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Download Food Science Seminar, 9-4-08 - Center for Advanced Ultrastructural
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Center Instrumentation www.uga.edu/caur/facility.htm Zeiss 1450EP Environmental SEM Peltier Stage (+50 to -25 C) EDX LEO 982 Field emission SEM Cryostage and prep chamber EDX Nabity E-beam lithography Skyscan Micro CT tomographic x-ray Center Instrumentation JEOL 100CX TEM Biological imaging FEI Tecnai20 analytical TEM Cryostage and prep station Heater stage EDX STEM Leica SP2 spectral scanning laser confocal Upright platform Leica SP5 live cell scanning laser confocal Two MP lasers attached Inverted platform Light Microscopy Suite UGA Student Technology Fee Leica inverted compound scope Leica upright compound scope with DIC and polarizing filters Leica dissecting scope Scale of Imaging TEM 0.25 um Confocal 40 um SEM 2 um Light 100 um Transmission Electron Microscopy Technai 20 200 KeV 1.4 Å Standard Preparation Tissue TEM Chem. Fixation SEM Cryo Fixation Chem. Fixation Cryo Fixation Rinse/store Substitution Rinse/store En bloc staining CryoDehydrationDehydration Dehydration sectioning Drying Resin Mounting infiltration Sectioning Post staining Coating Scanning Electron Microscopy Lenses and detectors SEM Setup Electron/Specimen Interactions When the electron beam strikes a sample, both photon and electron signals are emitted. X-rays Through thickness composition info Incident Beam Primary backscattered electrons Atomic number and topographical Cathodoluminescence Electrical Secondary electrons Topographical Auger electrons Surface sensitive compositional Specimen Specimen Current Electrical Specimen/Beam Interactions Monte Carlo simulation Beam Penetration Z represents molecular composition of material E represents energy of incident electron beam 3.0 KeV 20.0 KeV Effects of Accelerating Voltage Backscatter electron detector Conventional SEM Specimen at high vacuum – requires sample fixation and dehydration or freezing. Charging is minimized by coating sample with metal or carbon or lowering the operating kV. SEM Cryo-preservation Preserves sample in hydrated state Maintains structural integrity Ice crystal formation can be avoided Sublimation used to remove excess water Plunge Freeze and SEM Cryostage Specimen holder and transfer rod Nitrogen slushing and plunge station Leidenfrost effect Ice crystal formation Effects of Etching Cryofixed Feta Cryofixed Yogurt Both images courtesy Dr. Ashraf Hassan Correlation - Light Micrographs and CryoSEM CW S Whole Peanut P Peanut Butter Images courtesy Eyassu Abegaz Rice Uncooked Cooked Courtesy Aswin Amornsin Variable Pressure Scanning Electron Microscope - Vacuum in the sample chamber can range from high vacuum (< 10-6 Pascals) up to 3,000 Pa. - Gas in the sample chamber allows uncoated and unfixed samples to be imaged. -Detectors used at higher pressures are backscatter or special secondary detectors. - Moisture on the sample can be controlled by cooling/heating stage and water injection system. Variable Pressure SEM Variable pressure SEM – High Vacuum Mode VP SEM - Low Vacuum Mode Incident Electron Beam Zeiss VPSE Detector Principle VPSE Detector, Light Pipe and PMT. Photons BSE’s Light Pipe Photons are detected and amplified to provide the final image. Specimen Signal Detection with Variable Pressure Mode Peltier stage Heats to 50 C Cools to - 25 C SEM Control Interface Control water vapor and temperature Applications Live centipede Bacteria and biofilm on rock Kamchatka samples - Paul Schroeder Live Drosophila larva Pattern produced in silica gel Skyscan 1072 Micro-CT X-Ray Tomography Scanner MicroCT X-ray imaging that reconstructs images to form cross-sections and volumetric information. Resolution to 5 mm, 3D reconstruction, density measurements. Any sample works having differential density within sample (e.g. bone vs. tissue, or addition of x-ray contrast agents) Applications – Bone, insects, food science, material science, substrate/cell distribution. http://www.phoenix-xray.com Object is rotated 180 degrees. Images captured at one degree increments. Reconstructions done on aligned images to create volume data. Oak Ridge Natl Lab Confocal Scanning Laser Microscope -Confocal - Mutiphoton Sample Imaged by: - Fluorescent dyes - Autofluorescent compounds - Expressed fluorescent proteins (e.g. GFP) - Reflective surfaces Confocal Principle Laser Excitation Pinhole Excitation Filter PMT Objective Emission Filter Emission Pinhole Optical Sectioning with Confocal Laser Comparison with Flattened Cells Epifluorescence Confocal Thick Biofilms Fluorescence Confocal Change in structure over time Yogurt Images courtesy Dr. Ashraf Hassan Alternate Views from Z-Stack Reconstruction Reflectance mode - Yogurt Courtesy Dr. Ashraf Hassan Spatial information using stereo projections Coral zooxanthellae EPS on E. coli Labeling Cells Bacterial colonization on metal Reflectance metal Labelled bacteria Combined Multi-photon Excitation Single Photon Excitation Multi-Photon Excitation Depth penetration between multi-photon and confocal Multi-photon Confocal 3 microns 31 microns 55 microns Microtubule distribution in plant cells Micrograph courtesy David Burk Center for Ultrastructural Research (EM Lab) www.uga.edu/caur/ [email protected] 706-542-4080 Paul Schroeder, Geology John Shields, Cell Biology Jianguo Fan, Physics/Geology Sara Karlsson, Office manager
