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Resolving heterogeneity on the single molecular level with the photon-counting histogram
J D Müller1, Y Chen, E Gratton
1Laboratory for Fluorescence Dynamics, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801 USA. muller@uiuc.edu
Biophysical Journal
|January 5, 2000
Summary
Photon-counting histograms (PCH) analyze fluorescence fluctuations from diffusing particles. This method uniquely determines molecular brightness and particle number, enabling separation of mixed fluorescent species. Optimal conditions for resolving two species are detailed.
Area of Science:
- Biophysics
- Physical Chemistry
- Analytical Chemistry
Background:
- Fluorescence fluctuation spectroscopy relies on analyzing intensity variations caused by diffusing fluorescent particles.
- Photon-counting histograms (PCH) capture the distribution of photon counts from fluorescence fluctuations.
- Previous work established PCH theory for diffusing particles, identifying molecular brightness and particle number as key parameters.
Purpose of the Study:
- To explore the capabilities of PCH for separating mixtures of fluorescent species based on molecular brightness.
- To identify optimal conditions for resolving two-species mixtures using PCH.
- To experimentally validate PCH's ability to resolve molecular brightness and concentration in model systems and biomolecules.
Main Methods:
- Utilizing photon-counting histograms (PCH) to analyze fluorescence intensity fluctuations from diffusing particles.
- Convolving individual species' PCH to model the PCH of a mixture.
- Experimentally analyzing binary dye mixtures and fluorescently labeled proteins.
Main Results:
- PCH can uniquely determine molecular brightness and average particle number for each species.
- Optimal concentrations exist for maximizing signal statistics and resolving two-species mixtures.
- Experimental validation demonstrated successful resolution of molecular brightness and concentration in dye mixtures and a brightness ratio of two in proteins.
Conclusions:
- PCH is a powerful technique complementary to autocorrelation analysis for separating fluorescent mixtures based on molecular brightness.
- The method is sensitive to molecular brightness differences, enabling resolution of complex mixtures.
- The ability to resolve brightness ratios, even as low as two, has significant implications for biological applications involving labeled biomolecules.