy (TIRFM). Of these, TIRFM is the most precise, as it harnesses the generation of a spatially delimited evanescent wave at the interface of two surfaces with distinct refractive indices. The limited penetration of the evanescent wave illuminates a narrow specimen field, which facilitates the localization of fluorescently tagged proteins at the cell membrane but not inside of the cell. In addition to constraining the depth of the image, TIRFM also significantly enhances the signal-to-noise ratio, which is particularly valuable in the study of live cells. Here, we detail a protocol for micromirror TIRFM analysis of optogenetically activated protein kinase C-ε in HEK293-T cells, as well as data analysis to demonstrate the translocation of this construct to the cell-surface following optogenetic activation. Graphic abstract

PI(4,5)P2 regulates the gating of NaV1.4 channels

“…Upstream of the laser clean-up filters, the beams were conditioned for coherence using home-made Keplerian beam expanders. A high numerical-aperture apochromatic objective (60×, 1.5 NA; Olympus) installed on an RM21 microscope frame with a piezo-driven nano-positioning stage was illuminated by laser lines calibrated to provide 10 mW of incident light (MadCity Labs; Gada et al, 2023b ). The fluorescence from iRFP was captured using a back illuminated sCMOS camera from Teledyne Photometrics.…”