Setting up multicolour TIRF microscopy down to the single molecule level

Investigating biological mechanisms in ever greater detail requires continuous advances in microscopy techniques and setups. Total internal reflection fluorescence (TIRF) microscopy is a well-established technique for visualizing processes on the cell membrane. TIRF allows studies down to the single molecule level, mainly in single-colour applications. Instead, multicolour setups are still limited. Here, we describe our strategies for implementing a multi-channel TIRF microscopy system capable of simultaneous two-channel excitation and detection, starting from a single-colour commercial setup. First, we report some applications at high molecule density and then focus on the challenges we faced for achieving the single molecule level simultaneously in different channels, showing that rigorous optimizations on the setup are needed to increase its sensitivity up to this point, from camera setting to background minimization. We also discuss our strategies regarding crucial points of fluorescent labelling for this type of experiment: labelling strategy, kind of probe, efficiency, and orthogonality of the reaction, all of which are aspects that can influence the achievable results. This work may provide useful guidelines for setting up advanced single-molecule multi-channel TIRF experiments to obtain insights into interaction mechanisms on the cell membrane of living cells. © 2023 the author(s), published by De Gruyter.