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Observing secretory granules with a multiangle evanescent wave microscope.

A Rohrbach1

  • 1Max Planck Institute for Medical Research, 69120 Heidelberg, Germany. rohrbach@embl-heidelberg.de

Biophysical Journal
|April 25, 2000
PubMed
Summary
This summary is machine-generated.

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Total internal reflection fluorescence microscopy (TIRFM) allows precise measurement of secretory granule diameters up to 300 nm. This advanced technique uses evanescent waves for sub-diffraction limit imaging in biological samples.

Area of Science:

  • Biophysics
  • Cell Biology
  • Microscopy

Background:

  • Total internal reflection fluorescence microscopy (TIRFM) utilizes evanescent waves for surface excitation.
  • Evanescent waves decay exponentially, enabling depth-dependent fluorescence measurements.
  • Bovine chromaffin cells contain secretory granules crucial for cellular processes.

Purpose of the Study:

  • To investigate the quantitative capabilities of TIRFM for measuring secretory granule dimensions.
  • To explore the application of a novel telecentric multiangle evanescent wave microscope.
  • To determine the axial distribution and diameters of granules using fluorescence intensity and Laplace transforms.

Main Methods:

  • Utilized a novel telecentric multiangle evanescent wave microscope.

Related Experiment Videos

  • Varied laser incidence angle to control evanescent wave penetration depth (60-300 nm).
  • Applied Laplace transforms to fluorescence intensity data for fluorophore distribution analysis.

    • Main Results:

    • Successfully reconstructed diameters of isolated granules ranging from 70 nm to 300 nm.
    • Demonstrated granule diameter reconstruction beyond the resolution limit of confocal microscopy.
    • Observed evanescent wave propagation along surfaces and scattering at higher refractive index objects.

    Conclusions:

    • TIRFM, particularly with the novel multiangle system, offers high-resolution quantitative measurements of sub-micron structures like secretory granules.
    • Accurate selection of evanescent wave parameters is critical for reliable quantitative TIRFM measurements.
    • The technique provides insights into granule distribution and size, surpassing conventional microscopy limits.