Abstract
Large-scale electron microscopy (EM) allows analysis of both tissues and macromolecules in a semi-automated manner, but acquisition rate forms a bottleneck. We reasoned that a negative bias potential may be used to enhance signal collection, allowing shorter dwell times and thus increasing imaging speed. Negative bias potential has previously been used to tune penetration depth in block-face imaging. However, optimization of negative bias potential for application in thin section imaging will be needed prior to routine use and application in large-scale EM. Here, we present negative bias potential optimized through a combination of simulations and empirical measurements. We find that the use of a negative bias potential generally results in improvement of image quality and signal-to-noise ratio (SNR). The extent of these improvements depends on the presence and strength of a magnetic immersion field. Maintaining other imaging conditions and aiming for the same image quality and SNR, the use of a negative stage bias can allow for a 20-fold decrease in dwell time, thus reducing the time for a week long acquisition to less than 8 h. We further show that negative bias potential can be applied in an integrated correlative light electron microscopy (CLEM) application, allowing fast acquisition of a high precision overlaid LM-EM dataset. Application of negative stage bias potential will thus help to solve the current bottleneck of image acquisition of large fields of view at high resolution in large-scale microscopy.
Highlights
Mapping the full ultrastructural layout of complex biological systems at nanometer-scale resolution is a major challenge in cell biology
The bias is applied via an external power supply connected to a custom stage plate such that the sample is electrically isolated from the rest of the fluorescence microscope and electrical components of the stage (Vos et al, 2020)
By generating an electric field between the sample and the BSE detector, the bias potential accelerates signal electrons inwards away from their otherwise linear trajectories. Because of their lower energy, secondary electrons (50 eV) are redirected over a wider area depending on their initial emission angle and energy
Summary
Mapping the full ultrastructural layout of complex biological systems at nanometer-scale resolution is a major challenge in cell biology. While throughput is already a bottleneck for large-scale 2D imaging (Kuipers et al, 2015), most of these approaches have been developed under the framework of 3D imaging. Other approaches involve parallelizing the imaging load across multiple instruments. This has been employed in focused ion beam scanning electron microscopy (FIB-SEM) for the reconstruction of thick slices of Drosophila brain tissue at isotropic (8 nm × 8 nm × 8 nm) voxel resolution (Hayworth et al, 2015) as well as in serial section transmission electron microscopy (ssTEM) for the yearlong acquisition of a cubic millimetre of mouse brain tissue (Yin et al, 2019). Multi-beam instruments in which a sample is simultaneously imaged by multiple focused electron beams have been developed (Eberle et al, 2015; Ren and Kruit, 2016)
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