Single-molecule
imaging (SMI) has been widely utilized to investigate
biomolecular dynamics and protein–protein interactions in living
cells. However, multicolor SMI of intracellular proteins is challenging
because of high background signals and other limitations of current
fluorescence labeling approaches. To achieve reproducible intracellular
SMI, a labeling probe ensuring both efficient membrane permeability
and minimal non-specific binding to cell components is essential.
We developed near-infrared fluorescent probes for protein labeling
that specifically bind to a mutant β-lactamase tag. By structural
fine-tuning of cell permeability and minimized non-specific binding,
SiRcB4 enabled multicolor SMI in combination with a HaloTag-based
red-fluorescent probe. Upon addition of both chemical probes at sub-nanomolar
concentrations, single-molecule imaging revealed the dynamics of TLR4
and its adaptor protein, TIRAP, which are involved in the innate immune
system. Statistical analysis of the quantitative properties and time-lapse
changes in dynamics revealed a protein–protein interaction
in response to ligand stimulation.