To visualize subcellular ultrastructure it was necessary to incorporate high levels of heavy-atom contrast agents into the biological specimens of interest. This was achieved by treating the pelleted cells or dissected tissues with primary glutaraldehyde and formaldehyde fixation, followed by osmium tetroxide postfixation, and subsequent uranyl acetate and en-bloc lead aspartate staining. Blocks were mounted on aluminum pins after first trimming to ensure that the stained cells made electrical contact with the pin and the sides of the block were coated with sputtered gold to increase conductivity and to prevent charging when the block face was imaged. Specimens were loaded into a 3View Gatan serial block face imaging system installed on a Zeiss Sigma Variable Pressure SEM. Backscattered electron image series were collected with beam energies of between 1 and 3 kV. It was found that the choice of instrumental parameters for operating the SEM was critical to acquire optimal images. In particular, it was necessary to operate in variable pressure mode and to adjust the incident electron energy to minimize electrical charging of the specimen block. Application of serial block face SEM to preparations of stem cells revealed a heterogeneous presentation of glycogen bodies in hPSCs. Preliminary results were consistent with our laboratorys previous TEM data, which showed that glycogen synthesis was higher in undifferentiated hESCs than in iPSCs. Experiments are in progress to map the three-dimensional distributions of glycogen deposits in the different types of stem cells, as a potential marker to assess energetic states of stem cell populations.
|Shomorony, A; Pfeifer, C R; Aronova, M A et al. (2015) Combining quantitative 2D and 3D image analysis in the serial block face SEM: application to secretory organelles of pancreatic islet cells. J Microsc 259:155-64|