Post on 27-Jun-2020
transcript
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Introduction to
Electron Microscopy
Andres Kaech
Preparation
Center for Microscopy and Image Analysis
Biology Electron microscope
High vacuum
Electron beam
Sensitive to vibration/motion (High magnifications)
Biological samples need to be transferred into a solid state...
...which preserves the structures as a function of the living state…
…and not as a function of specimen preparation
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”
Physical demands of electron microscopy
2 cm
What is (was) this?
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Dehydration
Critical Point Drying
Freeze-fractured/etched specimenFreeze-dried
specimen
Freeze-fracturing/Freeze-drying/Coating
RT-SEM
Low temperature processing
Low-temperature embeddingRT-embedding
RT-TEM
Cryo-Ultramicrotomy
Cryo-TEM
Cryo thin section
FROZEN SPECIMEN
Freeze-substitution
Cryo-SEM
RT specimen processing
Coating
RT-SEM
Ultramicrotomy
Staining
WARM SPECIMEN
High pressure freezing
Propane jet freezing
Plunge freezing
Embedding
Chemical fixation
thawing
Immunolabeling
Replica
RT-TEM
Preparation pathways overview
Dehydration
Critical Point Drying
Freeze-fractured/etched specimenFreeze-dried
specimen
Freeze-fracturing/Freeze-drying/Coating
RT-SEM
Low temperature processing
Low-temperature embeddingRT-embedding
RT-TEM
Cryo-Ultramicrotomy
Cryo-TEM
Cryo thin section
FROZEN SPECIMEN
Freeze-substitution
Cryo-SEM
RT specimen processing
Coating
RT-SEM
Ultramicrotomy
Staining
WARM SPECIMEN
High pressure freezing
Propane jet freezing
Plunge freezing
Embedding
Chemical fixation
thawing
Immunolabeling
Replica
RT-TEM
Main preparation pathways for TEM
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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
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 lipidssome proteins & phenolic compounds
• Provides contrast
• Can solubilise some proteins
Os
OO
O O
Embedding
Fixation
Dehydration
Staining
Thin sectioning
TEM
Post-fixation with OsO4
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.
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
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 nm300 nm
Embedding
Fixation
Dehydration
Staining
Thin sectioning
TEM
Cutting sections of ca. 70 nm -> electron transparent
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
Embedding
Fixation
Dehydration
Staining
Thin sectioning
TEM
Room temperature processing for TEM
Aldehydes: Slow (seconds to minutes), lots of artefacts like shrinkage, osmotic effects, conformational changes of proteins, loss of ions and small molecules. OsO4: Depolimerisation of proteins
ShrinkageConformational changes of proteinsLoss of lipids
Mechanical effectsLoss of LipidsShrinkage 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-ImmobilizationStabilization of biological material by freezing
Liquid water and vitrified water
Frozen water with ice crystals
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)
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
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
Embedding
Fixation
Dehydration
Staining
Thin sectioning
TEM
Reduced extraction of cell constituentsReduced shrinkage
Mechanical effectsLoss of LipidsShrinkage during polymerisation
Compression, knife marks
Interaction of heavy metals with biology provides electron density
Interpretation/orientation
Cryo preparation for TEM
No RT fixation artefactsIce 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
Specimen courtesy of Bettina Sobottka, Neurologische Klinik, University of Zurich
Room temperature vs. cryo preparation
High-pressure frozen, freeze-substituted mouse cerebellum
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Dehydration
Critical Point Drying
Freeze-fractured/etched specimenFreeze-dried
specimen
Freeze-fracturing/Freeze-drying/Coating
RT-SEM
Low temperature processing
Low-temperature embeddingRT-embedding
RT-TEM
Cryo-Ultramicrotomy
Cryo-TEM
Cryo thin section
FROZEN SPECIMEN
Freeze-substitution
Cryo-SEM
RT specimen processing
Coating
RT-SEM
Ultramicrotomy
Staining
WARM SPECIMEN
High pressure freezing
Propane jet freezing
Plunge freezing
Embedding
Chemical fixation
thawing
Immunolabeling
Replica
RT-TEM
Main preparation pathways for SEM
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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Critical point drying
Fixation
Dehydration
Coating
SEM
SS Starting pointEE End pointCC 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
Critical point drying
Fixation
Dehydration
Coating
SEM
Air drying
Room temperature processing for SEM
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Critical point drying
Fixation
Dehydration
Coating
SEM
Room temperature processing for SEM
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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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
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 SEM
Cryo processing for SEM
Sublimation(partial freeze-
drying)
Fixation
Freeze-fracturing
Coating
Cryo-SEM
Cryo-Immobilization (same as for TEM)
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Cryo processing for SEM
Sublimation(partial freeze-
drying)
Fixation
Freeze-fracturing
Coating
Cryo-SEM
…under high vacuum and at low temperature
2 cm
-120°C…-150°C
Cryo processing for SEM
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 SEM
Sublimation(partial freeze-
drying)
Fixation
Freeze-fracturing
Coating
Cryo-SEM
EF…Exoplasmatic fracture facePF…Plasmatic fracture face
Ice
Cytoplasm
Cryo processing for SEM
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 SEM
Sublimation(partial freeze-
drying)
Fixation
Freeze-fracturing
Coating
Cryo-SEMElectron microscopy ETH Zurich
Freeze-fractured Vero cell: NO sublimation
Cryo processing for SEM
Sublimation(partial freeze-
drying)
Fixation
Freeze-fracturing
Coating
Cryo-SEMElectron microscopy ETH Zurich
Freeze-fractured mouse intestine: with sublimation
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Cryo processing for SEM
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
Cryo processing for SEM
Sublimation(partial freeze-
drying)
Fixation
Freeze-fracturing
Coating
Cryo-SEM Interpretation/orientation
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High-pressure frozen, freeze-fractured brain tissue
Electron microscopy ETH Zurich
Cryo processing for SEM
Cryo processing for SEM
Critical point dried, dry-fractured brain tissue
Deh
ydra
tion
Crit
ical
Poi
nt D
ryin
g
Fre
eze-
frac
ture
d/et
ched
spe
cim
enF
reez
e-dr
ied
spe
cim
en
Fre
eze-
frac
turi
ng/F
reez
e-dr
ying
/Coa
ting
RT
-SE
M
Lo
w t
emp
erat
ure
pro
cess
ing
Low
-tem
pera
ture
em
bedd
ing
RT
-em
bedd
ing
RT
-TE
M
Cry
o-U
ltram
icro
tom
y
Cry
o-T
EM
Cry
o th
in s
ect
ion
FR
OZ
EN
SP
EC
IME
N
Fre
eze-
subs
titut
ion
Cry
o-S
EM
RT
sp
ecim
en p
roce
ssin
g
Coa
ting
RT
-SE
M
Ultr
amic
roto
my
Sta
inin
g
WA
RM
SP
EC
IME
N
Hig
h pr
essu
re
free
zing
Pro
pane
jet
fre
ezi
ng
Plu
nge
free
zing
Em
bedd
ing
Ch
em
ica
l fix
atio
n
tha
win
g
Imm
un
ola
belin
g
Re
plic
a
RT
-TE
M
Pre
para
tion
path
way
s ov
ervi
ew