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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
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.
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).
- 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.