Establishing a cryogenic correlative light-electron microscopy hub for Oxford
Lead Research Organisation:
University of Oxford
Department Name: Biochemistry
Abstract
We are asking for equipment which will allow us to see inside cells in unprecedented detail.
Cells are extraordinary, varied and dynamic and they make up all living things. A major focus of biosciences research is to understand the varying structures of cells, allowing us to understand how they are constructed and how they change during fundamental processes, such as replication. We also need to understand how cells interact with each other, for example as the immune system detects a pathogen or as nerve cells connect to create a signalling synapse. To do this, we need to be able to observe the architecture and internal structures of cells, as well as to pin-point the locations of important molecules, and how their positions alter as cells change and interact.
Electron microscopes can be used to provide highly detailed views of cells and biological material and are much more precise than light microscopes. However, this brings challenges. The inside of an electron microscope is in a vacuum, in which living organisms cannot survive. It is therefore necessary to prepare a biological sample carefully before it can be studied in this way. The best solution is to freeze the sample and to maintain it in very cold conditions throughout imaging. This is called cryogenic electron microscopy, or cryo-EM. A second challenge is that cells are too thick to study in an electron microscope as the electrons cannot pass through a cell. To solve this, we make thin layers, which cut through frozen cells. These lamella are thin enough to image. Finally, cells are large and complex and the images taken on electron microscopes are therefore crowded. It can be hard to find what we want to look at. To solve this, we can use a technique called correlative light-electron microscopy (cryo-CLEM). Here, the things which we want to study are labelled using a fluorescent marker and we can observe the cells using a fluorescence microscope to see where the marker is. We can then make thin layers of the cell, focusing in on the region with the fluorescence signal and can image them in an electron microscope. By correlating the images from the fluorescence and electron microscope we can get much more information, combining the higher resolution of the electron microscopy with the targeted detected capability which comes from fluorescence labelling. Within our cryo-EM facility, we already have the equipment required to solve the first two of these challenges and we are asking for the microscope and associated equipment to allow us to conduct fluorescence microscopy under cryogenic conditions.
This equipment will be used by many researchers from across Oxford, to answer all kinds of questions about biology. They will image the sites where neurons contact each other and see how their molecules arrange as the cells are trying to find their way to make the right contacts. They will image the genomes of bacteria and see how they change when the bacteria are exposed to antibiotics. They will see how cells move their chromosomes around in processes which go wrong in cancer. They will understand how the compartments within cells contact and communicate with one another and they will observe what happens when parasites contact human cells and when immune cells contact pathogens. The capability provided by cryo-CLEM will allow us to see inside cells in a new way, to discover how they drive these, and many more, processes needed for life.
Cells are extraordinary, varied and dynamic and they make up all living things. A major focus of biosciences research is to understand the varying structures of cells, allowing us to understand how they are constructed and how they change during fundamental processes, such as replication. We also need to understand how cells interact with each other, for example as the immune system detects a pathogen or as nerve cells connect to create a signalling synapse. To do this, we need to be able to observe the architecture and internal structures of cells, as well as to pin-point the locations of important molecules, and how their positions alter as cells change and interact.
Electron microscopes can be used to provide highly detailed views of cells and biological material and are much more precise than light microscopes. However, this brings challenges. The inside of an electron microscope is in a vacuum, in which living organisms cannot survive. It is therefore necessary to prepare a biological sample carefully before it can be studied in this way. The best solution is to freeze the sample and to maintain it in very cold conditions throughout imaging. This is called cryogenic electron microscopy, or cryo-EM. A second challenge is that cells are too thick to study in an electron microscope as the electrons cannot pass through a cell. To solve this, we make thin layers, which cut through frozen cells. These lamella are thin enough to image. Finally, cells are large and complex and the images taken on electron microscopes are therefore crowded. It can be hard to find what we want to look at. To solve this, we can use a technique called correlative light-electron microscopy (cryo-CLEM). Here, the things which we want to study are labelled using a fluorescent marker and we can observe the cells using a fluorescence microscope to see where the marker is. We can then make thin layers of the cell, focusing in on the region with the fluorescence signal and can image them in an electron microscope. By correlating the images from the fluorescence and electron microscope we can get much more information, combining the higher resolution of the electron microscopy with the targeted detected capability which comes from fluorescence labelling. Within our cryo-EM facility, we already have the equipment required to solve the first two of these challenges and we are asking for the microscope and associated equipment to allow us to conduct fluorescence microscopy under cryogenic conditions.
This equipment will be used by many researchers from across Oxford, to answer all kinds of questions about biology. They will image the sites where neurons contact each other and see how their molecules arrange as the cells are trying to find their way to make the right contacts. They will image the genomes of bacteria and see how they change when the bacteria are exposed to antibiotics. They will see how cells move their chromosomes around in processes which go wrong in cancer. They will understand how the compartments within cells contact and communicate with one another and they will observe what happens when parasites contact human cells and when immune cells contact pathogens. The capability provided by cryo-CLEM will allow us to see inside cells in a new way, to discover how they drive these, and many more, processes needed for life.
Technical Summary
We seek to establish a platform for cryogenic-correlative light-electron microscopy (cryo-CLEM) for the central Oxford cryo-electron microscopy hub.
We already have access to three transmission electron microscopes, including a Titan Krios with a K3 detector and Bioquantum energy filter and a Talos Arctica with a falcon 4 detector. These have been used successfully to collect tomograms. We also have a Zeiss Crossbeam FIB-SEM, allowing sample thinning and lamella production. This is supported by a preparation room containing plunge freezing devices, incubators and microscopes to allow preparation of cellular samples. A computational cluster is available and has tomography software installed.
To enable cryo-CLEM, we now seek:
* A cryo-fluorescence microscope with cryo-stage and cryo-objective lens
* A laser system to upgrade this microscope to allow cryo-SOFI fluctuation microscopy. This will increase the resolution approximately three-fold, reaching state-of-the-art and allowing many more biological applications.
* A laser system to allow brief melting and refreezing, allowing the study of dynamics. For example, samples can be frozen with caged compounds which are activated by a microsecond pulse during laser melting.
* A micropatterning system which can be used to precisely place cells onto electron microscopy grids. This will ensure that cells are in suitable places on grids to allow FIB-milling and tomogram collection, as well as facilitating cell-cell contacts.
This equipment will be combined with our existing infrastructure to form a cryo-CLEM hub which is fully accessible to the Oxford research community. It will allow a range of experiments which use fluorescence to identify rare structures or events to guide FIB-milling, or which require correlation of fluorescence and electron microscopy images to confirm the presence of labelled structures within tomograms. This will allow a broad range of questions to be answered in fundamental bioscience
We already have access to three transmission electron microscopes, including a Titan Krios with a K3 detector and Bioquantum energy filter and a Talos Arctica with a falcon 4 detector. These have been used successfully to collect tomograms. We also have a Zeiss Crossbeam FIB-SEM, allowing sample thinning and lamella production. This is supported by a preparation room containing plunge freezing devices, incubators and microscopes to allow preparation of cellular samples. A computational cluster is available and has tomography software installed.
To enable cryo-CLEM, we now seek:
* A cryo-fluorescence microscope with cryo-stage and cryo-objective lens
* A laser system to upgrade this microscope to allow cryo-SOFI fluctuation microscopy. This will increase the resolution approximately three-fold, reaching state-of-the-art and allowing many more biological applications.
* A laser system to allow brief melting and refreezing, allowing the study of dynamics. For example, samples can be frozen with caged compounds which are activated by a microsecond pulse during laser melting.
* A micropatterning system which can be used to precisely place cells onto electron microscopy grids. This will ensure that cells are in suitable places on grids to allow FIB-milling and tomogram collection, as well as facilitating cell-cell contacts.
This equipment will be combined with our existing infrastructure to form a cryo-CLEM hub which is fully accessible to the Oxford research community. It will allow a range of experiments which use fluorescence to identify rare structures or events to guide FIB-milling, or which require correlation of fluorescence and electron microscopy images to confirm the presence of labelled structures within tomograms. This will allow a broad range of questions to be answered in fundamental bioscience
