Center Instrumentation uga/caur/facility.htm

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. Scale of Imaging TEM SEM 2 um 0.25 um Light Confocal 100 um 40 um

6. Transmission Electron Microscopy Technai 20 200 KeV 1.4 Å

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

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 Primary backscattered electrons X-rays Through thickness composition info Atomic number and topographical Cathodoluminescence Electrical Secondary electrons Auger electrons Surface sensitive compositional Topographical Specimen 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 KeVEffects 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. Plunge Freeze and SEM Cryostage Specimen holder and transfer rod Nitrogen slushing and plunge station

18. Leidenfrost effect Ice crystal formation

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

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

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

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 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.

36. 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.

37. Oak Ridge Natl Lab

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

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

41. Optical Sectioning with Confocal Laser

42. Comparison with Flattened Cells Epifluorescence Confocal

43. Thick Biofilms Fluorescence Confocal

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

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

46. Spatial information using stereo projections Coral zooxanthellae

47. EPS on E. coli Labeling Cells Bacterial colonization on metal Reflectance metal Labelled bacteria Combined

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

49. Depth penetration between multi-photon and confocal Multi-photon Confocal 3 microns 31 microns 55 microns

50. Microtubule distribution in plant cells Micrograph courtesy David Burk