Transcript Food Science Seminar, 9-4-08 - Center for Advanced Ultrastructural

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
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
Scale of Imaging
TEM
0.25 um
Confocal
40 um
SEM
2 um
Light
100 um
Transmission Electron Microscopy
Technai 20
200 KeV
1.4 Å
Standard Preparation
Tissue
TEM
Chem.
Fixation
SEM
Cryo
Fixation
Chem.
Fixation
Cryo
Fixation
Rinse/store Substitution
Rinse/store
En bloc
staining
CryoDehydrationDehydration
Dehydration
sectioning
Drying
Resin
Mounting
infiltration
Sectioning
Post staining
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.
X-rays
Through thickness
composition info
Incident Beam
Primary backscattered
electrons
Atomic number and
topographical
Cathodoluminescence
Electrical
Secondary electrons
Topographical
Auger electrons
Surface sensitive
compositional
Specimen
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
Plunge Freeze and SEM Cryostage
Specimen holder and
transfer rod
Nitrogen slushing
and plunge station
Leidenfrost effect
Ice crystal
formation
Effects of Etching
Cryofixed Feta
Cryofixed Yogurt
Both images courtesy Dr. Ashraf Hassan
Correlation - Light Micrographs and CryoSEM
CW
S
Whole Peanut
P
Peanut Butter
Images courtesy Eyassu Abegaz
Rice
Uncooked
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
Incident Electron Beam
Zeiss VPSE Detector
Principle
VPSE Detector, Light Pipe and
PMT.
Photons
BSE’s
Light Pipe
Photons are detected
and amplified to
provide the final image.
Specimen
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 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.
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.
Oak Ridge Natl Lab
Confocal Scanning Laser Microscope
-Confocal
- Mutiphoton
Sample Imaged by:
- Fluorescent dyes
- Autofluorescent compounds
- Expressed fluorescent proteins
(e.g. GFP)
- Reflective surfaces
Confocal
Principle
Laser
Excitation Pinhole
Excitation Filter
PMT
Objective
Emission
Filter
Emission Pinhole
Optical Sectioning with Confocal Laser
Comparison with Flattened Cells
Epifluorescence
Confocal
Thick Biofilms
Fluorescence
Confocal
Change in structure over time
Yogurt
Images courtesy
Dr. Ashraf Hassan
Alternate Views
from Z-Stack
Reconstruction
Reflectance mode - Yogurt
Courtesy Dr. Ashraf Hassan
Spatial information using
stereo projections
Coral zooxanthellae
EPS on E. coli
Labeling Cells
Bacterial colonization on metal
Reflectance metal
Labelled bacteria
Combined
Multi-photon Excitation
Single Photon
Excitation
Multi-Photon
Excitation
Depth penetration between
multi-photon and confocal
Multi-photon
Confocal
3 microns
31 microns
55 microns
Microtubule distribution in plant cells
Micrograph courtesy David Burk
Center for Ultrastructural Research (EM Lab)
www.uga.edu/caur/
[email protected]
706-542-4080
Paul Schroeder, Geology
John Shields, Cell Biology
Jianguo Fan, Physics/Geology
Sara Karlsson, Office manager