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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/[email protected] 706-542-4080
Paul Schroeder, GeologyJohn Shields, Cell BiologyJianguo Fan, Physics/GeologySara Karlsson, Office manager