Quantitative microlocalization of diffusible ions in normal and galactose cataractous rat lens by secondary ion mass spectrometry.

Secondary ion mass spectrometry (SIMS) is a surface analytical technique with high sensitivity for elemental detection and microlocalization capabilities within the micrometre range. Quantitative analysis of epoxy resins and gelatin have been reported (Burns-Bellhorn & File, 1979). We report here the first application of this technique to quantitative microlocalization in the context of a physiological problem--analyses of sodium, potassium and calcium in normal and galactose-induced cataract in rat lens. It is known that during the development of galactose-induced cataract the whole lens content of potassium is decreased, sodium is increased and, in late stages, calcium concentration increases. Whether these alterations in diffusible ions occur homogeneously or heterogeneously is not known. Standard curves were generated from epoxy resins containing known concentrations of sodium, potassium or calcium organometallic compounds using the Cameca IMS 300 Secondary Ion Mass Spectrometer. Normal and cataractous lenses were prepared by freezing in isopentane in a liquid nitrogen bath followed by freeze-drying at -30 degrees C. After dry embedding in epoxy resin, 10 microns thick sections of lens were pressure mounted on silicon wafers, overcoated with gold, and ion emission measured under the same instrumental conditions used to obtain the standard curves. Quantitative analysis of an area 27 microns in diameter, or a total analysed volume of 1.1 microns3, was performed by using a mechanical aperture in the ion optical system. Ion images provided qualitative microanalysis with a lateral resolution of 1 micron. Control rat lenses gave values for sodium and potassium content with a precision of +/- 17% or less. These values were compared to flame photometry and atomic absorption measurements of normal lenses and were accurate within 25%. Analysis of serum and blood also gave accurate and precise measurements of these elements. Normal rat lenses had a gradient of sodium, and, to a lesser degree, of potassium from the cortex to the nucleus. Development of galactose-induced cataract was heterogeneous by morphological criteria, beginning at the lens equator and spreading from the cortex into the nucleus. However, the loss of potassium and increase in sodium concentration occurred at early stages in both the cortex and nucleus cells, possibly because these cells are interconnected by gap junctions. There is a local alteration in elemental content prior to morphologically demonstrable cataract formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Matrix effects in secondary ion mass spectrometric analysis of biological tissue.

We have made several observations during the course of our studies that show the presence of matrix effects in soft biological tissue and standards. The sputtering rate of gelatin is approximately twice that of epoxy resin, but the ion yield of lithium in gelatin is an order of magnitude less than in epoxy. Osmium impregnation of freeze-dried material significantly alters the localization of calcium, but not potassium and barium. The absolute count rate for calcium in osmicated tissue is increased several-fold above that in freeze-dried tissue. Scanning electron microscopy of sputtered material shows the formation of cones during sputtering, which is particularly, but not exclusively, associated with melanin granules and red blood cells. These structures are known to be highly emissive for Na, K, and Ca. Boron implanted tissue also exhibits selective boron emission from melanin granules. Relative proportions of monoatomic and polyatomic emission vary in epoxy, gelatin and tissue. Ion images of carbon, chlorine and vanadium in tissue embedded with a vanadium-doped epoxy resin show variations in local regions that correspond to tissue structure. The energy distributions of common secondary ions differed somewhat in resin and two different tissue regions. These examples show the existence of potential matrix effects in soft biological tissue that involve both differential sputtering and ion yield effects.