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Related Experiment Videos

Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment

K M Berland1, P T So, E Gratton

  • 1Department of Physics, University of Illinois at Urbana-Champaign 61801.

Biophysical Journal
|February 1, 1995
PubMed
Summary

This study introduces two-photon excitation for fluorescence correlation spectroscopy, enabling precise measurements of molecular movement within living cells. Researchers tracked nanoparticle diffusion in cytoplasm, revealing initial slowdowns and subsequent immobilization.

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Area of Science:

  • Biophysics
  • Cell Biology
  • Microscopy

Background:

  • Fluorescence Correlation Spectroscopy (FCS) is a powerful technique for studying molecular dynamics.
  • Traditional FCS often requires confocal setups and can be limited in depth discrimination.
  • Investigating molecular mobility within the complex cellular environment presents significant challenges.

Purpose of the Study:

  • To apply two-photon excitation to Fluorescence Correlation Spectroscopy (FCS) for enhanced cellular analysis.
  • To perform the first measurements of translational mobility in the cytoplasm of living cells using this technique.
  • To accurately determine diffusion coefficients of nanoparticles within cellular environments.

Main Methods:

  • Utilized two-photon molecular excitation coupled with Fluorescence Correlation Spectroscopy (FCS).

Related Experiment Videos

  • Developed a method for depth-resolved measurements without emission pinholes.
  • Calibrated measurements using latex beads of known sizes in solutions of varying viscosity.
  • Applied the technique to measure the diffusion of 7-nm and 15-nm radius latex beads in mouse fibroblast cytoplasm.

    • Main Results:

    • Accurate diffusion constant (D) measurements were achieved for particles in bulk solutions.
    • Initial measurements in cytoplasm showed nanoparticles diffusing 2-5 times slower than in water.
    • Average diffusion rates were 18 x 10⁻⁸ cm²/s for 7-nm and 5 x 10⁻⁸ cm²/s for 15-nm beads.
    • Observed time-dependent diffusion, with motion slowing 10-100 times after a few hours due to cellular adhesion.

    Conclusions:

    • Two-photon excitation significantly enhances FCS capabilities for live-cell imaging and mobility studies.
    • This method provides depth discrimination and reduces phototoxicity.
    • The study demonstrates the feasibility of measuring nanoparticle translational diffusion within the cytoplasm of living cells.
    • Findings reveal complex, time-dependent dynamics of nanoparticles interacting with the cellular environment.