Microsc. Microanal. Microstruct. 6, 65-77 (1995)
DOI: 10.1051/mmm:1995100

Electron Spectroscopic Imaging of Organic Compounds Using PC-Based Energy Sequence Imaging Software

Andrew D. Johnson¹, Robert J. Mairs², Mark N. Gaze³, Gretel Sass⁴ et Ian M. Huxham<sup>1

1</sup>  Molecular & Cellular Biology Division, EM Centre Joseph Black Building; University of Glasgow, Glasgow, G12 8QQ, UK
²  Department of Radiation Oncology, University of Glasgow, Glasgow G12 8QQ, UK
³  Meyerstein Institute of Clinical Oncology, The Middlesex Hospital Medical School, London W11 8AA, UK
⁴  medac GmbH, Fehlandstrasse 3, D-20345 Hamburg, Germany

Abstract
We have designed, characterised and tested Windows based software packages for rapid elemental microanalysis using a sequence of energy filtered transmission electron microscopic images which combines electron spectroscopic imaging with electron energy loss spectroscopy (Image-EELS). Image sequences containing up to 45 images of pixels were recorded rapidly over an appropriate range of the energy spectrum for an analysis of boron, nitrogen, titanium and fluorine. Energy loss information was extracted from local variations in the energy loss spectrum with good energy resolution (5 eV) and a spatial resolution of 2 nm for the determination of the relative concentration of elements within a region of interest by grey level integration in consecutive images of an energy loss sequence, using the least mean squares method to calculate the parameters for an background model. The methods were used to define the optimum collections conditions and practical limitations of the system for qualitative and semi-quantitative elemental analysis relevant to biological specimens. Qualitative elemental analysis of images from Lowicryl HM23- embedded ultra-thin section of a boric acid polymer showed that boron was not distributed evenly. Semi-quantitative elemental analysis of the fluorine-containing organic polymer Nafion suggested a linear relationship between fluorine energy loss intensity, image area and specimen thickness. These techniques were applied to an examination of the reaction between purified RNA and the novel titanium-containing anti-cancer agent MKT-4, showing that ratios calculated from the cumulative electron energy loss intensity increase within regions of interest as a function of grey level intensity for titanium and nitrogen are directly proportional to the incubation concentration of the drug with RNA. Expression of data in this way is shown to be a useful indicator of the relative drug distribution within specific intracellular compartments of human cancer cells incubated with MKT-4 in vitro.

PACS
8780 - Biophysical instrumentation and techniques.
8280P - Electron spectroscopy for chemical analysis photoelectron, Auger spectroscopy, etc..
8725F - Physics of subcellular structures.
0780 - Electron and ion microscopes and techniques.
7320 - Physics and chemistry computing.
7330 - Biology and medical computing.
5260B - Computer vision and image processing techniques.

Key words
biological techniques -- biology computing -- cellular biophysics -- electron energy loss spectra -- electron spectroscopy -- image resolution -- image sequences -- microcomputer applications -- organic compounds -- spectroscopy computing -- transmission electron microscopy -- organic compounds -- PC based energy sequence imaging software -- electron spectroscopic imaging -- Windows based software packages -- rapid elemental microanalysis -- energy filtered transmission electron microscopic images -- electron energy loss spectroscopy -- titanium containing anti cancer agent MKT 4 -- image sequences -- energy resolution -- spatial resolution -- grey level integration -- least mean squares method -- optimum collections conditions -- semi quantitative elemental analysis -- quantitative elemental analysis -- biological specimens -- Lowicryl HM23 embedded ultra thin section -- boric acid polymer -- fluorine containing organic polymer -- relative drug distribution -- human cancer cells -- purified RNA -- Nafion -- Image EELS -- B -- N -- Ti -- F


© EDP Sciences 1995