Variable‐angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex

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Abstract
Summary: Live‐cell microscopy imaging of fluorescent‐tagged fusion proteins is an essential tool for cell biologists. Total internal reflection fluorescence microscopy (TIRFM) has joined confocal microscopy as a complementary system for the imaging of cell surface protein dynamics in mammalian and yeast systems because of its high temporal and spatial resolution. Here we present an alternative to TIRFM, termed variable‐angle epifluorescence microscopy (VAEM), for the visualization of protein dynamics at or near the plasma membrane of plant epidermal cells and root hairs in whole, intact seedlings that provides high‐signal, low‐background and near real‐time imaging. VAEM uses highly oblique subcritical incident angles to decrease background fluorophore excitation. We discuss the utilities and advantages of VAEM for imaging of fluorescent fusion‐tagged marker proteins in studying cortical cytoskeletal and membrane proteins. We believe that the application of VAEM will be an invaluable imaging tool for plant cell biologists.