A high resolution, multi-functional scanning electron microscope for a multiuser interdisciplinary BioEM facility.

Lead Research Organisation: University of Leicester
Department Name: Genetics

Abstract

Biological processes affect all aspects of our lives, from our health, the food we eat, and the environment around us. To understand these processes, we need to learn how organisms function, and interact with each other and the surrounding environment. Imaging is a very powerful tool to study these events.

Electron microscopes use electrons as an illumination source to observe cell surfaces and internal structures in extremely fine detail, and image even smaller structures such as viruses, proteins and nanoparticles. Exciting advances in computational and technical approaches are now leading to a new wave of biological EM imaging techniques with even greater resolution.

Novel detectors can inform on elemental and physical details and correlate images generated with other methodologies, such as light or fluorescent microscopes, to locate particular features, proteins and events within tissue or other biological materials. Improved software packages and computer programming power also enable reconstruction of 2D slices of tissue into 3D models that opens a world of opportunities in understanding complex events and features that have previously been difficult to interpret from two-dimensional imaging.

It is critical that we replace our current outdated EM with a new state of the art microscope that provides significantly higher resolution and the technologies of 3D reconstruction, and light and EM microscopy comparisons of biological samples. The current EM is limiting our ability to do innovative bioimaging research due to its low resolution and limited capabilities. Local EM facilities are focussed on inorganic materials and are not ideal for biological research.

Our aim is to establish a multiuser interdisciplinary bioimaging facility centred around the new multi-functional microscope. This centre of excellence for biological focussed electron microscopy (BioEM) will provide research and training support across the University of Leicester, and for other universities and industrial collaborators across the Midlands for which these biological applications are currently not available.

Our objectives are to (1) install a new Field Emission Scanning Electron Microscope with Array Tomography and Energy Dispersive Spectroscopy, (2) expand our interdisciplinary research integral to the BBSRC strategy of understanding the rules of life, (3) provide a multi-user core facility providing specialised training in EM of biological processes to support academic and industrial collaborations, while promoting transfer of technical skills across the Midlands and (4) grow our portfolio of industry-supported research.

Research output will be improved with nanoscale resolution and multiple analytical options. The new EM will enable greater understanding of cellular structures, elemental analysis of biological materials and 3D reconstruction of cells and tissues. Introducing these exciting new tools will give significant potential for innovative bioimaging collaborations and enhance career opportunities for technical research staff who will be able to develop practical skills and scientific knowledge on an exciting new platform.

The new EM will also complement and provide support to the local materials-based EM facilities, the Midlands Regional CryoEM Facility and integrate with the Leicester Advanced Imaging Facility and thus be transformative for biological research in Leicester and the Midlands. This core infrastructure will provide unprecedented teaching and training opportunities for undergraduate and postgraduate students.

Importantly BioEM will be transformative for our interdisciplinary academic and industrial research in microbial and human cell biology underpinning health and disease, antimicrobial resistance, food safety and sustainability, and research in environmental biology, geology and archaeology that together will inform on current food security and human and animal health issues.

Technical Summary

The FESEM will deliver high-resolution data for a range of applications. The electron beam generated by field emission gun technology delivers sub-nanometre resolution for imaging fine topographical detail at large depths of field. Because dried biological samples and plastic resin embedded materials are insulating in nature, they are particularly susceptible to charging which affects image quality. The FESEM will be configured with features including variable pressure mode, tandem deceleration stage and annular backscatter electron detectors (aBSD), that work at lower kV and high pressure settings to optimise secondary and backscattered electron detection from low yield biological samples reducing imaging artefacts.

A light element Energy Dispersive Spectroscopy (EDS) detector designed for elemental composition typical in biological samples will enable research in topics including elemental changes in bacteria, determining composition of pollution particulates, and analysis of nanomaterials. It also brings the option for researchers to explore alternative contrast methods, locate specific cellular features, or alternative approaches to segmentation in 3D EM. An airlock will be installed to protect the detectors from contamination and produce a cleaner environment for EDS analysis.

High-end image acquisition and data processing software will allow researchers to perform large spatial mapping of structures and use automated area selection and image acquisition features to collect serial resin section datasets for 3D EM and correlative workflows. An image analysis station with specialist software will provide a dedicated space for segmentation and 3D reconstruction of data. The option of basic array tomography, as opposed to the more bespoke choice of serial block face SEM, will allow exploration of 3D EM and Correlative Light and Electron Microscopy techniques while maintaining accessibility, which is vital for a multiuser, multi-methodology facility.

Publications

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