Search

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.

Riboswitches are novel regulatory elements that respond to cellular metabolites to control gene expression. They are constituted of highly conserved domains that have evolved to recognize specific metabolites. Such domains, so-called aptamers, are folded into intricate structures to enable metabolite recognition. Over the years, the development of ensemble and single-molecule fluorescence techniques has allowed to probe most of the mechanistic aspects of aptamer folding and ligand binding. In this review, we summarize the current fluorescence toolkit available to study riboswitch structural dynamics. We fist describe those methods based on fluorescent nucleotide analogues, mostly 2-aminopurine (2AP), to investigate short-range conformational changes, including some key steady-state and time-resolved examples that exemplify the versatility of fluorescent analogues as structural probes. The study of long-range structural changes by Forster resonance energy transfer (FRET) is mostly discussed in the context of single-molecule studies, including some recent developments based on the combination of single-molecule FRET techniques with controlled chemical denaturation methods. This article is part of a Special Issue entitled: Riboswitches.