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Related Experiment Videos

Position-sensitive scanning fluorescence correlation spectroscopy.

Joseph P Skinner1, Yan Chen, Joachim D Müller

  • 1School of Physics and Astronomy, University of Minnesota, Minneapolis, 55455, USA. josephs@physics.umn.edu

Biophysical Journal
|May 17, 2005
PubMed
Summary
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Position-sensitive scanning fluorescence correlation spectroscopy (PSFCS) extracts spatial information from fluorescence fluctuations. This technique analyzes moving observation volumes to determine sample dynamics, including flow and immobilization.

Area of Science:

  • Biophysics
  • Spectroscopy
  • Microscopy

Background:

  • Fluorescence correlation spectroscopy (FCS) analyzes temporal fluorescence fluctuations in a stationary volume.
  • Scanning FCS (SFCS) introduces spatial information by moving the observation volume.
  • Existing SFCS methods lack precise spatial localization of fluorescence dynamics.

Purpose of the Study:

  • To introduce position-sensitive scanning FCS (PSFCS) for analyzing spatial and temporal fluorescence dynamics.
  • To develop theoretical expressions for PSFCS in various conditions like diffusion, flow, and immobilization.
  • To validate PSFCS by comparing experimental data with theoretical predictions.

Main Methods:

  • Implementing a circular scan trajectory with synchronized data acquisition.

Related Experiment Videos

  • Calculating correlations as a function of lag time and phase angle to encode position.
  • Deriving theoretical models for diffusion, flow, and immobilization within PSFCS framework.

    • Main Results:

    • PSFCS successfully determines the direction and speed of a flowing dye solution.
    • The position of immobilized particles can be accurately identified using PSFCS.
    • Experimental data aligns well with the derived PSFCS theory.

    Conclusions:

    • PSFCS provides a novel method to access spatial information in fluorescence dynamics.
    • The technique is capable of characterizing complex sample behaviors, including flow and immobilization.
    • PSFCS shows promise for applications in living cells, as demonstrated with enhanced green fluorescent protein in COS cells.