Introduction to Electron Microscopy Andres Kaech Preparation › static › winterschool ›...
Transcript of Introduction to Electron Microscopy Andres Kaech Preparation › static › winterschool ›...
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Introduction to
Electron Microscopy
Andres Kaech
Preparation
Center for Microscopy and Image Analysis
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Biology Electron microscope
High vacuum
Electron beam
Sensitive to vibration/motion
(High magnifications)
Biological samples need to be
transferred into a solid state...
...keeping the sample close to the
native state
Not suitable for EM
Resistant to high vacuum
Resistant in electron beam
Thin – permeable for electrons
(for TEM)
Contrast
Physical demands of electron microscopy
Aqueous/hydrated
Soft
Light elements
(C, O, H, N, S, P etc.)
“Large”
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Biology Electron microscope
High vacuum
Electron beam
Sensitive to vibration/motion
(High magnifications)
Any treatment changes the
specimen!
Not suitable for EM
Resistant to high vacuum
Resistant in electron beam
Thin – permeable for electrons
(for TEM)
Contrast
Physical demands of electron microscopy
Aqueous/hydrated
Soft
Light elements
(C, O, H, N, S, P etc.)
“Large”
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Physical demands of electron microscopy
2 cm
What is (was) this?
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Critical Point Drying
Coating
RT-SEM
WARM SPECIMEN
Main preparation pathways for TEM
Dehydration
Chemical fixation
Freeze-fractured/etched specimenFreeze-dried
specimen
Freeze-fracturing/Freeze-drying/Coating
RT-SEM
Low temperature processing
Cryo-Ultramicrotomy
Cryo-TEM
Cryo thin section
Cryo-SEM
RT specimen processing
High pressure
freezing
Plunge freezing
thawing
Immunolabeling
Replica
RT-TEM
Bare grid technique
Staining
Ultramicrotomy
Embedding
Low-temperature embedding
RT-embedding
FROZEN SPECIMEN
Freeze-substitution
Propane jet
freezing
RT-TEM, FIB-SEM
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Main preparation pathways for TEM
Embedding
Fixation
Dehydration
Staining
Thin sectioning
TEM Requires thin specimen: 70 nm
Requires solid specimen (embedding in plastic)
Plastic only soluble in solvents (e.g. acetone)
Solvents dissolve biological matter
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Embedding
Fixation
Dehydration
Staining
Room temperature processing for TEM
Thin sectioning
TEM
Stabilization of biological material
Chemical fixation (cross-linking) with Aldehydes, OsO4, Ur2+…
Glutaraldehyde CH2 CH2 CH2 C
O
H
C
O
H
1 mm
1 mm
1 mm
1 mm3: penetration within 30-60 min at 20-37°C
Maximum size for good preservation
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Room temperature processing for TEM
Osmiumtetroxide
• Cross linker mainly of unsaturated lipids
some proteins & phenolic compounds
• Provides contrast
• Can solubilise some proteins
Os
OO
O O
Embedding
Fixation
Dehydration
Staining
Thin sectioning
TEM
Post-fixation with OsO4
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Chemical fixation
The specimen must be small to enable
quick fixation with chemicals
Chemical fixatives
penetrate small specimen
at sufficient speed
Chemical fixatives block
their penetration if specimen
is too large...slow or no
fixation
1 mm
1 mm
1 mm
G. Griffith, Fine structure immunocytochemistry, Springer 1993
1 mm3: penetration within 30-60 min at 20-37°C
Room temperature processing for TEM
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Embedding
Fixation
Dehydration
Staining
Thin sectioning
TEM
Room temperature processing for TEM
Well preserved Not well preserved
Liver tissue
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Embedding
Fixation
Dehydration
Staining
Thin sectioning
TEM
Room temperature processing for TEM
Substitution of water with solvent (ethanol, acetone)
Usually performed with gradient of different concentrations.
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Embedding
Fixation
Dehydration
Staining
Thin sectioning
TEM
Room temperature processing for TEM
Infusion with “plastic” formulation followed by polymerisation
Specimen embedded in Epon
• Plastic formulations consist of monomers, hardener, accelerator
• Polymerization by heat or UV light
• Epoxy resins, acrylic resins
• Note: Resins are toxic and allergenic
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Embedding
Fixation
Dehydration
Staining
Thin sectioning
TEM
Room temperature processing for TEM
Cutting sections of ca. 70 nm -> electron transparent
Ultramicrotomy
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Embedding
Fixation
Dehydration
Staining
Thin sectioning
TEM
Room temperature processing for TEM
Cutting sections of ca. 70 nm -> electron transparent
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Room temperature processing for TEM
30 nm
70 nm
100 nm
150 nm
200 nm
300 nm
Embedding
Fixation
Dehydration
Staining
Thin sectioning
TEM
Cutting sections of ca. 70 nm -> electron transparent
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Embedding
Fixation
Dehydration
Staining
Thin sectioning
TEM
Contrast enhancement with heavy metals
Room temperature processing for TEM
UAc H2O Pb-citrate H2O5 min 30 sec 5 min 30 sec
Parafilm
Droplet with staining solution
Grid with sections facing down
• Uranium ions: phosphate groups of lipids (membrane contrast)
• Lead ions preferably bind to proteins
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Embedding
Fixation
Dehydration
Staining
Thin sectioning
TEM
Interpretation/orientation
Room temperature processing for TEM
HEP2 cells infected with Chlamydia pneumoniae
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Embedding
Fixation
Dehydration
Staining
Thin sectioning
TEM
Room temperature processing for TEM
Aldehydes: Slow (seconds to minutes), lots of artefacts like shrinkage…
OsO4: Depolimerisation of proteins
Shrinkage
Conformational changes of proteins
Loss of lipids
Mechanical effects
Loss of Lipids
Shrinkage during polymerisation
Compression, knife marks
Staining artefacts (precipitation of heavy metals)
Interpretation mistakes
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Embedding
Fixation
Dehydration
Staining
Thin sectioning
TEM
Cryo preparation for TEM
Cryo-Immobilization
Stabilization of biological material by freezing
Liquid water and vitrified water
Frozen water with ice crystals
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Embedding
Fixation
Dehydration
Staining
Thin sectioning
TEM
Cryo preparation for TEM
Well frozen mouse cerebellum
Not well frozen mouse cerebellum
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Embedding
Fixation
Dehydration
Staining
Thin sectioning
TEM
Cryo preparation for TEM
High pressure freezing (HPM)
Freezing under high pressure (2100 bar)
Adequate freezing of samples up to 200 µm thickness without anti-freeze
Plunge freezing in liquid ethane/propane:
Only suspensions (< 1 µm) or thin tissues containing anti-freeze
Propane jet freezing (JFD):
Adequate freezing of suspensions not thicker than 15 µm
Thicker specimen require anti-freeze
Slam freezing:
Suspensions and thin tissues (few µm, only front well frozen ca. 1 µm)
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Embedding
Fixation
Dehydration
Staining
Thin sectioning
TEM
Cryo preparation for TEM
Relative sizes
Plunge/slam freezer Propane jet freezer
High-pressure freezer
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Embedding
Fixation
Dehydration
Staining
Thin sectioning
TEM
Freeze-substitution
Substitution of water/ice with solvent (ethanol, acetone)
Usually combined with simultaneous fixation with chemicals
(OsO4, Uranyl-acetate…)
Cryo preparation for TEM
-100
-90
-80
-70
-60
-50
-40
-30
-20
-10
0
0 5 10 15 20 25 30 35
Time (h)
Te
mp
era
ture
(°C
)
-90°Cacetone-90°Cacetone
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Embedding
Fixation
Dehydration
Staining
Thin sectioning
TEM
Infusion with “plastic” formulation followed by polymerisation at
low or room temperature
Cryo preparation for TEM
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Embedding
Fixation
Dehydration
Staining
Thin sectioning
TEM
Same procedure as RT
Cryo preparation for TEM
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Embedding
Fixation
Dehydration
Staining
Thin sectioning
TEM
Reduced extraction of cell constituents
Reduced shrinkage
Mechanical effects
Loss of Lipids
Shrinkage during polymerisation
Compression, knife marks
Interaction of heavy metals with biology provides electron
density
Interpretation/orientation
Cryo preparation for TEM
No RT fixation artefacts
Ice crystal damage possible
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Specimen courtesy of Bettina Sobottka, Neurologische Klinik, University of Zurich
Room temperature vs. cryo preparation
500 nm
Conventionally fixed (glutaraldehyde) High pressure frozen
Mouse cerebellum
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Specimen courtesy of Bettina Sobottka, Neurologische Klinik, University of Zurich
Room temperature vs. cryo preparation
500 nm
Conventionally fixed (glutaraldehyde) High pressure frozen
Mouse cerebellum
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RT-SEM
Low-temperature embedding
RT-embedding
RT-TEM, FIB-SEM
Cryo-Ultramicrotomy
Cryo-TEM
Cryo thin section
Freeze-substitution
Ultramicrotomy
Staining
WARM SPECIMEN
Embedding
thawing
Immunolabeling
Replica
RT-TEM
Main preparation pathways for SEM1 µm1 µm
Bare grid technique
Dehydration
Critical Point Drying
Coating
RT-SEM
Chemical fixation
Low temperature processingRT specimen processing
High pressure
freezing
Propane jet
freezing
Plunge freezing
Freeze-dried
specimen
Freeze-fracturing/Freeze-drying/Coating
Cryo-SEM
FROZEN SPECIMEN
Freeze-fractured/etched specimen
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Room temperature processing for SEM1 µm1 µm
Critical point
drying
Fixation
Dehydration
Coating
SEM
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1 µm1 µm
Critical point
drying
Fixation
Dehydration
Coating
SEM
Same as RT preparation for TEM
Room temperature processing for SEM
Sample finally in solvent like ethanol or acetone
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1 µm1 µm
Critical point
drying
Fixation
Dehydration
Coating
SEM
SS Starting point
EE End point
CC Critical point
liquid
gas
solid
CC
SS
EE
Temperature
PressurePhase diagram of CO2
Critical point of CO2: 31°C, 74 bar
Critical point of H2O: 374°C and 221 bar
Room temperature processing for SEM
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1 µm1 µm
Critical point
drying
Fixation
Dehydration
Coating
SEM
Air drying
Room temperature processing for SEM
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1 µm1 µm
Critical point
drying
Fixation
Dehydration
Coating
SEM
Room temperature processing for SEM
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1 µm1 µm
Critical point
drying
Fixation
Dehydration
Coating
SEM
Air dryingCritical point drying
Surface of rose blossom SPI
Electron Microscopy ETH ZurichSpider mite
Room temperature processing for SEM
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1 µm1 µm
Critical point
drying
Fixation
Dehydration
Coating
SEM
Platinum/Gold (1-10 nm)
Primary electron beam
• Sputter coating
• Resistance evaporation
Thin heavy metal layer applied to the specimen surface
Room temperature processing for SEM
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1 µm1 µm
Critical point
drying
Fixation
Dehydration
Coating
SEM Interpretation/orientation
Room temperature processing for SEM
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Critical point dried, fractured liver tissue
Center for microscopy and image analysis, University of Zurich
Room temperature processing for SEM1 µm1 µm
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Cryo processing for SEM1 µm1 µm
Sublimation
(partial freeze-
drying)
Fixation
Freeze-fracturing
Coating
Cryo-SEM
Cryo-Immobilization (same as for TEM)
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Cryo processing for SEM1 µm1 µm
Sublimation
(partial freeze-
drying)
Fixation
Freeze-fracturing
Coating
Cryo-SEM
…under high vacuum and at low temperature
2 cm
-120°C…-150°C
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Cryo processing for SEM1 µm1 µm
Sublimation
(partial freeze-
drying)
Fixation
Freeze-fracturing
Coating
Cryo-SEM
…under high vacuum and at low temperature
2 cm
-120°C…-150°C
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Cryo processing for SEM1 µm1 µm
Sublimation
(partial freeze-
drying)
Fixation
Freeze-fracturing
Coating
Cryo-SEM
EF…Exoplasmatic fracture face
PF…Plasmatic fracture face
Ice
Cytoplasm
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Cryo processing for SEM1 µm1 µm
Sublimation
(partial freeze-
drying)
Fixation
Freeze-fracturing
Coating
Cryo-SEM
Revealing the ultrastructure by removing the ice
embedding the biological material (under high vacuum)
Heating (for example: -100°C for 5 minutes)
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Cryo processing for SEM1 µm1 µm
Sublimation
(partial freeze-
drying)
Fixation
Freeze-fracturing
Coating
Cryo-SEM
Electron microscopy ETH Zurich
Freeze-fractured mouse intestine: with sublimation
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Cryo processing for SEM1 µm1 µm
Sublimation
(partial freeze-
drying)
Fixation
Freeze-fracturing
Coating
Cryo-SEM
Platinum/Gold (1-10 nm)
Primary electron beam
Thin heavy metal layer applied to the specimen surface
…at low temperature
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Cryo processing for SEM1 µm1 µm
Sublimation
(partial freeze-
drying)
Fixation
Freeze-fracturing
Coating
Cryo-SEM Interpretation/orientation
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Vero cells
Cryo processing for SEM1 µm1 µm
Nucleus
Outer nuclear membrane
Inner nuclear membrane
NP
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Vero cells HSV infected
Cryo processing for SEM1 µm1 µm
Virus particle in perinuclear space
NP
NP
NP
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High-pressure frozen, freeze-fractured brain tissue
Electron microscopy ETH Zurich
Cryo processing for SEM1 µm1 µm