C9940 3-Dimensional
Transmission electron microscopy
Lecture 2: Sample preparation
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Samples in electron microscopy
1 mm 1 um 1 nm
Tick (ESEM) Plant cell (TEM)
Plant (SEM)
Bacteria (SEM)
Virus (TEM)
Bacteriophage (TEM)
RNA polymerase (TEM)
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Interaction of electrons with matter
Mean free path
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Interaction of electrons with matter
SEM
TEM
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Transmission electron microscopy
BF image
Objective
aperture
- difference in intensity in two adjacent area - Transmitted and diffracted waves travel
through different distances
Amplitude contrast Phase contrast
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Scanning electron microscopy
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Applications in life-sciences
 SEM imaging
 Block face imaging
 Negative staining
 Cryo-EM techniques
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SEM imaging
Pros:
- imaging of sample morphology
at significant scale difference(1mm - 10nm)
- fast sample preparation
Cons:
- non-native (sample dehydrated)
Sample preparation:
- air drying
- metal sputtering (Pt, Au, Ir)
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SEM imaging
Pros:
- imaging of sample morphology
at significant scale difference(1mm - 10nm)
- fast sample preparation
Cons:
- non-native (sample dehydrated)
Sample preparation:
- freezing into LN2
- sublimation
- metal sputtering (Pt, Au, Ir)
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SEM imaging
Pros:
- imaging of sample morphology
at significant scale difference(1mm - 10nm)
- fast sample preparation
Cons:
- non-native (sample dehydrated)
Sample preparation:
- chemical fixation
- contrasting (Pt,U)
- dehydration (EtOH,aceton,HMDS)
- critical point drying
- metal sputtering (Pt, Au, Ir)
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Block face imaging
Chemical fixation (formaldehyd, glutaraldehyde, osmium tetraoxide)
Dehydration (EtOH, aceton)
Plastic embedding
Sectioning
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Block face imaging
Pros:
- 3D volume reconstruction at ultrastructural level
of detail
- high signal to noise
- low dose sensitivity
- robust (easy sample handling)
Cons:
- non-physiological conditions during sample prep
- artefacts (changes in cell structure, depression
of proteins)
- extremely toxic chemicals (OsO4)
- attainable level of detail limited
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Block face imaging
Pros:
- 3D volume reconstruction at ultrastructural level
of detail
- high signal to noise
- low dose sensitivity
- robust sample preparation
Cons:
- non-physiological conditions during sample prep
- artefacts (changes in cell structure, depression
of proteins)
- extremely toxic chemicals (OsO4)
- attainable level of detail limited
Sample preparation 1:
- formaldehyde, glutaraldehyde
- chemical fixation - ~2% solution in water
or buffer
- variable duration – 2-24 hours (sample
thickness)
- contrasting (OsO4, UAc, Pb)
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Block face imaging
Pros:
- 3D volume reconstruction at ultrastructural level
of detail
- high signal to noise
- low dose sensitivity
- robust sample preparation
Cons:
- non-physiological conditions during sample prep
- artefacts (changes in cell structure, depression
of proteins)
- extremely toxic chemicals (OsO4)
- attainable level of detail limited
Sample preparation 2:
Dehydration – EtOH or aceton series
(30% for 15mins, 50% for 15min, 70% for
15mins, 90% for 15mins, 100% - 3x)
- shrinking of protein and lipids
- sample shrinking up to 40%
- formation of various artefacts
Resin embedding – resin infiltration ( 2:1
propylen oxide: resin for 1h, 1:1 for 1h, 1:2
for 1h, 100% resin overnight
- polymerazation 24-72h at 60-70C
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Block face imaging
Mechanical sectioning for TEM
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Block face imaging
Mechanical sectioning for TEM
- 50 – 70 nm thick sections
- high-resolution imaging in TEM (tomography)
- 3D volume reconstruction
- resolution limited by sample preparation
- staining with EM contrasting agents (nanoparticles)
or fluorescent markers (CLEM) for targetting
NIH el. mic. facility
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Block face imaging
Mechanical or FIB sectioning for SEM
- detection of back scattered electrons
- mechanical sectioning either inside or outside SEM
- FIB sectioning (10nm)
- FIB-SEM tomography – correlative studies limited
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Thin section methods
Focused ion beam block-face for SEM
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Heavy metal staining
Stains: uranyl acetate (pH=4)
uranyl formate (pH=4)
ammonium molybdenate (pH=7)
phosphorus thungstanate (pH=7)
Negative staining
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Heavy metal staining
Pros: quick sample screening
high amplitude contrast
less prone to beam damage
Cons: limited resolution (20A)
flattening artefacts
denaturation of proteins
Negative staining
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Heavy metal staining
Negative staining
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Heavy metal staining
- DNA visualization
Metal shadowing
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- non-physiological conditions during sample preparationd
- artefacts (changes in cell structure, depression of proteins)
- usually toxic chemicals used during sample prep
- obtainable level of detail limited
+ high signal to noise
+ low dose sensitivity
+ robust (easy sample handling)
Heavy metal staining
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Plunge freezing

Rapid immersion of buffered sample into cry

Cryogens: liquid ethane

ethane:propane mixture

Vitrification has to be fast ~1000 K/s


Possible only for samples with thickness ~<1

=> amorphous ice

=> thin layer (200-600nm)
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Plunge freezing
3-4ul
0.1-1mg/ml for purified protein complexes
OD~0.5 for bacteria
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Plunge freezing

Sample frozen in hydrated state


Amorphous ice


Sample has to be kept at temperatures

above devitrification point (~-135C)


Internal structures can be visualized


High resolution information is retained

Possible problems: ice thickness

hexagonal ice, cubic ice
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Plunge freezing
Extrusion of particles from thin ice Denaturation at air water interface
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Plunge freezing

Cons:


Low signal to noise


Prone to radiation damage


More delicate sample handling required

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Cellular cryo-EM techniques
Plunge freezing:
- rapid immersion of buffered sample into
cryogen (liquid ethane, ethane:propane mix)
- vitrification has to be fast 10e4-10e5 K/s
- available only for samples ~<10um thick
High pressure freezing
- sample thickness <200um
- freezing with liquid nitrogen
- 2000 bars, 20 ms
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Cellular cryo-EM techniques
3-4ul
0.1-1mg/ml for purified protein complexes
OD~0.5 for bacteria
Plunge freezing
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Cellular cryo-EM techniques
High pressure freezing, freeze substitution
Freeze substitution
- reduction of ultrastructure changes compared to
dehydration at ambient temperature
- dehydration at temperatures <-70C
(aceton typically -90C)
- fixatives are evenly distributed before cross-linking
at ambient temperature
- resin embedding for ultramicrotomy at room temp.
www.leica-microsystems.com
Yamada et al. JMM 2010
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Cellular cryo-EM techniques
CEMOVIS – cryo-EM of vitrous sections
- no chemical fixation, dehydration or contrasting
- low contrast
- preservation of the sample in near-native conditions
- mechanical sectioning by ultramicrotome at LN2
conditions
- sectioning artefacts
Al-Amoudi et al. EMBO J 2004
Al-Amoudi et al. JSB 2005
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Thin section methods - note
Freeze substitution

Reduce ultra-structure changes at

due to dehydration as seen at amb

temperature


Dehydration at temperatures belo -7

(aceton typically -90C)

Fixatives are evenly distributed bef

cross-linking at ambient temperatur

Resin embedding for ultramicrotom

at room temperature
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Cellular cryo-EM techniques
Focused ion beam milling of cellular lamellas
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Cellular cryo-EM techniques
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Cellular cryo-EM techniques
Vaccinia virus inside cell HeLa cells
Pavel Plevka group