Resolving fluorescent species by their brightness and diffusion using correlated photon-counting histograms

Area of Science:

• Biophysics
• Spectroscopy
• Quantitative Biology

Background:

• Fluorescence fluctuation spectroscopy (FFS) analyzes fluorescence intensity variations from diffusing molecules in small volumes.
• Existing FFS techniques like fluorescence correlation spectroscopy (FCS) and photon counting histograms (PCH) resolve molecular species based on diffusion or brightness, respectively.
• A need exists for methods that can simultaneously differentiate molecules based on both diffusion and brightness characteristics.

Purpose of the Study:

• To introduce correlated photon counting histograms (cPCH), a novel FFS technique.
• To demonstrate that cPCH can simultaneously resolve fluorescent species by their brightness and diffusion properties.
• To show cPCH's potential for enhanced quantitative analysis in biology.

Main Methods:

• Definition of the cPCH distribution based on the probability of detecting photon counts at different time points.
• Development of expressions for cPCH applicable to dual-channel and dual-color measurements.
• Utilizing simulations to compare cPCH performance against FCS and PCH.

Main Results:

• Simulations show cPCH resolves two species differing in both diffusion and brightness more effectively than FCS or PCH alone.
• cPCH analysis can be extended to longer dwell times for improved signal-to-noise ratio.
• The cPCH methodology is adaptable for image analysis applications.

Conclusions:

• Correlated photon counting histograms (cPCH) offer a significant advancement in fluorescence fluctuation spectroscopy.
• This dual-parameter approach enhances the resolution of complex molecular populations.
• cPCH is poised to become a valuable tool for quantitative biology by maximizing information extraction from fluorescence signals.