Zeiss Gemini SEM 460 with L-shape 90 degree Ion-Sculptor Focused Ion Beam (FIB) column
Lead Research Organisation:
UNIVERSITY COLLEGE LONDON
Department Name: The Wolfson Inst for Biomedical Research
Abstract
Electron microscopes provide an extraordinarily powerful tool for studying biological mechanisms, allowing scientists to reveal key links between structural features and functional processes. Since they utilise electrons rather than light waves to scan tissue, this allows the properties of cells and cellular components to be examined in the nanometre size range.
Recently, a variant of the conventional transmission electron microscope - called a focused ion-beam scanning electron microscope (FIBSEM) - has been developed which allows researchers to gain an extremely accurate three-dimensional nanoscale view of a chosen biological sample. This technological advance works by carrying out repeated rounds of (1) imaging the surface of a tissue block with a scanning electron beam to collect a snapshot view of a thin surface layer, followed by (2) ablating ('milling') this nanometre-thin surface layer with a focused ion beam to expose the tissue layer directly below for the next imaging step. The leading-edge microscopes fully automate this repetitive imaging-and-milling process to collect 3D stacks of images, permitting the generation of ultra-high-resolution representations of the structure of volumes of tissue.
FIBSEM has significantly advanced the kinds of questions that can be addressed in biological and medical research, but has a key constraint: the tissue volumes that can be imaged using conventional commercial FIBSEM systems are still very small. Competing block-face imaging techniques, which use a diamond knife for sectioning, allow larger tissue volumes to be imaged, but do not provide the same resolution of the collected images in all directions, leading to potential interpretation errors in assessing structure. For many applications these limitations are highly problematic - for example, when attempting to map neuronal circuits in the brain - a major current challenge in neuroscience.
Very recently, a new design - referred to as an enhanced FIBSEM (eFIBSEM) - has been developed which fully addresses these limitations. With this microscope it is now possible to collect tissue volumes that are >100x larger than those accessible with earlier FIBSEM designs while maintaining the highest spatial resolution uniformly in all directions ('isotropic').
In this application, we request a novel eFIBSEM platform which will provide access to this ground-breaking technology for a huge number of researchers at University College London (UCL), the Francis Crick Institute and nationwide. There are very few of these microscopes available globally and at present, access typically relies on international collaborations with the Janelia Research Campus in the U.S. where the instrument platform was developed. The current proposal capitalises on key collaborations - by independent research teams both at UCL and the Crick - with these developers. This includes proof-of-concept experiments that demonstrate the viability of using eFIBSEM to measure neuronal connectivity and function in central brain circuits, and to map large areas of brain wiring. This custom-built instrument will thus provide major new insights into how neurons and circuits in the brain process information and drive behaviour, how brain circuits evolve, and how proteins and viruses associated with disease and infection are organised in cellular structures. The availability of this instrument in a state-of-the-art facility will give researchers at UCL, the Crick and other institutions nationally a powerful opportunity to advance their neuroscience and cell biology research.
Recently, a variant of the conventional transmission electron microscope - called a focused ion-beam scanning electron microscope (FIBSEM) - has been developed which allows researchers to gain an extremely accurate three-dimensional nanoscale view of a chosen biological sample. This technological advance works by carrying out repeated rounds of (1) imaging the surface of a tissue block with a scanning electron beam to collect a snapshot view of a thin surface layer, followed by (2) ablating ('milling') this nanometre-thin surface layer with a focused ion beam to expose the tissue layer directly below for the next imaging step. The leading-edge microscopes fully automate this repetitive imaging-and-milling process to collect 3D stacks of images, permitting the generation of ultra-high-resolution representations of the structure of volumes of tissue.
FIBSEM has significantly advanced the kinds of questions that can be addressed in biological and medical research, but has a key constraint: the tissue volumes that can be imaged using conventional commercial FIBSEM systems are still very small. Competing block-face imaging techniques, which use a diamond knife for sectioning, allow larger tissue volumes to be imaged, but do not provide the same resolution of the collected images in all directions, leading to potential interpretation errors in assessing structure. For many applications these limitations are highly problematic - for example, when attempting to map neuronal circuits in the brain - a major current challenge in neuroscience.
Very recently, a new design - referred to as an enhanced FIBSEM (eFIBSEM) - has been developed which fully addresses these limitations. With this microscope it is now possible to collect tissue volumes that are >100x larger than those accessible with earlier FIBSEM designs while maintaining the highest spatial resolution uniformly in all directions ('isotropic').
In this application, we request a novel eFIBSEM platform which will provide access to this ground-breaking technology for a huge number of researchers at University College London (UCL), the Francis Crick Institute and nationwide. There are very few of these microscopes available globally and at present, access typically relies on international collaborations with the Janelia Research Campus in the U.S. where the instrument platform was developed. The current proposal capitalises on key collaborations - by independent research teams both at UCL and the Crick - with these developers. This includes proof-of-concept experiments that demonstrate the viability of using eFIBSEM to measure neuronal connectivity and function in central brain circuits, and to map large areas of brain wiring. This custom-built instrument will thus provide major new insights into how neurons and circuits in the brain process information and drive behaviour, how brain circuits evolve, and how proteins and viruses associated with disease and infection are organised in cellular structures. The availability of this instrument in a state-of-the-art facility will give researchers at UCL, the Crick and other institutions nationally a powerful opportunity to advance their neuroscience and cell biology research.
Technical Summary
We request funding for an eFIBSEM (enhanced focused ion beam scanning electron microscope). Focused ion beam milling combined with scanning electron microscopy (FIBSEM) provides a means to achieve exquisite spatial resolution of samples in 3D, but until recently the size of the volumes that could be imaged using conventional FIBSEMs was severely constrained. A recent breakthrough led by scientists at Janelia Research Campus U.S. has revolutionised current technology by substantially re-engineering a commercially available FIBSEM, yielding a two-orders-of-magnitude increase in imaged volumes.
Specifically, the major modifications in the new design are: (1) mounting of the SEM and FIB at a 90-degree angle, (2) custom hard- and software to enable closed-loop control of the FIB beam, and (3) adding tools that allow seamless imaging over weeks and serve to maximise the reliability of the instrument. Together, these refinements substantially improve the conditions for both SEM imaging and FIB milling, as well as enabling both backscattered and secondary electrons to be used for SEM image formation, yielding a substantial increase in imaging speed while maintaining signal-to-noise ratio. Crucially, in overcoming previous design limitations, the eFIBSEM platform enables high-throughput, isotropic imaging of super-large tissue volumes (100x100x100 um3) at nm resolution, making large-scale, synaptic-resolution connectomes accessible for the first time. We outline a range of applications that will make immediate use of this system, including measuring synaptic weights in the context of structural connectomes of different brain circuits, disentangling the logic of inhibitory circuits in the olfactory bulb, unravelling the evolution of neural circuits and exploring ultrastructural organisation of protein and virion assemblies in disease and infection models. The specific Zeiss instrument has been chosen based on systems pioneered by our collaborators at Janelia.
Specifically, the major modifications in the new design are: (1) mounting of the SEM and FIB at a 90-degree angle, (2) custom hard- and software to enable closed-loop control of the FIB beam, and (3) adding tools that allow seamless imaging over weeks and serve to maximise the reliability of the instrument. Together, these refinements substantially improve the conditions for both SEM imaging and FIB milling, as well as enabling both backscattered and secondary electrons to be used for SEM image formation, yielding a substantial increase in imaging speed while maintaining signal-to-noise ratio. Crucially, in overcoming previous design limitations, the eFIBSEM platform enables high-throughput, isotropic imaging of super-large tissue volumes (100x100x100 um3) at nm resolution, making large-scale, synaptic-resolution connectomes accessible for the first time. We outline a range of applications that will make immediate use of this system, including measuring synaptic weights in the context of structural connectomes of different brain circuits, disentangling the logic of inhibitory circuits in the olfactory bulb, unravelling the evolution of neural circuits and exploring ultrastructural organisation of protein and virion assemblies in disease and infection models. The specific Zeiss instrument has been chosen based on systems pioneered by our collaborators at Janelia.
