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Related Concept Videos

Protein Dynamics in Living Cells01:19

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Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
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Combining Fluorescence Fluctuations and Photobleaching to Quantify Surface Density.

Julius Sefkow-Werner1,2, Elisa Migliorini1,2, Catherine Picart1,2

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Summary

We developed photobleaching fluctuation fluorescence spectroscopy (pbFFS) to quantify molecules with unknown fluorophore distributions. This method accurately measures molecular density and label distribution, applicable both in solution and on surfaces.

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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.