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Proceedings Paper

Dissecting key components of the Ca2+ homeostasis game by multifunctional fluorescence imaging
Author(s): Stefano Bastianello; Catalin D. Ciubotaru; Martina Beltramello; Fabio Mammano
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Paper Abstract

Different sub-cellular compartments and organelles, such as cytosol, endoplasmic reticulum and mitochondria, are known to be differentially involved in Ca2+ homeostasis. It is thus of primary concern to develop imaging paradigms that permit to make out these diverse components. To this end, we have constructed a complete system that performs multi-functional imaging under software control. The main hardware components of this system are a piezoelectric actuator, used to set objective lens position, a fast-switching monochromator, used to select excitation wavelength, a beam splitter, used to separate emission wavelengths, and a I/O interface to control the hardware. For these demonstrative experiments, cultured HeLa cells were transfected with a Ca2+ sensitive fluorescent biosensor (cameleon) targeted to the mitochondria (mtCam), and also loaded with cytosolic Fura2. The main system clock was provided by the frame-valid signal (FVAL) of a cooled CCD camera that captured wide-field fluorescence images of the two probes. Excitation wavelength and objective lens position were rapidly set during silent periods between successive exposures, with a minimum inter-frame interval of 2 ms. Triplets of images were acquired at 340, 380 and 430 nm excitation wavelengths at each one of three adjacent focal planes, separated by 250 nm. Optical sectioning was enhanced off-line by applying a nearest-neighbor deconvolution algorithm based on a directly estimated point-spread function (PSF). To measure the PSF, image stacks of sub-resolution fluorescent beads, incorporated in the cell cytoplasm by electroporation, were acquired under identical imaging conditions. The different dynamics of cytosolic and mitochondrial Ca2+ signals evoked by histamine could be distinguished clearly, with sub-micron resolution. Other FRET-based probes capable of sensing different chemical modifications of the cellular environment can be integrated in this approach, which is intrinsically suitable for the analysis of the interactions and cross-talks between different signaling pathways (e.g. Ca2+ and cAMP).

Paper Details

Date Published: 13 July 2004
PDF: 10 pages
Proc. SPIE 5324, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XI, (13 July 2004); doi: 10.1117/12.553731
Show Author Affiliations
Stefano Bastianello, Venetian Institute of Molecular Medicine (Italy)
Catalin D. Ciubotaru, Venetian Institute of Molecular Medicine (Italy)
Martina Beltramello, Venetian Institute of Molecular Medicine (Italy)
Fabio Mammano, Venetian Institute of Molecular Medicine (Italy)
Univ. degli Studi di Padova (Italy)

Published in SPIE Proceedings Vol. 5324:
Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XI
Jose-Angel Conchello; Carol J. Cogswell; Tony Wilson, Editor(s)

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