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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).
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