APPLICATIONS OF MEDIUM-VOLTAGE STEM FOR THE 3-D STUDY OF ORGANELLES WITHIN VERY THICK SECTIONS

Scanning transmission electron microscopy at 300 kV enables the visual ization of nucleolar silver-stained structures within thick sections ( 3-8 mum) of Epon-embedded cells at high tilt angles (-50-degrees; +50- degrees). Thick sections coated with gold particles were used to deter mine the best conditions for obtaining images with high contrast and g ood resolution. For a 6-mum-thick section the values of thinning and s hrinkage under the beam are 35 to 10%, respectively. At the electron d ensity used in these experiments (100 e-/angstrom2/s) it is estimated that these modifications of the section stabilized in less than 10 min . The broadening of the beam through the section was measured and calc ulations indicated that the subsequent resolution reached 100 nm for o bjects localized near the lower side of 4-mum-thick sections with a sp ot-size of 5.6 nm. Comparing the same biological samples, viewed alter nately in CTEM and STEM, demonstrated that images obtained in STEM hav e a better resolution and contrast for sections thicker than 3 mum. Th erefore, the visualization of densely stained structures, observed thr ough very thick sections in the STEM mode, will be very useful in the near future for microtomographic reconstruction of cellular organelles .