Principles and best practices of optimizing a micromirror-based multicolor TIRF microscopy system

TIRF (Total Internal Reflection Fluorescence) microscopy is a powerful tool for measuring the intra- and intermolecular dynamics of fluorescently-labeled single molecules. As TIRF measurements move to more complex biological systems with more fluorescent probes, the multi-band-pass dichroic that separates excitation from emission becomes limiting for the microscope’s detection efficiency. To avoid this, multicolor colocalization-based experiments can employ “micromirror” (mm)TIRF, which replaces the dichroic with two 45°-angled rod mirrors that control the TIR excitation beam(s). Whereas a dichroic spectrally separates excitation and emission wavelengths, the micromirrors act to spatially separate the excitation beams from the collected emission photons within the objective lens itself. Comprehensive control of the TIR beam in mmTIRF can yield excellent signal to noise, and hence data quality, but at the price of increased optical complexity. Here, we introduce the theory behind these additional optical components and provide practical advice from our experience on the best way to set up, align, optimize, and maintain a mmTIRF instrument. We also demonstrate the practical effects of small misalignments to illustrate both the optimized signal quality and the degree of accuracy required to achieve it. We hope that this guide increases the accessibility of this type of instrumentation and helps researchers use it to produce data of the highest quality possible. ### Competing Interest Statement The authors have declared no competing interest.