Fluorescence cross-correlation spectroscopy (FCCS) is a single-molecule technique used for protein-protein interactions in biochemistry, biology, biophysics, and biomedicine. Traditional fluorescence correlation spectroscopy (FCS) suffers from limited temporal resolution due to after-pulsing, which may hinder real-time observation of fast photophysical processes. In contrast, FCCS offers enhanced temporal resolution and direct evidence of association reaction (binding) based simply on the amplitude and diffusion time of the observed cross-correlation curves using two-channel detection. However, very limited applications of FCCS have been published in Förster resonance energy transfer (FRET) analysis and protein-protein interactions studies. Here, we developed different FCCS modalities to investigate the translational diffusion, FRET analysis, and fluorescence depolarization of the mEGFP-linker-mScarlet-I construct (or GE2.3) as a crowding sensor. The FCCS setup was calibrated using a photostable reference fluorophore (rhodamine-110). Using a 50/50 beam splitter, we investigated the translational diffusion coefficient and hydrodynamic radius of cleaved and intact GE2.3 as a flexible, non-spherical molecule within the context of the Stokes-Einstein model in Ficoll-70-enriched buffer. We also used FCCS with a dichroic mirror to simultaneously detect the fluorescence emissions of both the donor (mEGFP) and acceptor (mScarlet-I) separately for single-molecule FRET analysis of GE2.3. In addition, we used FCCS with a polarizing beam splitter to investigate the wavelength-dependent depolarization of the emitted fluorescence of GE2.3 due to both rotational dynamics and energy transfer from the donor to the acceptor. These results will ultimately open doors to the development of different FCCS modalities for a wide range of scientific questions related to protein-protein interactions at the single-molecule level in living cells under healthy and diseased conditions.
Rosenberg et al. (Sun,) studied this question.