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

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• 作者：Lorenz ; J&uuml ; rgen J. ; Lorenz ; Matthias G.O. ; Barker ; Jeffery L.
• 关键词：Pixel-by-pixel ; Criteria of interest ; Pattern detection ; Calcium regulation ; Calcium imaging ; Fluorescence microscopy ; Development ; Hippocampus
• 刊名：Journal of Neuroscience Methods
• 出版年：2003
• 出版时间：August 15, 2003
• 年：2003
• 卷：127
• 期：2
• 页码：157-166
• 全文大小：554 K

文摘

Since its inception, the analysis of time-lapse video-images acquired during Ca²⁺ 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_i²⁺) in attached cultured embryonic hippocampal cells under baseline and experimental conditions designed to evaluate the contribution of extracellular Ca²⁺ (Ca_e²⁺) to baseline Ca_i²⁺ levels. We discovered distinct groups of Ca_e²⁺-dependent Ca_i²⁺ 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_e²⁺-dependent Ca_i²⁺ levels and their corresponding subcellular distributions in developing hippocampal cells. Such a strategy should be useful in studying the emergence and distribution of Ca_i²⁺ signaling at subcellular levels of resolution using fluorescence microscopy.