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The Atomic Force Microscope can resolve structures less than a nanometer across, and so it is used for single particle analysis and 3D reconstructions. One application of the instrument is to image DNA molecules incubated with protein, in order to clarify the DNA binding properties of candidate nucleoid proteins. Another application is to image the regular arrays of membrane proteins in 2D crystals.
Various human cell lines are cultured in the Unit and are used for determining a range of aspects of mitochondrial biogenesis and function. Imaging the distribution of proteins is a key method for establishing the localisation of proteins and small molecules to mitochondria. It also allows us to study changes, induced by increased or decreased gene expression, either to structures, such as nucleoids, inside the mitochondrion, or to the structure of the mitochondrion itself. The Unit’s Zeiss LSM 510 Meta confocal microscope is capable of producing high quality 2D and 3D images, the latter via the stacking of multiple 2D images (z-stacks). Software is available to quantify the abundance and distribution of organelles or proteins.
An electron microscopy suite with a Phillips Tecnai 12 instrument allows us to screen and analyse 2D crystals and single particles. For high-resolution electron cryo-microscopy we have access to a Tecnai F20 200 kV Field Emission Gun microscope, at the MRC Laboratory of Molecular Biology. Regular arrays of membrane proteins in 2D crystals are identified by optical diffraction. We have a Vitrobot automated system for rapid cryofixation of samples.