Cryo focussed ion beam scanning electron microscope for correlative functional studies of biological systems in situ by tomography (360G-Wellcome-212916_Z_18_Z)

£820,000

Dramatic advances in cryo-electron microscopy (EM) have greatly enhanced macromolecular structure determination. The current frontier is in situ cellular structure determination to understand the machinery of life operating in the native biological environment. Visualising the native structure of cells and tissues by cryo-electron tomography requires the preparation of cryo-samples ~100-600 nanometers in thickness. The vitrified tissue sections must be thin enough to allow penetration by the electron beam, yet thick enough to contain complete structures of biological relevance. Although cells and tissues can be sectioned in the vitreous state by cryo-ultramicrotomy, both section thickness and quality are severely limited by mechanical damage including deformation, non-uniform compression and crevassing. The only available method able to precisely modify the thickness of frozen-hydrated samples and balance these conflicting requirements is focussed ion beam (FIB) milling in a scanning EM. FIB milling provides access to the cell interior, within otherwise unperturbed cellular environments, yielding vitrified lamellae suitable for electron tomography and unaffected by compression forces. Here we request a cryo FIB field emission gun scanning EM (FEG SEM) to produce lamellae targeted to fluorescent features of interest for transfer into a cryo-transmission EM (TEM) for three-dimensional (3D) structure determination by electron tomography.

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Grant Details

Amount Awarded 820000
Applicant Surname Fleck
Approval Committee Multi-User Equipment and Technology Development Committee
Award Date 2018-07-05T00:00:00+00:00
Financial Year 2017/18
Grant Programme: Title Multi-User Equipment Grant
Internal ID 212916/Z/18/Z
Lead Applicant Prof Roland Fleck
Other Applicant(s) Dr Anthony Roberts, Dr Giulia Zanetti, Prof Brian Stramer, Prof Gabriel Waksman, Prof Helen Saibil, Prof Juan Burrone
Partnership Value 820000
Planned Dates: End Date 2023-09-15T00:00:00+00:00
Planned Dates: Start Date 2018-09-16T00:00:00+00:00
Recipient Org: Country United Kingdom
Region Greater London