Proceedings of SPIE, Vol. 5324, pp 265-274, 2004. Dissecting key components of the Ca 2+ homeostasis game by multi-functional fluorescence imaging Stefano Bastianello a , Catalin Ciubotaru a , Martina Beltramello a , Fabio Mammano a,b,* a Venetian Institute of Molecular Medicine (VIMM), via G.Orus 2, 35129 Padova, Italy. a University of Padova, Dept. of Physics, via Marzolo 8, 35131 Padova, Italy. ABSTRACT Different sub-cellular compartments and organelles, such as cytosol, endoplasmic reticulum and mitochondria, are known to be differentially involved in Ca 2+ 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 Ca 2+ 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 Ca 2+ 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. Ca 2+ and cAMP). Keywords: Fluorescence microscopy, deconvolution, multi-spectral analysis, piezoelectric actuator, CCD camera, mitochondria, cameleon, FRET, Fura2. 1. INTRODUCTION Fluorescence imaging is important for investigating cell structure and function and continues to spur the improvement of optical methods 1 as well as the synthesis of new molecular probes 2 . Laser scanning confocal microscopy, in its many forms, is generally considered the technique of choice to reach diffraction-limited resolution and optical sectioning of (relatively) thick samples 3 . Alternative approaches, based on the conventional light microscope, exploit structured illumination 4 or deconvolution 5,6 to achieve comparable or superior performance (at a fraction of the cost) with the added bonus that excitation wavelengths can be selected out of a continuum. Here we show that deconvolution coupled to rapid wavelength changes can be used to achieve excellent optical sectioning while imaging Ca 2+ dynamics simultaneously in distinct sub-cellular compartments. * fabio.mammano@unipd.it ; phone +39 049 7923 231; fax +39 049 7923 266; www.vimm.it/cochlea