Lucilia cuprina pericardial cells are primarily involved in the filtration of hemolymph. Ratio images using fluorescent
pH indicator, DM-Nerf, were collected using a confocal microscope. The results support suggestions that there is
zonation of cellular activity that reflect organelle distribution. Statistical analysis of the excitation ratios indicate
significant spatial differences in pH of the three major zones- cortex, vacuole zone and endoplasm in pericardial cells.
The outer cortex was estimated to have a pH between 5.5 and 6.8, the vacuole zone between 4.5 and 5.5 and the
endoplasm between 4.0 and 5.0.
The genus Saprolegnia in the phylum Oomycota have intracellular structures that are distinct from that of filamentous fungi. The vacuolar reticulum for example in Saprolegnia consists of fine static tubules that taper towards the apex of the hypha and are connected to a large vacuole in the basal region. This paper discusses the contribution of the different microscopic techniques in observing ultrastructural changes resulting from modulating GTP binding proteins associated with vesicle production and placement. TEM, DIC and fluorescent observations complemented each other and provided valuable detailed information as to changes in the vacuolar reticulum and the arrangement of organelles. The use of comparative imaging was essential for obtaining sufficient information to make an accurate assessment of changes resulting from perturbation. Without comparison of multiple imaging techniques the resulting conclusions would have been limited with the added potential of being inaccurate. Imaging properties such as cellular detail, overview and specificity from the various forms of microscopy confirmed and contributed information to the analysis. The argument of whether Saprolegnia use a tubular or a vesicular network system to transfer nascent membrane to the growing tip would have been difficult to determine using only one or two imaging techniques. Comparative analysis has indicated that the vacuolar reticulum, previously considered to be static, is a membrane reservoir that allows for membrane transfer to the apical and subapical regions.
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.