Abstract
Endothelial cells perform a wide variety of fundamental functions for the cardiovascular system, their proliferation and migration being strongly regulated by their intracellular calcium concentration. Hence it is extremely important to carefully measure endothelial calcium signals under different stimuli. A proposal to automate the intracellular calcium profiles extraction from fluorescence image sequences is presented. Digital image processing techniques were combined with a multi-target tracking approach supported by Kalman estimation. The system was tested with image sequences from two different stimuli. The first one was a chemical stimulus, that is, ATP, which caused small movements in the cells trajectories, thereby suggesting that the bath application of the agonist does not generate significant artifacts. The second one was a mechanical stimulus delivered by a glass microelectrode, which caused major changes in cell trajectories. The importance of the tracking block is evidenced since more accurate profiles were extracted, mainly for cells closest to the stimulated area. Two important contributions of this work are the automatic relocation of the region of interest assigned to the cells and the possibility of data extraction from big image sets in efficient and expedite way. The system may adapt to different kind of cell images and may allow the extraction of other useful features.
Highlights
Vascular endothelium has long been considered as a homogeneous population of inert cells that forms a permeable barrier between flowing blood and surrounding tissues, regulating the exchange of nutrients, oxygen and catabolic waste at capillary level
With that purpose in mind, the application was tested with several image sequences obtained from two principal types of experiments: Endothelium under chemical (ATP) stimulus and Endothelium under mechanical stimulus
The upper left movie shows the inner wall of the aortic rings loaded with the Ca2+-sensitive fluorophore, Fura-2, which was fully covered by fluorescent endothelial cells (ECs)
Summary
Vascular endothelium has long been considered as a homogeneous population of inert cells that forms a permeable barrier between flowing blood and surrounding tissues, regulating the exchange of nutrients, oxygen and catabolic waste at capillary level. This rather conventional view has recently given the way to a more realistic interpretation of the multifaceted role played by endothelial cells (ECs) within the cardiovascular system. An increase in intracellular Ca2+ concentration ([Ca2+]i) represents one of the most important signaling pathways whereby ECs exert their sophisticated control of cardiovascular homeostasis [4,5,6]
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