The Thermo Fisher Apreo is a high resolution FESEM capable of operating in low vacuum conditions. The microscope is equipped with a VolumeScope Serial Block Face system. This consists of an ultramicrotome mounted inside the vacuum chamber of the SEM. Resin-embedded samples are prepared by methods similar to that in a TEM, though involving the use of more metals to enhance signal and provide conductivity to the sample. The surface of the block of resin-embedded sample is imaged by detection of back-scattered electrons. Following imaging the ultramicrotome is used to cut a thin section (typically around 50 nm or less) from the face of the block. After the section is cut, the sample block is raised back to the focal plane and imaged again. This sequence of sample imaging, section cutting and block raising can acquire hundreds to thousands of images in an automated fashion. This allows the interpretation of the complex 3D architecture of cells and tissues in their natural context.
The system offers a novel solution to improve the axial resolution by combining mechanical sectioning with optical sectioning, realized by Multi-Energy Deconvolution-SEM. Following the diamond knife sectioning of the block-face, the freshly exposed block face is imaged several times using increasing accelerating voltages. These images are subsequently used in a deconvolution algorithm to derive several optical subsurface layers, forming a 3D subset. By repeating this cycle, it is possible to achieve isotropic datasets with less than 10 nm z-resolution. The MAPSTM software makes it possible acquire wide-field tissue mapping (Tiling and Stitching) at each section to capture information over a large area.
The instrument was purchased through a grant from the Australian Cancer Research Foundation and is now part of the ACRF Cancer Ultrastructure and Function Facility, along with light microscopy facilities in the Institute for Molecular Biosciences.
If you would like further information on this instrument, please contact Rick Webb or Robyn Chapman.
