Pixel-based criteria-oriented analysis of time-lapse Ca2+-fluorescence images

Abstract Since its inception, the analysis of time-lapse video-images acquired during Ca 2+ imaging experiments using fluorescence microscopy has been progressively optimized for achieving a high temporal resolution. In contrast, the spatial resolution of the acquired images is often compromised during analysis to varying degrees by the need to draw regions of interest (ROI). We developed a strategy to analyze images at the acquired spatial resolution—pixel-by-pixel, grouping all pixels based on criteria of interest (COI) in regard to their associated fluorescence values over time and visualizing the distributions of the pixel-groups detected in a pseudo-colored map. We applied this pixel-based COI-strategy to the analysis of relative intracellular free calcium levels (Ca i 2+ ) in attached cultured embryonic hippocampal cells under baseline and experimental conditions designed to evaluate the contribution of extracellular Ca 2+ (Ca e 2+ ) to baseline Ca i 2+ levels. We discovered distinct groups of Ca e 2+ -dependent Ca i 2+ regulation patterns emergent during the earliest phases of hippocampal cell differentiation, which were not limited to inter-cell differences. Thus, pixel-based COI-analysis of time-lapse images can be used to disclose distinct patterns of Ca e 2+ -dependent Ca i 2+ levels and their corresponding subcellular distributions in developing hippocampal cells. Such a strategy should be useful in studying the emergence and distribution of Ca i 2+ signaling at subcellular levels of resolution using fluorescence microscopy.