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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.

Show MeSH
Comparison of epifluorescence versus total internal reflection fluorescence microscopy. The thin layer of illumination is an evanescent field produced by an excitation light beam in a glass cover slip that is incident at a high angle upon the solid-solution interface at which the cells adhere. Thus, an evanescent wave arises on the cell–substrate interface and penetrates a small distance (∼150 nm) into the cells.
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fig1: Comparison of epifluorescence versus total internal reflection fluorescence microscopy. The thin layer of illumination is an evanescent field produced by an excitation light beam in a glass cover slip that is incident at a high angle upon the solid-solution interface at which the cells adhere. Thus, an evanescent wave arises on the cell–substrate interface and penetrates a small distance (∼150 nm) into the cells.

Mentions: The principle of total internal reflection fluorescence (TIRF) microscopy, illustrated in Fig. 1, is based on the excitation of fluorophores by an evanescent field of totally internally reflected light. To achieve total internal reflection, the laser beam is directed obliquely at the interface between two media from a high (coverslip) to a low (cell adherent to the coverslip) refractive index with an incident angle greater than the critical angle of total internal reflection. Total internal reflection sets up a thin layer of light in the cell, called the evanescent field. The strength of this field (termed the evanescent wave) decreases exponentially and its effects extend only a few hundred nanometer into the cell. The portion of the specimen within the evanescent field can therefore be excited to emit fluorescence selectively.


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)

Comparison of epifluorescence versus total internal reflection fluorescence microscopy. The thin layer of illumination is an evanescent field produced by an excitation light beam in a glass cover slip that is incident at a high angle upon the solid-solution interface at which the cells adhere. Thus, an evanescent wave arises on the cell–substrate interface and penetrates a small distance (∼150 nm) into the cells.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Comparison of epifluorescence versus total internal reflection fluorescence microscopy. The thin layer of illumination is an evanescent field produced by an excitation light beam in a glass cover slip that is incident at a high angle upon the solid-solution interface at which the cells adhere. Thus, an evanescent wave arises on the cell–substrate interface and penetrates a small distance (∼150 nm) into the cells.
Mentions: The principle of total internal reflection fluorescence (TIRF) microscopy, illustrated in Fig. 1, is based on the excitation of fluorophores by an evanescent field of totally internally reflected light. To achieve total internal reflection, the laser beam is directed obliquely at the interface between two media from a high (coverslip) to a low (cell adherent to the coverslip) refractive index with an incident angle greater than the critical angle of total internal reflection. Total internal reflection sets up a thin layer of light in the cell, called the evanescent field. The strength of this field (termed the evanescent wave) decreases exponentially and its effects extend only a few hundred nanometer into the cell. The portion of the specimen within the evanescent field can therefore be excited to emit fluorescence selectively.

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