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Quantifying axial secretory-granule motion with variable-angle evanescent-field excitation
Dinah Loerke1, Walter Stühmer, Martin Oheim
1Department of Molecular Biology of Neuronal Signals, Max-Planck-Institute for Experimental Medicine, Hermann-Rein Str. 3, D-37075 Göttingen, Germany. dloerke@gwdg.de
Journal of Neuroscience Methods
|September 18, 2002
Summary
Evanescent-wave microscopy precisely tracks secretory vesicles, revealing distinct granule populations near the plasma membrane. This method quantifies vesicle transport and fusion dynamics, crucial for understanding exocytosis mechanisms.
Area of Science:
- Cell Biology
- Biophysics
- Microscopy
Background:
- Secretory vesicle transport to the plasma membrane is key to exocytosis.
- Evanescent-wave (EW) microscopy tracks vesicles with nanometer precision.
- Quantifying vesicle axial distance to the plasma membrane using EW microscopy has been challenging.
Purpose of the Study:
- To develop a method for estimating individual granule diameter and axial distance using EW microscopy.
- To analyze granule populations and their dynamics during exocytosis.
- To investigate the spatial distribution and movement of secretory vesicles relative to the plasma membrane.
Main Methods:
- Utilized image stacks from EW microscopy with varying penetration depths (80-125 nm).
- Estimated individual granule diameter and axial distance from fluorescence intensity data.
- Analyzed a population of 90 granules, including measurements before and after stimulation of exocytosis.
Main Results:
- Determined an average granule diameter of 305 +/- 47 nm, smaller than diffraction-limited xy measurements.
- Observed selective loss of 18 +/- 5% of granules closest to the plasma membrane upon stimulation.
- Identified recruitment of deeper granules (> or = 120 nm) to a population 60 nm from the membrane.
Conclusions:
- EW microscopy can accurately estimate granule diameter and axial distance, overcoming previous limitations.
- Demonstrated the existence of functionally distinct granule populations based on their proximity to the plasma membrane.
- Provided insights into the dynamic reorganization of secretory vesicle populations during exocytosis.