Deceleration of probe beam by stage bias potential improves resolution of serial block-face scanning electron microscopic images

Abstract

Serial block-face scanning electron microscopy (SBEM) is quickly becoming an important imaging tool to explore three-dimensional biological structure across spatial scales. At probe-beam-electron energies of 2.0 keV or lower, the axial resolution should improve, because there is less primary electron penetration into the block face. More specifically, at these lower energies, the interaction volume is much smaller, and therefore, surface detail is more highly resolved. However, the backscattered electron yield for metal contrast agents and the backscattered electron detector sensitivity are both sub-optimal at these lower energies, thus negating the gain in axial resolution. We found that the application of a negative voltage (reversal potential) applied to a modified SBEM stage creates a tunable electric field at the sample. This field can be used to decrease the probe-beam-landing energy and, at the same time, alter the trajectory of the signal to increase the signal collected by the detector. With decelerated low landing-energy electrons, we observed that the probe-beam-electron-penetration depth was reduced to less than 30 nm in epoxy-embedded biological specimens. Concurrently, a large increase in recorded signal occurred due to the re-acceleration of BSEs in the bias field towards the objective pole piece where the detector is located. By tuning the bias field, we were able to manipulate the trajectories of the primary and secondary electrons, enabling the spatial discrimination of these signals using an advanced ring-type BSE detector configuration or a standard monolithic BSE detector coupled with a blocking aperture.

A carbon nanotube tape for serial-section electron microscopy of brain ultrastructure

“…There is sufficient metal content in the tissue for SEM imaging, as images are captured within small pixel dwell times, and signals are collected from a small interaction volume within a shallow depth. Collectively, all of these changes result in an improved axial resolution and better signal-to-noise ratio 49 , 50 .…”

Serial block face scanning electron microscopy in cell biology: Applications and technology

“…This technique has been known to improve SEM BSE imaging (Ohta et al, 2012). Bouwer et al (Bouwer et al, 2017) observed that applying a negative sample stage bias reduced beam landing energies, reduced sample damage and increased BSE signal-to-noise ratio by accelerating BSE towards the BSE detector above the sample. In conjunction with a special BSE detector they were also able to alter the trajectories of primary and secondary electrons and control beam penetration depth.…”

Optimization of negative stage bias potential for faster imaging in large-scale electron microscopy

“…Faster imaging could also be achieved by increasing signal collection in established thin sections approaches, which would allow for reduced acquisition time while maintaining a sufficient signal to noise ratio (SNR). It has previously been shown that the use of a retarding field increases SNR in SEM (Phifer et al, 2009), but for biological imaging the use of a retarding field has thus far been investigated in detail only for serial blockface scanning EM (SBF-SEM) (Bouwer et al, 2016;Ohta et al, 2012).…”

“…1). The ensuing acceleration results in an increase to the collected signal (Šakić et al, 2011) and-if the detector geometry, landing energy, and potential bias are tuned properlycan be used to filter out secondary electrons (Bouwer et al, 2016). The same signal can in principle then be obtained with a shorter acquisition time.…”