OPTIMIZATION OF PHOSPHORUS LOCALIZATION BY EFTEM OF NUCLEIC-ACID CONTAINING STRUCTURES

Energy Filtered Transmission Electron Microscopy (EFTEM) has been used to study nucleic acids localization in unstained thin sections of vir us-infected cells. For this purpose, phosphorus maps (P-maps) have bee n obtained by applying the N-windows Egerton model for background subt raction from data acquired by a non-dedicated TEM Jeol 1200EXII equipp ed with a post-column PEELS Gatan 666-9000 and a Gatan Image Filter (G IF-100). To prevent possible errors in the evaluation of elemental map s and thus incorrect nucleic acid localization, we have studied differ ent regions of swine testis (ST) cells with similar. local density con taining either high concentration of nucleic acids (condensed chromati n and ribosomes) or a very low concentration (mitochondria). Special c are was taken to optimize the sample preparation conditions to avoid a s much as possible the traditional artifacts derived from this source. Selection of the best set of pre-edge images for background fitting w as also considered in order to produce ''true P-maps''. A new software for interactive processing of images series has been applied to estim ate this set. Multivariate Statistical Analysis was used as a filterin g tool to separate the ''useful information'' present in the inelastic image series (characteristic signal) from the ''non-useful informatio n'' (noise and acquisition artifacts). The reconstitution of the origi nal image series preserving mainly the useful information allowed the computation of P-maps with improved signal-to-noise ratio (SNR). This methodology has been applied to study the RNA content of maturation in termediate coronavirus particles found inside infected cells. (C) 1998 Elsevier Science Ltd. All rights reserved.