Research Highlights
High-performance serial block-face SEM of non-conductive biological samples enabled by focal gas injection-based charge compensation
Direct comparison of charge compensation (A) and variable pressure SEM (B) of the same cultured cell expressing APEX2 targeted to the endomembrane system by KDEL. Gas injection was 80% N2 and chamber pressure was ~5 x 10-3 mbar. VP-SEM was 0.3 mbar chamber pressure (maximum pressure allowed using Gemini SEM) using N2 with otherwise identical imaging conditions (2.5 keV and 1 msec pixel dwell time). Bar = 2 microns.
01-12-2017 La Jolla
A longstanding limitation of imaging with serial block-face scanning electron microscopy is specimen surface charging. This charging is largely due to the difficulties in making biological specimens and the resins in which they are embedded sufficiently conductive. Local accumulation of charge on the specimen surface can result in poor image quality and distortions. Even minor charging can lead to misalignments between sequential images of the block-face due to image jitter. Typically, variable-pressure SEM is used to reduce specimen charging, but this results in a significant reduction to spatial resolution, signal-to-noise ratio and overall image quality due to the large number of gas molecules present in the entire specimen chamber.
In collaboration with Carl Zeiss Microscopy (www.zeiss.com), NCMIR researchers developed a new technology to solve this problem, greatly improving the performance of serial blockface scanning EM (SBEM) using the Gatan 3View. This technology allows for the focused and highly synchronized delivery of nitrogen gas to the sample blockface during the imaging cycle to eliminate charging, greatly extending the utility of the volume imaging technique for even the most-charge-prone samples.
This technology involves the use of a device, designed and optimized by NCMIR engineers, that is fastened to the Gatan 3View ultramicrotome without need for platform modification. This specialized device enables a very small, localized stream of gas to be injected directly over the specimen surface during imaging while maintaining the chamber at high vacuum (<7x10-3 mbar). While the gas ionization charge neutralization is similar to variable pressure SEM, electron scattering is greatly reduced, resulting in a significant improvement in signal-to-noise and resolution. The action of the ultramicrotome drives the nozzle retraction, automatically moving it away from the specimen area during the cutting cycle of the knife, so the workflow is uninterrupted and high acquisition rates are maintained.
This Focal Charge Compensation (FocalCC) system expands SBEM versatility and considerably increases data quality without prolonging acquisition times and enables easy imaging of previously intractable samples. 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.”
Relevant Publication: Deerinck, T. J., T. M. Shone, E. A. Bushong, R. Ramachandra, S. T. Peltier, and M. H. Ellisman. "High‐performance serial block‐face SEM of nonconductive biological samples enabled by focal gas injection‐based charge compensation." Journal of microscopy (2017).
Funding source: This study was supported by the National Institutes of Health grant P41GM103412, supporting the National Center for Microscopy and Imaging Research.