Center Instrumentationwww.uga.edu/caur/facility.htm

Zeiss 1450EP Environmental SEMPeltier Stage (+50 to -25 C)EDX

LEO 982 Field emission SEMCryostage and prep chamberEDXNabity E-beam lithography

Skyscan Micro CT tomographic x-ray

Center InstrumentationJEOL 100CX TEM

Biological imaging

FEI Tecnai20 analytical TEMCryostage and prep stationHeater stageEDXSTEM

Leica SP2 spectral scanning laser confocalUpright platform

Leica SP5 live cell scanning laser confocalTwo MP lasers attachedInverted platform

Light Microscopy SuiteUGA Student Technology Fee

Leica inverted compound scope

Leica upright compound scope with DIC and polarizing filters Leica dissecting

scope

TEM SEM

Confocal Light

2 um

40 um

0.25 um

100 um

Scale of Imaging

Transmission Electron Microscopy

Technai 20200 KeV 1.4 Å

TEM SEM

TissueStandard Preparation

Chem.Fixation

CryoFixation

Chem.Fixation

CryoFixation

Rinse/storeRinse/storeEn bloc

staining

Substitution

Cryo-sectioningDehydration

DehydrationDehydration

Resin infiltration

SectioningPost staining

Drying

Mounting

Coating

Scanning Electron MicroscopyScanning Electron Microscopy

Lenses and detectors

SEM SetupElectron/Specimen Interactions

When the electron beam strikes a sample, both photon and electron signals are emitted.

Incident Beam

Specimen

X-raysThrough thickness composition info

Auger electronsSurface sensitive compositional

Primary backscattered electronsAtomic number and topographical

Cathodoluminescence

Electrical

Secondary electrons

Topographical

Specimen CurrentElectrical

Specimen/Beam Interactions

Monte Carlo simulation

Beam PenetrationZZ represents represents molecular molecular composition of composition of materialmaterial

EE represents represents energy of incident energy of incident electron beamelectron 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 crystalformation

Leidenfrost effect

Cryofixed YogurtBoth images courtesy Dr. Ashraf Hassan

Cryofixed Feta

Effects of Etching

Correlation - Light Micrographs and CryoSEM

Whole Peanut Peanut ButterImages courtesy Eyassu Abegaz

P

CW

SS

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 Zeiss VPSE Detector PrinciplePrinciple

Incident Electron BeamIncident Electron Beam

SpecimenSpecimen

BSE’sBSE’s

Photons are detected Photons are detected and amplified to and amplified to provide the final image.provide the final image.

PhotonsPhotons

VPSE Detector, Light Pipe and VPSE Detector, Light Pipe and PMT.PMT.

Light Pipe

Signal Detection with Variable Pressure Mode

Peltier stage

Heats to 50 CCools to - 25 C

SEM Control Interface

Control water vapor and temperature

Applications

Live centipede

Bacteria and biofilm on rockKamchatka samples - Paul Schroeder

Live Drosophila larva

Pattern produced in silica gel

Skyscan 1072Micro-CT

X-RayTomographyScanner

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.

http://www.phoenix-xray.com

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 PrincipleConfocal Principle

Objective

Laser

Emission Pinhole

Excitation Pinhole

PMT

EmissionFilter

Excitation Filter

Optical Sectioning with Confocal Laser

Epifluorescence Confocal

Comparison with Flattened Cells

Fluorescence Confocal

Thick Biofilms

Change in structure over time

Images courtesyDr. Ashraf Hassan

Yogurt

Alternate Views from Z-Stack Reconstruction

Reflectance mode - YogurtCourtesy Dr. Ashraf Hassan

Coral zooxanthellae

Spatial information using stereo projections

Labeling Cells EPS on E. coli

Bacterial colonization on metal

Reflectance metal Labelled bacteria Combined

Single Photon Excitation

Multi-PhotonExcitation

Multi-photon Excitation

ConfocalMulti-photon

3 microns 31 microns 55 microns

Depth penetration betweenmulti-photon and confocal

Microtubule distribution in plant cellsMicrograph courtesy David Burk

Center for Ultrastructural Research (EM Lab)www.uga.edu/caur/caur@uga.edu 706-542-4080

Paul Schroeder, GeologyJohn Shields, Cell BiologyJianguo Fan, Physics/GeologySara Karlsson, Office manager