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Related Experiment Videos

Multifunctional fluorescence correlation microscope for intracellular and microfluidic measurements.

Xiaotao Pan1, Willy Foo, Wanrong Lim

  • 1NUS Graduate Program in Bioengineering, National University of Singapore, 28 Medical Drive, Singapore 117456, Singapore.

The Review of Scientific Instruments
|June 8, 2007
PubMed
Summary
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This study presents a modified fluorescence correlation microscope (FCM) for advanced cellular analysis. The new system enables precise positioning for fluorescence correlation spectroscopy (FCS) measurements, enhancing biological research capabilities.

Area of Science:

  • Biophysics
  • Cell Biology
  • Microscopy

Background:

  • Confocal laser scanning microscopy (CLSM) is a powerful imaging technique.
  • Fluorescence correlation spectroscopy (FCS) provides quantitative information about molecular dynamics.
  • Integrating CLSM and FCS can enhance spatial resolution and analytical capabilities.

Purpose of the Study:

  • To develop a modified fluorescence correlation microscope (FCM) integrated with a commercial confocal laser scanning microscope (CLSM).
  • To enable accurate positioning of the FCS observation volume for precise molecular analysis.
  • To demonstrate the system's versatility in biological and microfluidic applications.

Main Methods:

  • Modification of a commercial CLSM by incorporating two sensitive detectors for FCS.

Related Experiment Videos

  • Utilization of a single pinhole for both CLSM imaging and FCS, ensuring positional accuracy.
  • Implementation of a slider switch for seamless transition between imaging and spectroscopy modes.
  • Application of two-photon excitation FCS for flow direction characterization in microchannels.

    • Main Results:

    • The FCM system allows precise positioning of the FCS observation volume within the field of view.
    • Spatial fluorescence cross-correlation spectroscopy was proposed and utilized for microfluidic flow angle determination.
    • Translational diffusion coefficients were accurately measured on Chinese hamster ovary cell membranes.
    • The system demonstrated versatility in characterizing microfluidic flow using two-photon excitation FCS.

    Conclusions:

    • The developed FCM system offers a robust platform for combining CLSM imaging with FCS.
    • The single-pinhole design guarantees the identity of imaged and spectroscopically observed positions.
    • This integrated system enhances the study of molecular dynamics in biological samples and microfluidic systems.