Seminars at Neuroscience 2018

Monday, November 5, 2018 – 12 PM - 1 PM

High-Performance Serial Block-face SEM of Nonconductive Biological Samples Enabled by Focal Gas Injection-Based Charge Compensation

Thomas Deerinck and Mark Ellisman, The National Center for Microscopy and Imaging Research, UCSD, La Jolla, California

Serial block-face scanning electron microscopy (SBEM) is rapidly becoming the method of choice for obtaining large-scale 3-D imaging data of biological specimens at nanometer-scale resolution. One of the principle limitations of this method is that specimens must be embedded in non-conductive epoxy resin prior to imaging, and this can lead to substantial specimen charging. Typically, cells and tissues are intensely heavy-metal stained in order to improve backscattered electron yield at low accelerating voltages and to reduce specimen charging. However, not all specimen charging can be eliminated in charge-probe samples such as those containing large open spaces or with low lipid content. Most commonly, variable-pressure SEM (VP-SEM) is used for many specimens to minimize charging, but at a significant loss of signal-to-noise and resolution owing to electron-gas interactions. Here we introduce an approach to adapt the method of focal gas injection-based charge compensation to SBEM. This approach, which we call Focal Charge Compensation (Focal CC), uses a fine needle to inject nitrogen gas directly over the surface of the sample block-face. Secondary electrons emitted from the sample surface ionize the gas molecules and the resulting ions dissipate the charge. Since the needle must be retracted during the cutting cycle, a simple device was designed to precisely deliver the gas over the sample during imaging and automatically retract during the cutting cycle. Since the overall chamber pressure is at high vacuum (<7x10-3 mbar), the signal-to-noise and resolution at the low accelerating voltages normally used for SBEM are vastly superior to VP-SEM.

Focal CC represents a major step forward in block-face imaging, as now even the most charge-prone samples can be imaged without charging artifacts. Because the primary electrons only have to traverse a thin layer of nitrogen gas molecules near the sample surface, beam current is only marginally decreased when compared to VP-SEM. This means that shorter pixel dwell times can be used (0.5-1 µsec), reducing specimen beam damage and resulting in faster data acquisition times. Additionally, beam skirting is also greatly reduced, resulting in superior spatial resolution. Finally, image jitter due to residual specimen charging is eliminated, making post-processing of image stacks to correct for this phenomenon unnecessary.