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Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells
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Adaptive optics for fluorescence correlation spectroscopy.

Charles-Edouard Leroux1, Irène Wang, Jacques Derouard

  • 1Université Grenoble 1/CNRS, LIPhy UMR 5588, Grenoble, France.

Optics Express
|January 26, 2012
PubMed
Summary
This summary is machine-generated.

Optical aberrations significantly bias Fluorescence Correlation Spectroscopy (FCS) measurements. An adaptive optics system corrects these aberrations, stabilizing FCS parameters and improving accuracy for molecular analysis.

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Area of Science:

  • Biophysics
  • Optical Spectroscopy
  • Microscopy

Background:

  • Fluorescence Correlation Spectroscopy (FCS) quantifies molecular concentration and kinetics.
  • Accurate FCS requires precise calibration of the observation volume, sensitive to optical conditions.
  • Optical aberrations can introduce significant errors in FCS parameter estimation.

Purpose of the Study:

  • To investigate the impact of optical aberrations on single-photon FCS measurements.
  • To demonstrate the effectiveness of an adaptive optics system for aberration correction in FCS.
  • To analyze the relationship between molecular brightness and optical quality.

Main Methods:

  • Gradually introducing optical aberrations by varying focal depth in fluorescent solutions.
  • Implementing a simple adaptive optics system with a deformable mirror for aberration correction.
  • Comparing FCS parameters before and after aberration correction.

Main Results:

  • Depth-dependent optical aberrations cause substantial bias in FCS-derived parameters (e.g., molecule number, diffusion time).
  • Adaptive optics correction stabilizes FCS parameters within tens of micrometers into the solution.
  • Molecular brightness was experimentally and theoretically shown to scale with the Strehl ratio squared.

Conclusions:

  • Optical aberrations are a critical factor affecting FCS accuracy, especially at increasing depths.
  • Adaptive optics provides an effective solution for mitigating aberrations and improving the reliability of FCS measurements.
  • The Strehl ratio is a key indicator for assessing optical quality and its impact on molecular brightness in FCS.