Sub-diffusion decays in fluorescence correlation spectroscopy: dye photophysics or protein dynamics?

Area of Science:

• Photophysics and Spectroscopy
• Biophysical Chemistry
• Fluorescence Microscopy

Background:

• Rhodamine dyes are widely used fluorescent labels in biological research.
• Understanding dye photophysical transitions is crucial for accurate interpretation of fluorescence data.
• Sub-diffusion dynamics and dark states can complicate fluorescence correlation spectroscopy (FCS) analysis.

Purpose of the Study:

• To investigate the transitions between bright and dark fluorescent states of rhodamine dyes.
• To characterize the nature of these dark states and their dependence on experimental conditions.
• To clarify the relationship between dye photophysics and biomolecular dynamics in FCS.

Main Methods:

• Fluorescence Correlation Spectroscopy (FCS) was employed.
• Experiments were conducted on free rhodamine dyes in aqueous solutions and on rhodamine-labeled proteins and DNA.
• Data analysis involved fitting correlation curves with multiple exponential decays and varying experimental parameters (viscosity, oxygen, laser power, geometry).

Main Results:

• Two sub-diffusion exponential decays were resolved for free rhodamines, with the slower component dependent on solution viscosity.
• Flickering dynamics on a microsecond timescale were observed for labeled biomolecules, fitted with three to four exponential decays.
• Dark states were assigned to triplet, twisted-intramolecular-charge-transfer/lactonic hybrid, lactonic, and photoionized states based on their population and lifetime changes.
• No direct correlation was found between sub-diffusion decays and biomolecular intramolecular dynamics.

Conclusions:

• The observed sub-diffusion decays in FCS are primarily attributed to the photophysics of the rhodamine dye label, not the intrinsic dynamics of the biomolecule.
• Biomolecular conformational dynamics can influence or modulate the photophysical behavior of the dye.
• Accurate modeling of dye photophysics and control experiments are essential for reliable interpretation of FCS data, cautioning against direct assignment of dark-state relaxation to biomolecular folding kinetics.