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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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Arrangement for objective lens-type TIRF in an inverted microscope. Laser illumination through a side port requires a dichroic mirror cube facing the side. At the P1 position, the laser beam is focused to lead the critical angle propagation into the coverslip (total internal reflection). Moving the lens L transversely changes the angle of incidence, thus the laser beam moves from P1 (TIRF) position to P2 position (epifluorescence). This system allows to switch between both mode of illumination (TIRF and epifluorescence).
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fig2: Arrangement for objective lens-type TIRF in an inverted microscope. Laser illumination through a side port requires a dichroic mirror cube facing the side. At the P1 position, the laser beam is focused to lead the critical angle propagation into the coverslip (total internal reflection). Moving the lens L transversely changes the angle of incidence, thus the laser beam moves from P1 (TIRF) position to P2 position (epifluorescence). This system allows to switch between both mode of illumination (TIRF and epifluorescence).

Mentions: To build an objective lens type TIRF microscopy system, the incident laser light has to pass through the edge of the high numerical aperture (⩾1.45) objective lens. This can be achieved simply by placing the focused incident laser light at edge of back focal plane of the high NA objective lens [25] (Fig. 2).


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)

Arrangement for objective lens-type TIRF in an inverted microscope. Laser illumination through a side port requires a dichroic mirror cube facing the side. At the P1 position, the laser beam is focused to lead the critical angle propagation into the coverslip (total internal reflection). Moving the lens L transversely changes the angle of incidence, thus the laser beam moves from P1 (TIRF) position to P2 position (epifluorescence). This system allows to switch between both mode of illumination (TIRF and epifluorescence).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2597054&req=5

fig2: Arrangement for objective lens-type TIRF in an inverted microscope. Laser illumination through a side port requires a dichroic mirror cube facing the side. At the P1 position, the laser beam is focused to lead the critical angle propagation into the coverslip (total internal reflection). Moving the lens L transversely changes the angle of incidence, thus the laser beam moves from P1 (TIRF) position to P2 position (epifluorescence). This system allows to switch between both mode of illumination (TIRF and epifluorescence).
Mentions: To build an objective lens type TIRF microscopy system, the incident laser light has to pass through the edge of the high numerical aperture (⩾1.45) objective lens. This can be achieved simply by placing the focused incident laser light at edge of back focal plane of the high NA objective lens [25] (Fig. 2).

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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