Applications of medium‐voltage STEM for the 3‐D study of organelles within very thick sections

Abstract

can be visualized through thick sections (several micro-Scanning transmission electron microscopy at 300 kV enables the visualization of nucleolar silver-stained structures within thick sections (3-8pm) of Epon-embedded cells at high tilt angles (-50"; + 50"). Thick sections coated with gold particles were used to determine the best conditions for obtaining images with high contrast and good resolution. For a 6-pm-thick section the values of thinning and shrinkage under the beam are 35 to 10% respectively. At the electron density used in these experiments ( 1 0 0 e -/~~/ s ) it is estimated that these modifications of the section stabilized in less than 10min. The broadening of the beam through the section was measured and calculations indicated that the subsequent resolution reached 100nm for objects localized near the lower side of 4-pm-thick sections with a spot-size of 5.6 nm. Comparing the same biological samples, viewed alternately in CTEM and STEM, demonstrated that images obtained in STEM have a better resolution and contrast for sections thicker than 3 pm. Therefore, the visualization of densely stained structures, observed through very thick sections in the STEM mode, will be very useful in the near future for microtomographic reconstruction of cellular organelles.

Electron Tomography of Metaphase Nucleolar Organizer Regions: Evidence for a Twisted-Loop Organization

“…Sections 1000 to 2000 nm thick were obtained, and no counterstaining was performed to analyze the silver deposits more easily (Beorchia et al, 1992). These sections were studied at 200 kV in a 200 CX electron microscope and images were obtained on negatives.…”

“…For the high-resolution tomographic studies, 3000-nm-thick sections were observed in a medium-voltage electron microscope working at 300 kV in the scanning and transmission electron microscope (STEM) mode (CM 30 Philips, Eindhoven, The Netherlands) as previously described (Beorchia et al, 1992). The specimen holder was mounted on a eucentric goniometer stage that can be tilted from Ϫ60°to ϩ60°.…”

“…8, November 1997anisotropic thinning of the specimen during data collection (Beorchia et al, 1992). Moreover, to position the specimen correctly relative to the axis of the goniometer stage, it was necessary to choose several fiducial markers.…”

Three-dimensional Organization of pKi-67: A Comparative Fluorescence and Electron Tomography Study Using Fluoronanogold

“…All ultrathin sections were observed with a 200 CX electron microscope at 100 kV (JEOL). Sections 0.5 m or 2 m in thickness were observed in a medium voltage CM30 electron microscope working at 250 kV in the STEM mode (Philips), as described previously (Beorchia et al 1992;Héliot et al 1997;Cheutin et al 2002). Before starting a tilt series, the whole-mounted cells were irradiated for 10 min at 100 e Ϫ /(Å 2 ϫ sec).…”

“…After acquiring a series of optical sections by confocal microscopy or collecting projections at different angles with a scanning and transmission electron microscope (STEM), volume reconstructions and tomographic analyses were performed (Beorchia et al 1992;Lucas et al 1996;Héliot et al 1997;Perkins et al 1997;Baumeister et al 1999;Cheutin et al 2002;Frank et al 2002). The results reveal both the fine localization and the different levels of organization of pKi-67.…”