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Observer-Independent Quantification of Insulin Granule Exocytosis and Pre-Exocytotic Mobility by TIRF Microscopy

Published online by Cambridge University Press:  13 November 2013

Magnus Matz ,
Kirstin Schumacher ,
Kathrin Hatlapatka ,
Dirk Lorenz ,
Knut Baumann  and
Ingo Rustenbeck
Magnus Matz
Affiliation:
Institute of Medicinal and Pharmaceutical Chemistry, University of Braunschweig, Braunschweig D38106, Germany
Kirstin Schumacher
Affiliation:
Institute of Pharmacology and Toxicology, University of Braunschweig, Braunschweig D38106, Germany
Kathrin Hatlapatka
Affiliation:
Institute of Pharmacology and Toxicology, University of Braunschweig, Braunschweig D38106, Germany
Dirk Lorenz
Affiliation:
Institute of Analysis and Algebra, University of Braunschweig, Braunschweig D38106, Germany
Knut Baumann*
Affiliation:
Institute of Medicinal and Pharmaceutical Chemistry, University of Braunschweig, Braunschweig D38106, Germany
Ingo Rustenbeck*
Affiliation:
Institute of Pharmacology and Toxicology, University of Braunschweig, Braunschweig D38106, Germany
*
*Corresponding author. E-mail: k.baumann@tu-bs.de
**Corresponding author. E-mail: i.rustenbeck@tu-bs.de
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Abstract

Total internal reflection fluorescence microscopy of fluorescently labeled secretory granules permits monitoring of exocytosis and the preceding granule behavior in one experiment. While observer-dependent evaluation may be sufficient to quantify exocytosis, most of the other information contained in the video files cannot be accessed this way. The present program performs observer-independent detection of exocytosis and tracking of the entire submembrane population of insulin granules. A precondition is the exact localization of the peak of the granule fluorescence. Tracking is based on the peak base radius, peak intensity, and the precrossing itineraries. Robustness of the tracking was shown by simulated tracks of original granule patterns. Mobility in the X–Y dimension is described by the caging diameter which in contrast to the widely used mean square displacement has an inherent time resolution. Observer-independent detection of exocytosis in MIN6 cells labeled with insulin-EGFP is based on the maximal decrease in fluorescence intensity and position of the centroid of the dissipating cloud of released material. Combining the quantification of KCl-induced insulin exocytosis with the analysis of prefusion mobility showed that during the last 3 s pre-exocytotic granules had a smaller caging diameter than control granules and that it increased significantly immediately before fusion.

Keywords

automated granule detectionfluorescence quantificationexocytosisinsulin secretionTIRF microscopy
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 20 , Issue 1 , February 2014 , pp. 206 - 218
Copyright
Copyright © Microscopy Society of America 2014 

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