Electron Microscopy and You in
the New Millennium
The Challenges in Electron Microscopy Concerning
Negative Stain and CryoEM Techniques for Virology
Reseach
With a General Review of The Baker Lab Paper Too
Bob Ashley AAS SMFW
Overview
• Free will vs. Determinism in electron microscopy
Grids and Support Film
• Usually Copper
• How does it react with the specimen?
• Mesh indicates amount of open area
• Higher number less open 50-1000
• We use 400 mesh square
• Very thin films and specimens
• Hexagonal shape openings
• Very delicate
• Even mild bend or buckle can cause distortion of specimen or focus aberration
To Glow or Not
Glow Discharge renders continuous carbon coated grids hydrophilic by applying
a negative charge.
• Aids stain spread more uniformly (increases wettability)
• Helps particles in specimin to adhere to the substrate
• Decreases likelihood of the virus particles being held in aggregates as a result of
the interaction between the virus particles and the surface charge of film
All Sides Being Equal?
• Dull Side (coppery)
• More area for film to
adhere
• Shiny side (polished)
• Not as much surface
area for films to adhere
• Can cause movement
under e- beam exposure
Support Film Concerns
• Mass thickness influences contrast while mechanical stability increases image clarity
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Films in general must have
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High transparency
Adequate strength to withstand E- beam and support specimen
• Carbon coated only (6-10 nm)
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Uniformly amorphous
More elastic scattering events with e- beam
Can be stable very thin 1-2nm
More stable than plastic alone
Hydrophobic, fragile, time consuming to make
• Plastic only 10-20nm (10-20 nm)
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Formvar
More clarity with less background than Carbon alone
Not as thermally stable and can cause charging and drift
• Carbon Coated with Plastic
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Stability of carbon but will have a thickness that may impede resolution
The Concept of Negative Stain
• Heavy metal atoms act as barrier to the e- beam
• Allows passage through specimen
• Stain penetration into hydrophilic specimen
• Dries faster than specimen
• Mostly hydrated regions replacing water
• Lipoproteins and proteins
• Stains form around hydrophobic
regions including lipid
• Contrast dependent on stain thickness
• Absolute resolution to about 2 nm
Simple Microscopy
• Lighter areas have more protein and exclude stain
• Darker areas are where the stain pools
• Indent in support mechanism
Stain Film and Particle
Interactions
A. Hydrophilic specimen
hydrophilic film
B. Hydrophilic specimen on
hydrophobic film
C. Hydrophobic specimen on
hydrophilic film
D. Hydrophobic specimen on
hydrophobic film
Negative and Positive Staining
A. 4% PTA Negative Stained
B. 4% UA Positive Stained
C. 4% UA Negative Stained
• Three types of staining visible
• Negative staining appearing white
• Negative staining appearing grey
• Positive staining appearing black
• Severe structural distortion
Factors Controlling Appearance
• Specimin
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pH
Isoelectric Point
Fixation
Concentration
• Stain
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pH
Charge
Buffer and specimen interaction
Osmolality
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Can influence structure and volume of particle
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Highest Ammonium Molybdate: 288 UA: 59 PTA:39
• Interaction with the support mechanism
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Grid film
Thickness of stain on film
Charge and beam interactions
The Effect of the Isoelectric Point of
Protein and Stain
• Isoelectric point (pI) or (IEP)
• The pH at which a particular molecule or surface carries no net electrical charge
• Can be time dependent and is not absolute
• Fixation with glutaraldehyde increases net negative charge
• The presence of a fixed negative or positive charge influences the deposition
of any given stain
• In general proteins
• Combine (positive stain) with cations (UA+) on the akaline side of the pI
• Combine with anions (PTA-) on the acidic side of the pI
• Protein pI
• Stain pH greater than pI applies negative charge
• Stain pH lower than pI applies positive charge
• Ex. Protein with a pI of 5.0 is negatively charged at pH 7.0 with PTA- which is higher
than the pI of the protein therefore the stain repels and is excluded by the protein
Other Effects of pH
• Optimal pH for stains is not known but each has a
satisfactory range
• At high pH the stain penetration is usually enhanced with
long stain time
• At low pH the surface detail is usually highlighted due to
acidic environment
• May change with stain storage and with stain drying
• Use fresh stain preferable and check before staining
specimen
Ex. stained with PTA at 5.0 pH, influenza virus surface spikes well preserved
same sample stained with 7.5 pH PTA stain penetrates virus envelope
Ex. PTA with pH of 4.5 recommended for resolving antibody particles bound to
rotavirus
Negative Stains
• Negative stain should:
• Have minimal interaction with specimin (pos. stain)
• High soluability in solution (precipitates and crystalizes in
e- beam)
• High density (must be at least twice the density of the
specimin to be visualized)
• High melting point to avoid beam damage
• Small grain size
• Chemical pH stability
Types of Stains
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Uranyl Acetate+ (cation)
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Uranyl Formate+(cation)
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Most widely used
Density of 2.87 g/cm^3
Ion diameter .4-.8 nm
pH of 4.0-5.5 (usually used at 4.5 unstable at 6.0)
Concentrations .5-5% ideal as 1%
Can act as fixative
Higher contrast than PTA
Stabilizes lipids therefore may minimize drying effect of virus particles
Smallest grain size for better penetration of interstices of sample
Useful for high-res
pH of 4.0-5.5 (usually used around 4.5)
Density of 3.68 g/cm^3
Ideal as .75%
Uranyl Oxalate+(cation)
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Can be used in pH from 5.0-7.0 (ideal at 6.5-6.8)
Desirable for pH sensitive specimins
Provides the contrast and penetration of UA without the acidity
Desirable for virus proteins below the pI or low molecular weight
Types of Stains Continued
• PTA-(anion)
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Along with UA most widely used
Density 4 g/cm^3
Grain size of 1.2 nm (not useful for high res work)
At neutral pH very little interaction with the specimen (avoids most positive
staining)
Very stable in e- beam
Will not fix a specimen
Not stable over time with storage <1 month
May dissociate quaternary proteins into small units
• Ammonium Molybdate
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Used for osmotically sensitive specimins
pH from 5.0-8.0 useful at 7.0-7.4
Higher contrast than PTA
• Methylamine Tungstate (NanoW)
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pH 6.4-7.0
Tolerates concentrated buffers
Drawbacks of Negative Stain
• Fixation
• Tends to concentrate sample
• Cellular debris and other junk
• Positive staining
• Beam irradiation
• Lower kV=more damage potential
• Drying
• Leads to distortion of particle
• Flattening
• Will usually happen perpendicular to support mechanism
• Makes sample typically larger in diameter to known size
Chill Out
Negative Stain
Light areas indicate density
• Cryo Advantages
• Keeps sample in natural state
• Higher resolution
• No artifacts from stain
Cryo
Contrast reversal of Negative Stain,
dark areas indicate density
CryoEM
• Amorphous or glassy ice
• Liquid nitrogen -195° C
• Liedenfrost Effect
• Liquid ethane
• Liquid propane
• Cubic ice
• Water in crystalline lattice obscures beam
• Usually around -140° C
• Vanilla Ice
• Too hot to handle yet too cold to hold
The Grid in Cryo
The Mechanism
Plunge Freezing
Manual Blotting
Advanced Grid
Processing
Visualizing Ice on the
Grid
Search for suitable area on grid
Low Dose Theory
• Ice is beam sensitive
• E- cause irradiated sample
• High res data can be lost in a matter of seconds
• Focus on area that is not photographed and
correct for astigmatism
• Keep levels to around 5-20 E/A^2
• Can be an software automated process
Irradiation Damage
Defocus
• Focus adjacent region of interest to true focus
• No inherent contrast from sample in ice
• No tone ring visible in FFT
• Reset to range desirable -2 to -5 ųm
The End Result
Try the best
you can to
achieve your
EM dreams!
Thank You