10 July 2003 Measuring calcium levels in Saprolegnia ferax using the two-photon laser scanning microscope
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Abstract
xThe genus Saprolegnia in the phylum Oomycetes contains a number of parasitic species that can cause a range of important animal diseases. The aim of this study was to measure the calcium gradient, one of the growth regulating mechanisms, in Saprolegnia ferax. The two-photon laser scanning microscope allowed for detailed physiological measurements of calcium levels along the fungus-like hyphae of S.ferax. Calcium concentration was determined by making ratiometric calculation of emission levels of the calcium-sensitive fluorochrome Indo-1 at 485nm to 405nm. The calculated values were compared to the intracellular calibration values. The advantage of the two-photon laser scanning microscope is that it allows minor changes in concentration to be detected in highly localized regions of the hyphae. The technique used in this study minimized background and autofluorescence and therefore allowed for more accurate changes in intracellular Ca2+ concentration to be detected. The calcium concentration at the hyphal tip and 5, 10 and 40μm distal to the tip were calculated to be 65, 17, 38 and 20nM respectively, confirming other studies that suggest a tip-high calcium gradient.
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Osu Lilje, "Measuring calcium levels in Saprolegnia ferax using the two-photon laser scanning microscope", Proc. SPIE 4963, Multiphoton Microscopy in the Biomedical Sciences III, (10 July 2003); doi: 10.1117/12.478036; https://doi.org/10.1117/12.478036
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