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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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Related in: MedlinePlus

Epifluorescence versus objective lens-type total internal reflection fluorescence. Images were captured with Olympus 1.45 NA 100× objective lens and an argon ion laser source of wavelength 488 nm, using the side-port configuration depicted in Fig. 2. (A and B) PC12 cells containing secretory vesicle marker neuropeptide Y-Venus (NPY-Venus). (C and D) PC12 cells containing secretory vesicle marker rabphilin-mRFP. The images were recorded by a cooled monochrome CCD camera (Imago, Till Photonics) in A and B, and recorded by EM-CCD camera (C9100-02, Hamamatsu Photonics) in C and D.
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fig3: Epifluorescence versus objective lens-type total internal reflection fluorescence. Images were captured with Olympus 1.45 NA 100× objective lens and an argon ion laser source of wavelength 488 nm, using the side-port configuration depicted in Fig. 2. (A and B) PC12 cells containing secretory vesicle marker neuropeptide Y-Venus (NPY-Venus). (C and D) PC12 cells containing secretory vesicle marker rabphilin-mRFP. The images were recorded by a cooled monochrome CCD camera (Imago, Till Photonics) in A and B, and recorded by EM-CCD camera (C9100-02, Hamamatsu Photonics) in C and D.

Mentions: As an example, in Fig. 1, the selective excitation of fluorescent labeled vesicles in a single cell (refractive index, n2 = 1.38) adherent to a glass coverslip (refractive index, n1 = 1.53), wave fronts from a laser excitation source pass trough the coverslip and are reflected from the coverslip–cell interface at a critical angle α, generating an evanescent wave. Fluorescent labeled vesicles (∼200 nm diameter) at the coverslip interface are excited by the evanescent wave and emit fluorescence which can be recorded, while those located farther away are not excited (Fig. 1, TIRF and Fig. 3B and D). With a conventional fluorescence microscope (Fig. 1, Epifluorescence and Fig. 3A and C) essentially all fluorescent vesicles in the cell are excited, so the emitted fluorescence from the vesicles at the interface of the coverslip is overwhelmed by the considerable number of the other vesicles which are not bound to the glass coverslip.


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)

Epifluorescence versus objective lens-type total internal reflection fluorescence. Images were captured with Olympus 1.45 NA 100× objective lens and an argon ion laser source of wavelength 488 nm, using the side-port configuration depicted in Fig. 2. (A and B) PC12 cells containing secretory vesicle marker neuropeptide Y-Venus (NPY-Venus). (C and D) PC12 cells containing secretory vesicle marker rabphilin-mRFP. The images were recorded by a cooled monochrome CCD camera (Imago, Till Photonics) in A and B, and recorded by EM-CCD camera (C9100-02, Hamamatsu Photonics) in C and D.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Epifluorescence versus objective lens-type total internal reflection fluorescence. Images were captured with Olympus 1.45 NA 100× objective lens and an argon ion laser source of wavelength 488 nm, using the side-port configuration depicted in Fig. 2. (A and B) PC12 cells containing secretory vesicle marker neuropeptide Y-Venus (NPY-Venus). (C and D) PC12 cells containing secretory vesicle marker rabphilin-mRFP. The images were recorded by a cooled monochrome CCD camera (Imago, Till Photonics) in A and B, and recorded by EM-CCD camera (C9100-02, Hamamatsu Photonics) in C and D.
Mentions: As an example, in Fig. 1, the selective excitation of fluorescent labeled vesicles in a single cell (refractive index, n2 = 1.38) adherent to a glass coverslip (refractive index, n1 = 1.53), wave fronts from a laser excitation source pass trough the coverslip and are reflected from the coverslip–cell interface at a critical angle α, generating an evanescent wave. Fluorescent labeled vesicles (∼200 nm diameter) at the coverslip interface are excited by the evanescent wave and emit fluorescence which can be recorded, while those located farther away are not excited (Fig. 1, TIRF and Fig. 3B and D). With a conventional fluorescence microscope (Fig. 1, Epifluorescence and Fig. 3A and C) essentially all fluorescent vesicles in the cell are excited, so the emitted fluorescence from the vesicles at the interface of the coverslip is overwhelmed by the considerable number of the other vesicles which are not bound to the glass coverslip.

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