A QUANTITATIVE, COMPARATIVE STUDY OF ELEMENTAL PROFILES IN THE BLOOD CELLS OF THE ASCIDIAN PHALLUSIA PHILIPPINENSIS USING ELECTRON AND NUCLEAR MICROSCOPY

Abstract

The origin for much of our knowledge relating to vanadium accumulation, transport and its biological significance in animals dates from its discovery by Henze in 1911 within a group of exclusively marine aquatic organisms, the ascidiacea. However since 1911, further studies on accumulation mechanisms and physiological significance of vanadium have made little progress, probably because solutions to many of the problems require interdisciplinary collaboration. In this study, element profiles were obtained for a range of cryofixed/freeze-dried blood cell types, extracted from the ascidian Phallusia philippinensis. The quantitative and qualitative analysis was performed using the National University of Singapore nuclear microscope, a Leica 440 Series scanning electron microscope (SEM) and a JSM T220A SEM, each equipped with a LINK energy dispersive solid state detector and multi LINK-Oxford multi channel analyzer system. Initially, quantitative analysis was performed on seven cryofixed/freeze-dried cell types, using nuclear microscopy. Morula cells were found to be predominantly involved in vanadium accumulation. Signet ring cells, lymphocytes, macrogranular amoebocytes and carotenoid pigment cells all contained vanadium at levels in the same order of magnitude, while compartment cells contained the smallest concentration. Bromine was found at levels with the same order of magnitude within all the cells analyzed, as was sulphur. Bromine may be incorporated into complexes utilized for anti-fouling, and the presence of high levels of sulphur could indicate its involvement in the vanadium concentration process. The apparent tendency of vanadium, bromine and sulphur to occur together may also indicate the presence of complexes such as metalloenzymes that act to bind together the trace elements. The variability of trace element concentration within morula cells was then examined using the nuclear microscope in conjunction with a PC based masking technique. The element profiles for seven 'vacuoles' within a morula cell were obtained, and considerable variation in cellular composition was found to occur. Calcium levels were comparable in all regions of the cell, indicating that minimal elemental flux had occurred during the preparation process. However vanadium, sulphur and bromine concentrations varied over one order of magnitude. This variability in distribution of biologically active elements within the cell indicated the possible cause of discrepancies seen between previous studies, involving electron probe analysis techniques. A comparison of Leica 440 SEM and JEOL JSM T220A SEM quantitative analysis was then made with nuclear microscope analysis. Nuclear microscopy produced better quantitative analysis at the parts per million level. However the spatial resolution range obtained using nuclear analysis is presently the limiting factor. As it is in the range of 1 µm, compared to STEM analysis, which can achieve resolutions of 2nm and produces clearer images at the sub cellular level. Cellular and sub cellular analysis is therefore placed on the limits of nuclear microprobe spatial resolution capabilities. Many chemical preparations required in electron microscopy to produce clarity in ultrastructural fidelity, compromise the quantitative analysis. Therefore the two methods can be seen to be complimentary. With electron microscopy giving clear precise images and nuclear microscopy providing more accurate quantitative analysis.