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Abstract: Calcium (Ca2+) signalling is essential for T cell activation, the on-switch for the adaptive immune system. It is assumed to start by localized short-lived initial Ca2+ signals \- which, yet, have not been characterized. Initial signal formation can be examined by fluorescence microscopy but requires imaging with temporal and spatial resolutions being as high as possible. This, in turn, poses challenges from the perspective of image and signal processing, which will be discussed on the basis of our current workflow [1]. Primary and Jurkat T cells were loaded with two dyes (Fluo-4, FuraRed), stimulated by anti-CD3 or anti\-CD3/CD28-coated beads, and imaged by ratiometric fluorescence microscopy (acquisition velocity up to 48fps; nominal spatial resolution 368nm). Different strategies for deconvolution and bleaching correction and their influence on quantitative measures for local Ca2+ activity were evaluated; approaches for SNR estimation and noise filtering were compared; cell shape/orientation normalization for cell population based Ca2+ signal analysis was proposed and applied. Deconvolution and bleaching correction techniques significantly influence quantitative Ca2+ dynamics measures on a single cell level. Lucy-Richardson deconvolution combined with fit\-based additive bleaching correction is assumed to be an acceptable trade\-off between computation time and image quality. Optimal noise filtering is, however, an open issue. Applied techniques range from moving averaging to more sophisticated low\-pass filtering. Cell shape/orientation normalization, population\-based Ca2+ dynamics analysis and the introduction of specific Ca2+ activity\/responsiveness measures mitigates to some degree against the influence of specific post\-processing block implementations. Fluorescence microscopy imaging of subcellular Ca2+ signals with a spatial resolution close to Abbe\'s resolution limit and rapid image acquisition is possible, but efficient processing of resulting large data sets and especially handling the trade\-off between SNR and temporal resolution remains challenging. [1] D Schetelig et al. Proc BVM 401-6 (2015); Funded by Forschungszentrum Medizintechnik Hamburg, DFG (GU360/15-1) and Landesforschungsfoerderung Hamburg.