High-performance serial block-face SEM of nonconductive biological samples enabled by focal gas injection-based charge compensation

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. Here we show the development and application of a simple system that effectively mitigates specimen charging by using focal gas injection of nitrogen over the sample block-face during imaging. A standard gas injection valve is paired with a precisely positioned but retractable application nozzle, which is mechanically coupled to the reciprocating action of the serial block-face ultramicrotome. This system enables the application of nitrogen gas precisely over the block-face during imaging while allowing the specimen chamber to be maintained under high vacuum to maximise achievable SEM image 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. The device described was added to a Gatan 3View system with minimal modifications, allowing high-resolution block-face imaging of even the most charge prone of epoxy-embedded biological samples. Lay description Serial block-face scanning electron microscopy is rapidly becoming the method of choice for obtaining 3-dimensional volume imaging data of cells and tissues at nanometre-scale resolution. However, one of the main drawbacks of this approach is the problem of specimen charging of the nonconductive epoxy resin used to embed specimens, causing moderate to severe image distortions. The most common method used to mitigate specimen charging is the use of variable-pressure SEM, in which the entire specimen chamber is held at low vacuum (typically 0.1-0.5 mbar) and the residual gas molecules are ionised, mainly by secondary electrons emitted from the sample surface, and the resulting ions are attracted to and neutralise specimen charge. This approach, while effective, results in signal loss and resolution degradation due to electron scattering caused by the large number of gas molecules present in the entire specimen chamber. Here we describe a simple device that enables a very small, localised stream of gas to be injected directly over the specimen surface during imaging while maintaining the chamber at high vacuum (<7 x 10(-3) mbar). While the gas ionisation charge neutralisation is similar to variable pressure SEM, electron scattering is greatly reduced, resulting in a significant improvement in signal-to-noise and resolution. The key to making this method work with serial block-face scanning electron microscopy was development of a device that allows the precise positioning of the small stream of gas to the very small space between the backscatter detector and the specimen block; a space which must also be occupied by the ultramicrotome arm in a different part of the imaging cycle. The nozzle composition and mechanism for precise repositioning required special design considerations and is described below. The resulting device and local gas injection method allows for high-resolution imaging of even the most charge-prone samples by serial block-face scanning electron microscopy.