A plasma focused ion beam microscope for Structural Cell Biology at the Astbury Biostructure Laboratory
Lead Research Organisation:
University of Leeds
Department Name: Astbury Centre
Abstract
Structural biology is an enormously powerful tool in biological discovery. It can reveal the molecular details of how proteins and other molecules interact, and so help us understand fundamental biological processes that have enormous implications in areas as diverse as human/animal health and wellbeing, food security and biotechnology. One of the cornerstones of modern structural biology is a technique called cryo-electron microscopy, where powerful microscopes are used to take highly magnified images of biological material such as proteins, from which we can work out the three-dimensional structure of the protein complexes. CryoEM (imaging frozen samples) can now address structural problems of unprecedented scale and complexity, and the results are transforming our understanding of biology.
Since the state of the art Titan Krios microscopes were installed in the Astbury Biostructure Laboratory (2016), we have become very adept at solving these type of structures, and our community, both in Leeds and beyond, has solved 100's of high-resolution structures. This approach has been enormously successful and impactful, and while these structures are immensely informative in their own right, it is becoming increasingly clear that the true power of structural biology will only be unlocked when we are able to understand these structures in their native contexts, within cells and tissues.
This application is to bring a new capability to ABSL by purchasing a new instrument called a plasma focused ion beam (pFIB) scanning EM (SEM). This machine will additionally have a fluorescence microscope integrated into the pFIB. A pFIB will help realise the potential of in situ structural biology workflows by (a) identifying areas of interest in cells and tissues using fluorescent markers that we can introduce, (b) milling lamellae (very thin sections) that encompass these fluorescent markers, both in cells, and in tissue, for cryoEM studies, and (c) undertake a different technique called block face milling of plant/animal tissues. With our existing state-of-the-art Titan Krios EMs at ABSL, this will allow us to understand biology across lengths scales from millimetres to nanometers, transforming structural discovery at the Astbury Biostructure Laboratory, and providing a vital resource for UK bioscience.
Since the state of the art Titan Krios microscopes were installed in the Astbury Biostructure Laboratory (2016), we have become very adept at solving these type of structures, and our community, both in Leeds and beyond, has solved 100's of high-resolution structures. This approach has been enormously successful and impactful, and while these structures are immensely informative in their own right, it is becoming increasingly clear that the true power of structural biology will only be unlocked when we are able to understand these structures in their native contexts, within cells and tissues.
This application is to bring a new capability to ABSL by purchasing a new instrument called a plasma focused ion beam (pFIB) scanning EM (SEM). This machine will additionally have a fluorescence microscope integrated into the pFIB. A pFIB will help realise the potential of in situ structural biology workflows by (a) identifying areas of interest in cells and tissues using fluorescent markers that we can introduce, (b) milling lamellae (very thin sections) that encompass these fluorescent markers, both in cells, and in tissue, for cryoEM studies, and (c) undertake a different technique called block face milling of plant/animal tissues. With our existing state-of-the-art Titan Krios EMs at ABSL, this will allow us to understand biology across lengths scales from millimetres to nanometers, transforming structural discovery at the Astbury Biostructure Laboratory, and providing a vital resource for UK bioscience.
Technical Summary
Structural biology is a uniquely powerful tool in modern biological research, and cryo-electron microscopy and cryo-electron tomography are now two of its cornerstones. CryoEM/ET can address structural problems of unprecedented scale and complexity, and the results are transforming our understanding of biology. The Astbury Biostructure Laboratory (ABSL) at the University of Leeds is an internationally-recognised cryoEM/ET facility that supports large numbers of scientists from Universities and Industry, and from the UK and beyond. We currently operate two Titan Krios EMs, recently upgraded to F4i and F4i/selectris detectors (Q2/2022).
This application is to bring a new capability to ABSL by purchasing a plasma focused ion beam (pFIB) scanning EM with an integrated fluorescence microscope (FM) and cryo lift out capability. A pFIB will help realise the potential of in situ structural biology workflows, allowing us to understand atomic resolution structures from cryoEM in the cell/tissue context delivered by FM and cryoET. Specifically, we will be able to a) identify areas of interest in cells and tissues using fluorescent markers that we can introduce, (b) mill lamellae that encompass these fluorescent foci, both in cells, and in tissue, for cryoET studies; (c) for tissues, mill bespoke geometries in tissue and lift out lamella for polishing and cryoET studies; and (d) undertake a different technique called block face milling of plant/animal tissues, delivering volume EM reconstructions on frozen and ambient tissue.
With our existing state-of-the-art Titan Krios EMs at ABSL, this will allow us to understand biology across lengths scales from millimetres to Angstroms , transforming structural discovery at the Astbury Biostructure Laboratory, and providing a vital resource for UK bioscience.
This application is to bring a new capability to ABSL by purchasing a plasma focused ion beam (pFIB) scanning EM with an integrated fluorescence microscope (FM) and cryo lift out capability. A pFIB will help realise the potential of in situ structural biology workflows, allowing us to understand atomic resolution structures from cryoEM in the cell/tissue context delivered by FM and cryoET. Specifically, we will be able to a) identify areas of interest in cells and tissues using fluorescent markers that we can introduce, (b) mill lamellae that encompass these fluorescent foci, both in cells, and in tissue, for cryoET studies; (c) for tissues, mill bespoke geometries in tissue and lift out lamella for polishing and cryoET studies; and (d) undertake a different technique called block face milling of plant/animal tissues, delivering volume EM reconstructions on frozen and ambient tissue.
With our existing state-of-the-art Titan Krios EMs at ABSL, this will allow us to understand biology across lengths scales from millimetres to Angstroms , transforming structural discovery at the Astbury Biostructure Laboratory, and providing a vital resource for UK bioscience.
