Real-time fluorescence imaging with 20 nm axial resolution has revolutionized the study of protein structures near the plasma membrane of live cells. The intricate assembly of proteins to form nanoscale structures has been a challenge for researchers, especially when these structures are dynamic. The development of a two-wavelength total internal reflection fluorescence method allows for real-time imaging of cellular structure height with nanometer resolution. This new technique involves tagging a protein of interest with two different fluorophores and imaging to obtain the ratio of emission in the two channels.
Super-resolution imaging has significantly advanced our understanding of various biological structures, from focal adhesions to the neuronal cytoskeleton. However, the ability to resolve real-time dynamics along the z-direction has been limited. Total internal reflection fluorescence microscopy is a simple method to improve axial resolution. By utilizing an evanescent excitation field that decays exponentially from the coverslip, the illumination depth can be limited to around 100–200 nm. Several TIRF-based axial imaging approaches have been developed to enhance axial resolution, including differential interference nanometry (DiNa), which is robust for studying processes involving protein oligomerization and trafficking.
Existing techniques are suitable for imaging relatively static structures but are limited in studying dynamic protein complexes. Therefore, there is a significant need for methods to acquire the nanometer position of protein structures in real-time. The simultaneous two-wavelength axial ratiometry (STAR) technique presented in this study offers a solution. By exciting the sample with two wavelengths and measuring the ratio of fluorescence emission intensity between the two channels, the nanometer height of assembling or disassembling structures can be measured in real-time with nanometer resolution.
The STAR technique was validated through experiments with fluorescently labeled silica microspheres and microtubules in fixed cells. The method accurately measured the height of these structures with nanometer precision. Additionally, the technique was used to visualize the nanoscale organization of microtubules and endocytosis of the epidermal growth factor receptor in live cells. The dynamic imaging capability of STAR allowed for the measurement of the nanometer z-dynamics of the epidermal growth factor receptor during ligand binding, clustering, and internalization at the plasma membrane.
The study demonstrates that STAR is a simple, turn-key strategy that can determine the z-position of proteins within a cell with nanometer resolution in real-time. This technique offers new insights into the mechanisms of trafficking and assembly of membrane-associated proteins crucial to essential cellular processes. STAR’s compatibility with widely available organic dyes and conventional fluorescent proteins makes it a valuable tool for studying dynamic protein complexes in living systems.
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