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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.

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Presentation on theme: "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."— Presentation transcript:

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2 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

3 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

4 Light Microscopy Suite UGA Student Technology Fee Leica inverted compound scope Leica upright compound scope with DIC and polarizing filters Leica dissecting scope

5 TEMSEM Confocal Light 2 um 40 um 0.25 um 100 um Scale of Imaging

6 Transmission Electron Microscopy Technai 20 200 KeV 1.4 Å

7 TEM SEM Tissue Standard Preparation Chem. Fixation Cryo Fixation Chem. Fixation Cryo Fixation Rinse/store En bloc staining Substitution Cryo- sectioning Dehydration Resin infiltration Sectioning Post staining Drying Mounting Coating

8 Scanning Electron Microscopy

9 Lenses and detectors

10 SEM Setup Electron/Specimen Interactions When the electron beam strikes a sample, both photon and electron signals are emitted. Incident Beam Specimen X-rays Through thickness composition info Auger electrons Surface sensitive compositional Primary backscattered electrons Atomic number and topographical Cathodoluminescence Electrical Secondary electrons Topographical Specimen Current Electrical

11 Specimen/Beam Interactions Monte Carlo simulation

12 Beam Penetration Z represents molecular composition of material E represents energy of incident electron beam

13 3.0 KeV 20.0 KeV Effects of Accelerating Voltage

14 Backscatter electron detector

15 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.

16 SEM Cryo-preservation Preserves sample in hydrated state Maintains structural integrity Ice crystal formation can be avoided Sublimation used to remove excess water

17 Specimen holder and transfer rod Nitrogen slushing and plunge station Plunge Freeze and SEM Cryostage

18 Ice crystal formation Leidenfrost effect

19 Cryofixed Yogurt Both images courtesy Dr. Ashraf Hassan Cryofixed Feta Effects of Etching

20 Correlation - Light Micrographs and CryoSEM Whole PeanutPeanut Butter Images courtesy Eyassu Abegaz P CW S

21 Uncooked Rice Cooked Courtesy Aswin Amornsin

22 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.

23 Variable Pressure SEM

24 Variable pressure SEM – High Vacuum Mode

25 VP SEM - Low Vacuum Mode

26 Zeiss VPSE Detector Principle Incident Electron Beam Specimen BSE’s Photons are detected and amplified to provide the final image. Photons VPSE Detector, Light Pipe and PMT. Light Pipe

27 Signal Detection with Variable Pressure Mode

28 Peltier stage Heats to 50 C Cools to - 25 C

29 SEM Control Interface

30 Control water vapor and temperature

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32 Applications Live centipede Bacteria and biofilm on rock Kamchatka samples - Paul Schroeder Live Drosophila larva

33 Pattern produced in silica gel

34 Skyscan 1072 Micro-CT X-Ray Tomography Scanner

35 MicroCT X-ray imaging that reconstructs images to form cross-sections and volumetric information. Resolution to 5  m, 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.

36 Object is rotated 180 degrees. Images captured at one degree increments. Reconstructions done on aligned images to create volume data. http://www.phoenix-xray.com

37 Oak Ridge Natl Lab

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39 - -Confocal - Mutiphoton Sample Imaged by: - Fluorescent dyes - Autofluorescent compounds - Expressed fluorescent proteins (e.g. GFP) - Reflective surfaces Confocal Scanning Laser Microscope

40 Confocal Principle Objective Laser Emission Pinhole Excitation Pinhole PMT Emission Filter Excitation Filter

41 Optical Sectioning with Confocal Laser

42 EpifluorescenceConfocal Comparison with Flattened Cells

43 FluorescenceConfocal Thick Biofilms

44 Change in structure over time Images courtesy Dr. Ashraf Hassan Yogurt

45 Alternate Views from Z-Stack Reconstruction Reflectance mode - Yogurt Courtesy Dr. Ashraf Hassan

46 Coral zooxanthellae Spatial information using stereo projections

47 Labeling Cells EPS on E. coli Bacterial colonization on metal Reflectance metalLabelled bacteriaCombined

48 Single Photon Excitation Multi-Photon Excitation Multi-photon Excitation

49 ConfocalMulti-photon 3 microns31 microns55 microns Depth penetration between multi-photon and confocal

50 Microtubule distribution in plant cells Micrograph courtesy David Burk

51 Center for Ultrastructural Research (EM Lab) www.uga.edu/caur/ caur@uga.educaur@uga.edu706-542-4080 Paul Schroeder, Geology John Shields, Cell Biology Jianguo Fan, Physics/Geology Sara Karlsson, Office manager

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