Optimization of negative stage bias potential for faster imaging in large-scale electron microscopy
| Autor: | Judith Klumperman, Nalan Liv, Jacob P. Hoogenboom, Yoram Vos, Ryan Lane, Ben N G Giepmans, Anouk H G Wolters, S. Elisa Chen, Luc van Kessel |
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| Přispěvatelé: | Basic and Translational Research and Imaging Methodology Development in Groningen (BRIDGE), Center for Liver, Digestive and Metabolic Diseases (CLDM) |
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Materials science
Image quality QH301-705.5 High-throughput imaging Correlative light and electron microscopy Scale (descriptive set theory) Signal Volume electron microscopy Bottleneck Article Stage bias Optics Structural Biology Microscopy Electron microscopy PROGRAM Large-scale electron microscopy Biology (General) FLUORESCENCE FIELD Penetration depth ComputingMethodologies_COMPUTERGRAPHICS business.industry Dwell time RESOLUTION INTEGRATED LIGHT Stage (hydrology) business |
| Zdroj: | Journal of Structural Biology: X, Vol 5, Iss, Pp 100046-(2021) Journal of structural biology: X, 5:100046 Journal of Structural Biology: X, 5 Journal of Structural Biology: X |
| ISSN: | 2590-1524 |
| Popis: | Graphical abstract Highlights • The use of a negative bias potential was empirically optimized for tissue imaging with SEM. • Optimized bias potential leads to a factor 20 increase in imaging speeds as well as an order of magnitude improvement to SNR. • SNR increase results from a combination of BSE acceleration and detector response. • Similar increases to SNR can be obtained when a magnetic immersion field is combined with a negative bias potential. • Stage bias can be applied within an integrated fluorescence and electron microscope allowing for fast correlative imaging of tissue sections. 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. |
| Databáze: | OpenAIRE |
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