Development of a novel workflow for the efficient volume imaging of thick tissue samples with correlative light and electron microscopy
Lead Research Organisation:
UK Health Security Agency
Department Name: Science
Abstract
ConnectomX proposes to join an existing relationship between the UK Health Security Agency (UKHSA) and the Central Laser Facility (CLF) to collaboratively develop a novel correlative light and electron microscopy (CLEM) workflow. This workflow will be applied to a UKHSA project imaging brain tissue for the presence of environmental pollutant particles. Together, the workflow will be tested, and proof of concept data will be collected to prove the utility of the Katana ultramicrotome as a universal solution to volume CLEM of tissue samples. The ConnectomX Katana ultramicrotome will be installed onto an existing scanning electron microscope (SEM) at the CLF. This will allow versatile switching between serial block face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM). ConnectomX will produce software and hardware solutions as part of an ongoing and iterative research and development collaboration to improve and build on the CLEM workflow pioneered in this proposal.
More specifically, the proposed workflow is to image entire brain tissue sections using multiphoton microscopy (MPM) at low resolution to identify smaller regions of interest (ROIs) impacted by nanoparticle infiltration. These ROIs will be identified related to blood brain barrier (BBB) permeability and will later be relocated in the SEM for high-resolution imaging. The ConnectomX Katana ultramicrotome can be mounted within the chamber of the SEM at the CLF to perform serial SBF-SEM on selected brain tissue sections containing ROIs identified in MPM. SBF-SEM utilises a diamond knife to cut away a thin layer of the sample, after which the surface is imaged. This process is repeated thousands of times to build up 3D visualisations of the structure of a sample. In this proposal, the Katana ultramicrotome will be used to acquire a low-resolution volume dataset within each brain tissue sample. Blood vessels or branding marks (made using MPM) in these low-resolution datasets will be used to relocate smaller ROIs with BBB permeability. Incorporation of the Katana into the CLEM workflow would ensure the imaging is performed in a more efficient and robust way by significantly reducing experiment time and increasing sample throughput. In addition, SBF-SEM data collected during the proposed study will be used to facilitate development of an automated ROI recognition software, which can be used to stop cutting once the ROI has been located. This can significantly reduce operator time at the SEM as well as reduce unnecessary data collection. For each sample, once the final ROI has been located using SBF-SEM, the Katana ultramicrotome will be unmounted from the SEM and FIB-SEM will be used to explore the nanoparticles on a subcellular level.
Data collected from this project will contribute towards the development of modified hardware and workflows that allow for FIB-SEM data to be acquired with the Katana still on the sample stage in the microscope. In-situ SBF-SEM and FIB-SEM will lead the way to automating CLEM from the electron microscopy point of view. This will truly push forward the boundaries of bioimaging at the nanoscale which is vital to fully understand how organelles, cells, and tissues function.
More specifically, the proposed workflow is to image entire brain tissue sections using multiphoton microscopy (MPM) at low resolution to identify smaller regions of interest (ROIs) impacted by nanoparticle infiltration. These ROIs will be identified related to blood brain barrier (BBB) permeability and will later be relocated in the SEM for high-resolution imaging. The ConnectomX Katana ultramicrotome can be mounted within the chamber of the SEM at the CLF to perform serial SBF-SEM on selected brain tissue sections containing ROIs identified in MPM. SBF-SEM utilises a diamond knife to cut away a thin layer of the sample, after which the surface is imaged. This process is repeated thousands of times to build up 3D visualisations of the structure of a sample. In this proposal, the Katana ultramicrotome will be used to acquire a low-resolution volume dataset within each brain tissue sample. Blood vessels or branding marks (made using MPM) in these low-resolution datasets will be used to relocate smaller ROIs with BBB permeability. Incorporation of the Katana into the CLEM workflow would ensure the imaging is performed in a more efficient and robust way by significantly reducing experiment time and increasing sample throughput. In addition, SBF-SEM data collected during the proposed study will be used to facilitate development of an automated ROI recognition software, which can be used to stop cutting once the ROI has been located. This can significantly reduce operator time at the SEM as well as reduce unnecessary data collection. For each sample, once the final ROI has been located using SBF-SEM, the Katana ultramicrotome will be unmounted from the SEM and FIB-SEM will be used to explore the nanoparticles on a subcellular level.
Data collected from this project will contribute towards the development of modified hardware and workflows that allow for FIB-SEM data to be acquired with the Katana still on the sample stage in the microscope. In-situ SBF-SEM and FIB-SEM will lead the way to automating CLEM from the electron microscopy point of view. This will truly push forward the boundaries of bioimaging at the nanoscale which is vital to fully understand how organelles, cells, and tissues function.
