FB820
Lecture 4
Sample Preparation
Jiri Novacek
Content
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● sample preparation for SEM (2D imaging)
● structural TEM sample preparation
● volume EM sample prep.
Scales attainable with electron microscopy
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1 mm 1 um 1 nm 1A
Tick (ESEM) Plant cell (TEM)
Plant (SEM)
Bacteria (SEM)
Virus (TEM)Bacteriophage (TEM)
RNA polymerase (TEM)
apoferritin @1.2A (TEM)
Interaction of an electron with a matter
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Interaction of an electron with a matter
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SEM
TEM
Scanning electron microscopy
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SEM imaging
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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)
SEM imaging
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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)
SEM imaging
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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)
Structural TEM sample preparation
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BF image
Objective
aperture
Amplitude contrast Phase contrast
● intensity difference in two adjacent area
● minor contribution in life-science TEM
● phase shift between transmitted and diffracted wave
● primary source of contrast in life-science TEM
Transmission electron microscopy
Rotary shadowing
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Pros:
- high signal to noise
- fast sample preparation
- potentially high-resolution - single vs. double stranded
nucleic acid
Cons:
- non-native (sample dehydrated on surface)
- limited applicability (primarily filamentous structure)
- limited information content (imaging thickness of metal layer
not the studied molecule)
Negative staining
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contrasting with heavy metal stains (typically 0.5-2.0% water solution)
● uranyl acetate (pH~4)
○ Pros:
■ high contrast
■ fixative effect
○ Cons:
■ disintegration of sensitive samples (e.g. enveloped viruses)
● uranyl formate (pH~4.5)
○ Pros:
■ high contrast
■ fixative effect
■ smaller grain (suitable for smaller proteins)
○ Cons:
■ low stability
■ soluble in very narrow pH range
■ disintegration of some sample
● ammonium molybdenate, phosphorus thungstanate
○ Pros:
■ pH~7
■ more suitable for fragile complexes (e.g. enveloped viruses)
○ Cons:
■ slightly lower contrast than UAc
■ low fixative effect (fragile complexes may be disassembled)
Negative staining
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Pros:
- sample preparation quick and robust
- high contrast
- efficient method for sample quality control
- initial structural data
- low sensitivity to radiation damage
Cons:
- resolution limited (10-20A)
- non-native conditions (air drying, high salt)
- flattening artifacts
- denaturation of proteins and NA
Negative staining
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Pros:
+ high signal to noise
+ low dose sensitivity
+ robust (easy sample handling)
Cons:
- non-physiological conditions during sample preparation
- artefacts (changes in cell structure, depression of
proteins)
- usually toxic chemicals used during sample prep
- obtainable level of detail limited
Plunge freezing - electron cryo-microscopy
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● Rapid immersion of buffered sample into cryogen
● Cryogens:
■ liquid ethane
■ ethane:propane mixture
● Vitrification has to be fast ~1000 K/s
● Possible only for samples with thickness ~<10um
● => amorphous ice
● => thin layer (50-400nm)
● near-native conditions
■ difficult to reverse the process and defrost the
sample back to functional state
■ LDA water - 0.94 g/l; HDA - 1.17 g/l
Plunge freezing
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3-4ul
0.1-1mg/ml for purified protein complexes
OD~0.5-20 for bacteria
Plunge freezing
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● 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
Plunge freezing
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Extrusion of particles from thin ice Denaturation at air water interface
Plunge freezing
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Pros:
+ near-native state of molecule
+ attainable resolution not limited by sample prep.
+ no toxic chemicals in the process
+ applicable not only to protein but usually also to
cellular monolayer
Cons:
- low signal to noise
- sample handling only under LN2 conditions (risk of
devitrification and sample surface contamination)
- prone to radiation damage (sample is insulator)
- obtainable level of detail limited
Volume EM - block face imaging
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Workflow
● Chemical fixation (formaldehyd, glutaraldehyde, osmium tetraoxide)
● Dehydration (EtOH, aceton)
● Resin embedding
● Sectioning
Block face imaging
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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
Block face imaging
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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)
● lysine
● arginine
Block face imaging
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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
Block face imaging
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Mechanical sectioning for TEM
Block face imaging
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- 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
Mechanical sectioning for TEM
Block face imaging
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- detection of back scattered electrons
- mechanical sectioning either inside or outside SEM
- FIB sectioning (10nm)
- FIB-SEM tomography – correlative studies limited
- FIB sectioning - destructive vs. mechanical sectioning - non-destructive
- FIB sectioning - easier image registration vs. mechanical sectioning - image
registration may become cumbersome
Mechanical sectioning of FIB sectioning for SEM
Block face imaging
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- detection of back scattered electrons
- mechanical sectioning either inside or outside SEM
- FIB sectioning (10nm)
- FIB-SEM tomography – correlative studies limited
- FIB sectioning - destructive vs. mechanical sectioning - non-destructive
- FIB sectioning - easier image registration vs. mechanical sectioning - image
registration may become cumbersome
Mechanical sectioning of FIB sectioning for SEM
Volume EM - cryo-EM techniques
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www.leica-microsystems.com
High pressure freezing 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
Volume EM - cryo-EM techniques
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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
High pressure freezing & freeze substitution
Volume EM - cryo-EM techniques
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- sectioning for TEM (tomography)
- section thickness ~70nm
- 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
CEMOVIS - cryo-EM of vitreous sections
Volume EM - cryo-EM techniques
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Focused ion beam micromachining of cellular lamellae
- only single section per cell
- section thickness ~100-300nm
- ablation with Ga+, Xe+, O+, Ar+
- minimal artefacts
- complex (FIB/SEM microscope as sample
preparation device
Volume EM - cryo-EM techniques
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Focused ion beam micromachining of cellular lamellae
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