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Cryo soft X-ray tomography (cryoSXT) is a technique that bridges the gap between light microscopy and electron microscopy. Whole cells are frozen in a near-native state; meaning without dehydration, fixation or staining. They are then imaged using soft X-rays in the “water window”, the photon energy range where biologically relevant elements such as carbon and nitrogen absorb X-rays ten-times better than the surrounding water. These conditions allow for imaging through ~10 microns in depth, with ~40 nm resolution, providing whole cell context and the ability to visualise cellular compartments, organelles and some cytoskeletal elements.
HEK293 cells, a type of mammalian cell culture line, will be grown on grids, treated with fluorescent organelle specific dyes, such as MitoTracker, and plunge frozen. Next, the student will image these cells in both cryo-fluorescent light microscopy and cryoSXT. The student will then process these datasets into three-dimensional volumes. Next, the student will correlate the fluorescence and X-ray data to identify the organelles of interest (mitochondria, lysosomes, endosomes). The student will then segregate the organelles of interest and measure their characteristics throughout the three-dimensional datasets.
Characteristics will include size, shape, volume, location, intensity of pixel values, etc. Additional characteristics will be developed as needed to accurately identify each organelle of interest. Statistical measures will be applied to ensure high accuracy and unambiguous identification of organelles. If possible, time will be spent creating figures and movies to explain the process and findings of this study.
Suitable Subjects: Biology, Other Biochemistry, Molecular Biology, Cell Biology
Project duration: 12 weeks
TO APPLY PLEASE FOLLOW THE LINK BELOW
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