3D-EM Staining & Imaging
Renovo’s 3D-EM infrastructure consists of a Zeiss Sigma VP scanning electron microscope (SEM) with a 3View® door by Gatan. The 3View® door is mounted with a high precision ultramicrotome which sits directly under the SEM column within the chamber, allowing automated acquisition of images by sequentially cutting and imaging the block face of a resin-embedded substrate. After imaging the block face, the sample is moved up 10µms to the cutting plane of the knife where the ultramicrotome cuts a specified thickness (50-200 nm) off the top of the sample and retracts, exposing a new block face and restarting the cycle. Thus, image stacks containing hundreds, or even thousands, of sequential slices may be consistently generated and a 3D profile of the sample is built at nanometer resolution. Our 3D-EM services provide researchers an opportunity to obtain high resolution volumetric datasets by removing the difficult step of cutting and collecting ultrathin sections manually.
Sample preparation and selection of fixative are critical for ultrastructural preservation and image acquisition. We accept pre-processed, fixed biological tissues, and select embedded material substrates for imaging to generate a 3D stack of images for further analysis. We have altered previously developed specific protocols for sample preparation to obtain reliable datasets with high accuracy and incredible detail, which facilitate image analysis and 3D-reconstruction.
We have developed several imaging applications for biological samples to make cellular ultrastructure in 3D routinely possible. Imaging can be customized to different modalities, one image per section or multiple images from different regions of interest (ROI) at different resolutions within the same block-face per section. Our EM experts will work closely with you to determine imaging parameters for each individual project in order to ensure you get the most out of your data. In addition to regular EM imaging, we also perform correlative confocal/light EM imaging for specific studies.