Serial Block-Face SEM
Highly Automated Sectioning and Volume Data Imaging
Schematic Representation of a Typical Workflow
A resin-embedded sample is cut with an ultramicrotome mounted inside the SEM chamber. The exposed sample surface is imaged. This cutting and imaging process is repeated until the structure of interest is completely imaged.
The acquired EM images are processed and digitally aligned into a 3D data set. Cell compartments can be identified and segmented.
The segmented 3D data set can be visualized, investigated, and statistically analyzed.
Understanding the Relationship between Structure and Function
Imaging Ultrastructural Details of Neurons
The brain is a complex organ with millions of neuronal connections and signaling pathways. Understanding the relationship between structure and function of brain tissue helps in unravelling some of this complexity to better understand how neural networks are organized and, in the long term, how to treat certain pathologies with medical interventions.
Imaging of Neurons in Cell Culture
SBF-SEM is the appropriate solution to image and follow neurons with long and thin protrusions such as dendrites and axons. Especially neurons in cell culture are difficult to image. The high proportion of the non-conductive resin makes the samples prone to charging. Focal Charge Compensation mitigates charging effects and ensures a high image quality. Ultrastructural details of neurons can be easily imaged and resolved with SBF-SEM in combination with Focal Charge Compensation.
The images show a single slice from a 3D data set of cultured hippocampal neurons expressing PSD95-APEX2 to stain post-synaptic densities (arrows). Images were acquired using a ZEISS FESEM, integrated ultramicrotome and Focal Charge Compensation. Ultrastructure such as thin dendrites and connections are visible with high resolution due to the removal of charging effects.
Single Neurons and Cellular Compartments in Mouse Brain Tissue
The video shows the cross sections of a mouse brain specimen captured using Serial Block-Face SEM. The high resolution this approach provides can be clearly seen in each of the single block-face images. Single neurons and cellular compartments can be identified and followed along the z-dimension.
Investigation of Axon Myelinization to Understand Multiple Sclerosis and Parkinson’s Disease
Electron micrographs provide high resolution information sufficient to count the number of single myelin lamellae and measure overall sheath thickness.
The sparse nature of structures in these samples leads to significant charging effects. Using Focal Charge Compensation eliminates these effects – you can now image with highest resolution in all three dimensions.
The animation shows a run through single slices (x-y) of rat spinal cord using 3View® and Focal Charge Compensation. Single lamellae within the myelin sheaths of the axons are clearly visible as well as microtubules and other cellular organelles