New Focal Charge Compensation Mode for ZEISS Field Emission Scanning Electron Microscopes Improves Image Quality

High-resolution 3D block face imaging for biological samples with fast acquisition rates and minimal sample damage

14 Nov 2017

Lui Terry

Administrator / Office Personnel

In collaboration with the National Center for Microscopy and Imaging Research (NCMIR) at the University of California San Diego, ZEISS has announced a new Focal Charge Compensation module for block face imaging with ZEISS GeminiSEM and 3View^® from Gatan, Inc. Focal Charge Compensation expands versatility and considerably increases data quality without prolonging acquisition times and enables easy imaging of even the most charge-prone samples.

Images courtesy of NCMIR Charging effects are clearly visible under high vacuum, in contrast to images taken with Focal Charge Compensation, which show no charging effects even in large expanses of bare resin. The images show a ~300 micron diameter axon bundle at different magnifications.

Resin-embedded tissues and cells can be imaged without charging artifacts, while the pixel dwell time is reduced. Decreasing beam exposure time not only ensures fast acquisition rates, but also guards against sample damage, which is key to acquiring reliable and reproducible 3D data. Professor Mark H. Ellisman, Director of NCMIR, says “Focal Charge Compensation will breathe new life into block face scanning electron microscopy by allowing high quality imaging of previously intractable specimens, including legacy samples prepared with traditional electron microscopy stains.”

Preventing Charging Effects

This extension of the 3View system from Gatan, Inc eliminates specimen charging. A gas injection system consisting of a tiny capillary needle is precisely located above the sample. Nitrogen is guided through this needle directly onto the block face surface while the chamber is maintained under high vacuum. This eliminates charging without degrading image quality. The needle retracts automatically during the cutting cycle so the workflow is uninterrupted and high acquisition rates are maintained.

Producing Thousands of Serial Images in a Single Day

The 3View system consists of an ultramicrotome directly integrated into the vacuum chamber of the ZEISS Sigma and ZEISS GeminiSEM field emission scanning electron microscopes. It enables automated serial block face imaging of embedded samples (e.g. cells or tissue) with a slice thickness down to 15 nanometers. The sample is continuously cut and imaged, and a three-dimensional rendering of the sample with nanometer-scale resolution can be reconstructed.