Optimized Two‐Color Single‐Molecule Tracking of Fast‐Diffusing Membrane Receptors

Single particle tracking (SPT) combined with total internal reflection fluorescence microscopy (TIRFm) is an outstanding approach to decipher mechanisms on the cell membrane at the nanoscale. Multicolor configurations, needed to investigate interactions, are still hindered by several challenges. This work systematically and quantitatively analyzes the impact of necessary elements of SPT‐TIRFm setups on the signal‐to‐noise ratio (SNR), which must be optimized especially in dynamic studies needing minimally invasive dyes for biomolecule labeling. Autofluorescence originating from commonly used optical glass results in the dominant limiting factor in TIRFm, and a cover glass material is tested yielding significant SNR improvements in multichannel TIRFm. Moreover, methodologies are optimized for reducing fluorophore photobleaching in multicolor implementations requiring simultaneous stabilization of multiple dyes. The developed strategies are applied to the fast p75NTR receptors labeled by two fluorophores on the membrane of living cells, achieving reliable, simultaneous two‐color SPT, contrary to configurations using standard cover glasses. This work highlights the importance of optical materials suitable for microscopy and with reduced autofluorescence for increasing sensitivity toward ultimate spatiotemporal resolutions. In particular, the present protocols can pave the way for multicolor super‐resolved localization and tracking of single molecules by TIRFm, greatly expanding the potential of SPT. Strategies for achieving two‐color single‐particle tracking on fast diffusing molecules labeled with small organic dyes are discussed Typical coverglasses generate significant fluorescence background in these experiments, so an alternative optical material is introduced, with the needed aberration corrections; moreover, other autofluorescence sources and photobleaching are analyzed and reduced.