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Imaging a target of Ca2+ signalling: dense core granule exocytosis viewed by total internal reflection fluorescence microscopy.

Ravier MA, Tsuboi T, Rutter GA - Methods (2008)

Bottom Line: A brief summary of this approach is provided, as well as a description of the physical basis for the technique and the tools to implement TIRF using a standard fluorescence microscope.We also detail the different fluorescent probes which can be used to detect secretion and how to analyze the data obtained.A comparison between TIRF and other imaging modalities including confocal and multiphoton microscopy is also included.

View Article: PubMed Central - PubMed

Affiliation: Unit of Endocrinology and Metabolism, University of Louvain Faculty of Medicine, UCL 55.30 Avenue Hippocrate 55, B-1200 Brussels, Belgium.

ABSTRACT
Ca2+ ions are the most ubiquitous second messenger found in all cells, and play a significant role in controlling regulated secretion from neurons, endocrine, neuroendocrine and exocrine cells. Here, we describe microscopic techniques to image regulated secretion, a target of Ca2+ signalling. The first of these, total internal reflection fluorescence (TIRF), is well suited for optical sectioning at cell-substrate regions with an unusually thin region of fluorescence excitation (<150 nm). It is thus particularly useful for studies of regulated hormone secretion. A brief summary of this approach is provided, as well as a description of the physical basis for the technique and the tools to implement TIRF using a standard fluorescence microscope. We also detail the different fluorescent probes which can be used to detect secretion and how to analyze the data obtained. A comparison between TIRF and other imaging modalities including confocal and multiphoton microscopy is also included.

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Analysis of secretion by TIRF microscopy. (A) Evanescent wave image of NPY-Venus fluorescence in a live MIN6 pancreatic β cell. The scale bar represents 5 μm. (B, top) Sequential images of a single vesicle observed after high [K+] stimulation. The third image shows a diffuse cloud of the NPY-Venus fluorescence, and the final image shows an abrupt disappearance of the fluorescent spot. (B and C, bottom) Time course of the fluorescence changes measured in the small circles enclosing fluorescent spots (filled symbols) and for concentric annuli around the circles (open symbols) of two different vesicles. The ordinate is given in arbitrary units of brightness. (C, top) Sequential images of a single vesicle observed after stimulation with 50 mM KCl without showing any diffuse cloud of the NPY-Venus fluorescence. The third image does not show any cloud of the dye, whereas the final image shows an abrupt disappearance. These events typically exhibited a much slower time course (approximately 3 s to reach peak fluorescence) than those showing in B and reflects the approach of vesicles to the plasma membrane without exocytosis (i.e. vesicle movement or retrieval). The scale bars represents 1 μm. (D) Effect of glucose on exocytosis as reported with NPY-Venus in pancreatic β cells. The NPY-Venus spots shown in B was counted manually as secretion events every 60 s and plotted against time. Stimulation with high glucose concentrations (30 mM) caused a marked increase in the number of secretion.
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fig4: Analysis of secretion by TIRF microscopy. (A) Evanescent wave image of NPY-Venus fluorescence in a live MIN6 pancreatic β cell. The scale bar represents 5 μm. (B, top) Sequential images of a single vesicle observed after high [K+] stimulation. The third image shows a diffuse cloud of the NPY-Venus fluorescence, and the final image shows an abrupt disappearance of the fluorescent spot. (B and C, bottom) Time course of the fluorescence changes measured in the small circles enclosing fluorescent spots (filled symbols) and for concentric annuli around the circles (open symbols) of two different vesicles. The ordinate is given in arbitrary units of brightness. (C, top) Sequential images of a single vesicle observed after stimulation with 50 mM KCl without showing any diffuse cloud of the NPY-Venus fluorescence. The third image does not show any cloud of the dye, whereas the final image shows an abrupt disappearance. These events typically exhibited a much slower time course (approximately 3 s to reach peak fluorescence) than those showing in B and reflects the approach of vesicles to the plasma membrane without exocytosis (i.e. vesicle movement or retrieval). The scale bars represents 1 μm. (D) Effect of glucose on exocytosis as reported with NPY-Venus in pancreatic β cells. The NPY-Venus spots shown in B was counted manually as secretion events every 60 s and plotted against time. Stimulation with high glucose concentrations (30 mM) caused a marked increase in the number of secretion.

Mentions: To analyze TIRF imaging data, single exocytotic events are selected manually, and the average fluorescence intensity of individual vesicle in a 0.7 × 0.7 μm2 placed over the vesicle center calculated using MetaMorph software (version 6.3, Universal Imaging Corporation, Downingtown, PA). To distinguish between fusion events and vesicle movement (i.e. vesicles pause at the plasma membrane and then move back inside the cell without fusing), we focus on fluorescence changes just before the disappearance of fluorescent signals. When there is a fusion event, a rapid transient increase in fluorescence intensity (to a peak intensity 1.5 times greater than the original fluorescence intensity within 1 s) is usually observed, whereas when vesicles move, the fluorescence intensity gradually decreases to the background level (Fig. 4). The number of fusion events during a 5-min period can be counted manually, based on the above criteria [9,17,53].


Imaging a target of Ca2+ signalling: dense core granule exocytosis viewed by total internal reflection fluorescence microscopy.

Ravier MA, Tsuboi T, Rutter GA - Methods (2008)

Analysis of secretion by TIRF microscopy. (A) Evanescent wave image of NPY-Venus fluorescence in a live MIN6 pancreatic β cell. The scale bar represents 5 μm. (B, top) Sequential images of a single vesicle observed after high [K+] stimulation. The third image shows a diffuse cloud of the NPY-Venus fluorescence, and the final image shows an abrupt disappearance of the fluorescent spot. (B and C, bottom) Time course of the fluorescence changes measured in the small circles enclosing fluorescent spots (filled symbols) and for concentric annuli around the circles (open symbols) of two different vesicles. The ordinate is given in arbitrary units of brightness. (C, top) Sequential images of a single vesicle observed after stimulation with 50 mM KCl without showing any diffuse cloud of the NPY-Venus fluorescence. The third image does not show any cloud of the dye, whereas the final image shows an abrupt disappearance. These events typically exhibited a much slower time course (approximately 3 s to reach peak fluorescence) than those showing in B and reflects the approach of vesicles to the plasma membrane without exocytosis (i.e. vesicle movement or retrieval). The scale bars represents 1 μm. (D) Effect of glucose on exocytosis as reported with NPY-Venus in pancreatic β cells. The NPY-Venus spots shown in B was counted manually as secretion events every 60 s and plotted against time. Stimulation with high glucose concentrations (30 mM) caused a marked increase in the number of secretion.
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fig4: Analysis of secretion by TIRF microscopy. (A) Evanescent wave image of NPY-Venus fluorescence in a live MIN6 pancreatic β cell. The scale bar represents 5 μm. (B, top) Sequential images of a single vesicle observed after high [K+] stimulation. The third image shows a diffuse cloud of the NPY-Venus fluorescence, and the final image shows an abrupt disappearance of the fluorescent spot. (B and C, bottom) Time course of the fluorescence changes measured in the small circles enclosing fluorescent spots (filled symbols) and for concentric annuli around the circles (open symbols) of two different vesicles. The ordinate is given in arbitrary units of brightness. (C, top) Sequential images of a single vesicle observed after stimulation with 50 mM KCl without showing any diffuse cloud of the NPY-Venus fluorescence. The third image does not show any cloud of the dye, whereas the final image shows an abrupt disappearance. These events typically exhibited a much slower time course (approximately 3 s to reach peak fluorescence) than those showing in B and reflects the approach of vesicles to the plasma membrane without exocytosis (i.e. vesicle movement or retrieval). The scale bars represents 1 μm. (D) Effect of glucose on exocytosis as reported with NPY-Venus in pancreatic β cells. The NPY-Venus spots shown in B was counted manually as secretion events every 60 s and plotted against time. Stimulation with high glucose concentrations (30 mM) caused a marked increase in the number of secretion.
Mentions: To analyze TIRF imaging data, single exocytotic events are selected manually, and the average fluorescence intensity of individual vesicle in a 0.7 × 0.7 μm2 placed over the vesicle center calculated using MetaMorph software (version 6.3, Universal Imaging Corporation, Downingtown, PA). To distinguish between fusion events and vesicle movement (i.e. vesicles pause at the plasma membrane and then move back inside the cell without fusing), we focus on fluorescence changes just before the disappearance of fluorescent signals. When there is a fusion event, a rapid transient increase in fluorescence intensity (to a peak intensity 1.5 times greater than the original fluorescence intensity within 1 s) is usually observed, whereas when vesicles move, the fluorescence intensity gradually decreases to the background level (Fig. 4). The number of fusion events during a 5-min period can be counted manually, based on the above criteria [9,17,53].

Bottom Line: A brief summary of this approach is provided, as well as a description of the physical basis for the technique and the tools to implement TIRF using a standard fluorescence microscope.We also detail the different fluorescent probes which can be used to detect secretion and how to analyze the data obtained.A comparison between TIRF and other imaging modalities including confocal and multiphoton microscopy is also included.

View Article: PubMed Central - PubMed

Affiliation: Unit of Endocrinology and Metabolism, University of Louvain Faculty of Medicine, UCL 55.30 Avenue Hippocrate 55, B-1200 Brussels, Belgium.

ABSTRACT
Ca2+ ions are the most ubiquitous second messenger found in all cells, and play a significant role in controlling regulated secretion from neurons, endocrine, neuroendocrine and exocrine cells. Here, we describe microscopic techniques to image regulated secretion, a target of Ca2+ signalling. The first of these, total internal reflection fluorescence (TIRF), is well suited for optical sectioning at cell-substrate regions with an unusually thin region of fluorescence excitation (<150 nm). It is thus particularly useful for studies of regulated hormone secretion. A brief summary of this approach is provided, as well as a description of the physical basis for the technique and the tools to implement TIRF using a standard fluorescence microscope. We also detail the different fluorescent probes which can be used to detect secretion and how to analyze the data obtained. A comparison between TIRF and other imaging modalities including confocal and multiphoton microscopy is also included.

Show MeSH
Related in: MedlinePlus