Challenges in Electron Microscopy for Virology Research: Negative Stain and CryoEM Techniques

1. 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

2. Overview • Free will vs. Determinism in electron microscopy

3. 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

4. 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

5. 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

6. Support Film Concerns • Mass thickness influences contrast while mechanical stability increases image clarity • Films in general must have • High transparency • Adequate strength to withstand E- beam and support specimen • Carbon coated only (6-10 nm) • 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) • Formvar • More clarity with less background than Carbon alone • Not as thermally stable and can cause charging and drift • Carbon Coated with Plastic • Stability of carbon but will have a thickness that may impede resolution

7. 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

8. Simple Microscopy • Lighter areas have more protein and exclude stain • Darker areas are where the stain pools • Indent in support mechanism

9. 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

10. 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

11. Factors Controlling Appearance • Specimin • pH • Isoelectric Point • Fixation • Concentration • Stain • pH • Charge • Buffer and specimen interaction • Osmolality • Can influence structure and volume of particle • Highest Ammonium Molybdate: 288 UA: 59 PTA:39 • Interaction with the support mechanism • Grid film • Thickness of stain on film • Charge and beam interactions

12. 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

13. 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

14. 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

15. Types of Stains • Uranyl Acetate+ (cation) • 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 • UranylFormate+(cation) • 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) • 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

16. Types of Stains Continued • PTA-(anion) • 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 • Used for osmotically sensitive specimins • pH from 5.0-8.0 useful at 7.0-7.4 • Higher contrast than PTA • Methylamine Tungstate (NanoW) • pH 6.4-7.0 • Tolerates concentrated buffers

17. 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

18. Chill Out Negative Stain Cryo Contrast reversal of Negative Stain, dark areas indicate density Light areas indicate density • Cryo Advantages • Keeps sample in natural state • Higher resolution • No artifacts from stain

19. 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

20. The Grid in Cryo

21. The Mechanism Plunge Freezing Manual Blotting

22. Advanced Grid Processing

23. Visualizing Ice on the Grid Search for suitable area on grid

24. 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

25. Irradiation Damage

26. 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

27. The End Result

28. Try the best you can to achieve your EM dreams! Thank You