Center Instrumentation 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

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

Transmission Electron Microscopy Technai KeV 1.4 Å

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

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. 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

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

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

Ice crystal formation Leidenfrost effect

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

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

Uncooked Rice 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

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

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  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.

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

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

Optical Sectioning with Confocal Laser

EpifluorescenceConfocal Comparison with Flattened Cells

FluorescenceConfocal Thick Biofilms

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

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

Coral zooxanthellae Spatial information using stereo projections

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

Single Photon Excitation Multi-Photon Excitation Multi-photon Excitation

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

Microtubule distribution in plant cells Micrograph courtesy David Burk

Center for Ultrastructural Research (EM Lab) Paul Schroeder, Geology John Shields, Cell Biology Jianguo Fan, Physics/Geology Sara Karlsson, Office manager