Unstained Sample: The compartmentalization of some of the secretory granules (g) is obvious, with circular or oval profiles being brighter than the rest of the granular matrix.
Examination of ultra-thin (30-nm) sections of non-osmicated, stain-free pancreatic tissue sections by low-voltage electron microscopy revealed the existence of granules with non-homogeneous matrix and sub-compartments having circular or oval profiles of different electron densities and sizes. Such partition is completely masked when observing tissues after postfixation with osmium tetroxide by low-voltage transmission electron microscopy at 5 kV and/or when thicker sections (70 nm) are examined at 80 kV. This morphological partition reflects an internal compartmentalization of the granule content that was previously predicted by morphological, physiological, and biochemical means.
The main advantage of conducting TEM observation of biological tissues with the LVEM5 microscope is the enhanced level of contrast achieved in the images of unstained tissues at beam energies below 10 keV. Any conventional TEM technique nowadays employs an accelerating voltage of 80 to 120 kV or higher, and modern electron microscopes are generally not designed for TEM imaging with beam energies below 40 keV. Increasing accelerating voltage is a prerequisite to achieve higher spatial resolutions. At the same time, it has an adverse effect on contrast in the biological tissues, as there is very little difference in the scattering of high-energy electrons from most cellular components. That leads to a very low contrast when imaging a tissue unless a contrasting agent is added.
Osmicated Sample: The secretory zymogen granules (g) matrix appears quite dense and homogeneous.
Consequently, staining tissues with heavy metal compounds in pre- or postembedding stages became established and routine steps in sample preparation before TEM observation. However, the use of stains has its disadvantages for the correct interpretation of fine cellular morphology as this can be modified or shadowed by chemical reactions with stain components. To avoid staining steps before TEM imaging, significantly lower incident beam energy should be employed to increase contrast. It was shown that for practical purposes of transmission electron microscopy in organic materials, accelerating voltages below 10 kV are required. In this case, the variations in scattering “slow” incident electrons by different tissue features are sufficient to produce adequate contrast without any stain while maintaining a satisfactory resolution level.
As illustrated in the paper, images of completely non-stained tissue reveal substructures within certain cell compartments, such as the secretory granules. Those substructures do not appear on the images of stained tissues, both in conventional and in low-voltage TEM. The osmium postfixation, although improving contrast on membranes and other elements, does not reveal any substructural elements within the granules, which appear as homogeneous, electron-dense vesicles. Differences between images generated by low-voltage microscopy and those acquired with tissues postfixed with osmium tetroxide seem to indicate that impregnation with heavy metals does in fact mask subtle substructures present in some cellular compartments.
Bendayan M, Londono I, & Paransky E (2011). Compartmentalization of pancreatic secretory zymogen granules as revealed by low-voltage transmission electron microscopy. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, 59 (10), 899-907 PMID: 21832147
