Combining Fluorescence Fluctuations and Photobleaching to Quantify Surface Density

Area of Science:

• Biophysics
• Analytical Chemistry
• Surface Science

Background:

• Characterizing molecules with variable fluorophore labeling is challenging.
• Accurate quantification of molecular density and label distribution is crucial for various applications.

Purpose of the Study:

• To establish a self-calibrated method, pbFFS, for characterizing molecules with unknown fluorophore distributions.
• To provide a reliable estimation of absolute density or concentration of molecules.
• To investigate label distribution and molecular oligomerization.

Main Methods:

• Photobleaching fluctuation fluorescence spectroscopy (pbFFS) was developed and theoretically derived.
• Experimental application to measure surface density of streptavidin and biotin-Atto molecules.
• Application to molecules diffusing in solution to confirm label distribution.

Main Results:

• pbFFS accurately measures surface density, consistent with spectroscopic ellipsometry.
• The method enables in situ characterization and analysis of low-mass adsorbed molecules.
• Quantification of biotin-Atto molecule density binding to streptavidin demonstrated the technique's sensitivity.

Conclusions:

• pbFFS is a versatile tool for investigating molecules with variable fluorophore labeling.
• It allows for quantification of molecular number and label distribution.
• The technique is valuable for surface science, biomimetic systems, and oligomerization studies.