Optimization of phosphorus localization by EFTEM of nucleic acid containing structures

Abstract

Energy Filtered Transmission Electron Microscopy (EFTEM) has been used to study nucleic acids localization in unstained thin sections of virus-infected cells. For this purpose, phosphorus maps (P-maps) have been obtained by applying the N-windows Egerton model for background subtraction from data acquired by a non-dedicated TEM Jeol 1200EXII equipped with a post-column PEELS Gatan 666-9000 and a Gatan Image Filter (GIF-100). To prevent possible errors in the evaluation of elemental maps and thus incorrect nucleic acid localization, we have studied different regions of swine testis (ST) cells with similar local density containing either high concentration of nucleic acids (condensed chromatin and ribosomes) or a very low concentration (mitochondria). Special care was taken to optimize the sample preparation conditions to avoid as much as possible the traditional artifacts derived from this source. Selection of the best set of pre-edge images for background fitting was also considered in order to produce "true P-maps". A new software for interactive processing of images series has been applied to estimate this set. Multivariate Statistical Analysis was used as a filtering tool to separate the "useful information" present in the inelastic image series (characteristic signal) from the "non-useful information" (noise and acquisition artifacts). The reconstitution of the original 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 intermediate coronavirus particles found inside infected cells.

Visualization of proteins in intact cells with a clonable tag for electron microscopy

“…The level of defocus was $1-2 lm depending on the specimen. For studies with sectioned cells fast-freezing, cryosubstitution and cryo-embedding of intact cells were done as described (Quintana et al, 1998(Quintana et al, , 2001Risco et al, 2002). A volume of 2-3 ll of live cell suspensions were applied to small (1 Â 1 mm) pieces of Whatman filter paper and frozen in liquid ethane cooled with liquid nitrogen using a dedicated cryofixation and cryopreparation system (CPC, Leica, Vienna, Austria) (Risco et al, 2002).…”

“…Samples were then subjected to a controlled increase of temperature (from À90°C to À40°C in 2.5 h) before embedding in increasing concentrations of the acrylic resin Lowicryl HM23 (Taab Laboratories, Aldermaston, UK) diluted in metanol at À40°C. Lowicryl HM23 is a resin of very low density, particularly suited when a maximum contrast is needed (Quintana, 1994;Quintana et al, 1998Quintana et al, , 2001. After infiltration during 29 h, samples were polymerized with UV light, 2 days at À40°C and 2 more days at 25°C.…”

Chemical mapping of DNA and counter-ion content inside phage by energy-filtered TEM

“…Energy-filtered images were recorded and elemental maps constructed, either by the three energy window method [17,22] (with two images recorded below and one image recorded above the ionization edge energy), or with the jump ratio method [19,23] (just using one image recorded at energies below, and one image recorded above the edge energy of interest).…”

“…For EFTEM, it is thus most important to make the samples as thin as possible and omit additions of any heavy elements during the preparation. EFTEM has previously been used to study viral morphogenesis in infected cells [13,[17][18][19] but has to our knowledge never been applied in the study of viral structures without extensive preparations for imaging including embedding, staining, and sectioning.…”

Some trends in microscope image processing

“…Then, the eigen-components that contain only noise or artefacts are discarded and the data set is reconstructed using useful components only. Applications of this procedure can be found in Quintana and Bonnet (1994) and Quintana et al (1998).…”