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Specimen Preparation and Imaging for Macromolecular Electron Microscopy Gina Sosinsky Neu 259 May 29, 2012. Topics. Range of sample sizes studied by macromolecular microscopy Types of samples (Repeating assemblies) Never-ending quest for higher resolution What limits resolution?

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Specimen Preparationand Imaging forMacromolecular Electron Microscopy

Gina Sosinsky

Neu 259

May 29, 2012


  • Range of sample sizes studied by macromolecular microscopy

  • Types of samples (Repeating assemblies)

  • Never-ending quest for higher resolution

  • What limits resolution?

  • Techniques

    • Metal shadowing

    • Negative/positive staining

    • Embedding in sugars or tannic acid

    • Cryo-Electron microscopy

    • Cryo-Negative staining

    • Cryo-tomography

  • Low dose microscopy

Range of sample sizes studied by macromolecular microscopy
Range of Sample Sizes Studiedby Macromolecular Microscopy

Paramecium bursariaChlorella Virus 1~1900 Å, ~1 GDa


crescentus~0.6 X 2 µm

Bacteriorhodopsin~58 Å, ~26 kDa

70S E. coli ribosome~250 Å

Theoretical Biophysics Group

Beckman Institute

University of Illinois at Urbana-Champaign

Timothy S. Baker Group


Briegel, et al. (2006)

Courtesy J. Frank

100 Å

1000 Å

1 µm

Types of samples studied by macromolecular microscopy
Types of Samples Studiedby Macromolecular Microscopy

Repeating Assemblies

70S E. coliribosome

Hepatitis B virus core




2D crystal

Single particles

with little or no


Single particles with

icosahedral or other


Helical symmetry

Two-dimensional crystals

Baker and Henderson (2001)

Types of samples studied by macromolecular microscopy1
Types of Samples Studiedby Macromolecular Microscopy

Cells, Organelles, Pleomorphic Viruses, etc.

Human Immunodeficiency Virus - 1Zhu, et al. (2006)

Baker and Henderson (2001)

Nuclear Pore ComplexBeck, et al. (2007)

The never ending quest for higher resolution in em
The Never-Ending Quest for Higher Resolution in EM

Points of Resolution in Structural Information of Proteins

Fujiyoshi (1998) Adv. Biophys 35:25-80

Limits of resolution of various imaging technologies
Limits of Resolution of Various Imaging Technologies

The Never-Ending Quest for Higher Resolution in EM


Currently achieved resolution

Leis, et al. (2009)

Prediction for resolution improvement

The never ending quest for higher resolution in em1
The Never-Ending Quest for Higher Resolution in EM

Resolution of Selected Solved Structures

The never ending quest for higher resolution in em2
The Never-Ending Quest for Higher Resolution in EM

Visualizing Helices in Penicillium stoloniferum Virus

~ 350 Å Diameter~7.3 Å resolution

X-eyed Stereo

What limits resolution
What Limits Resolution?

  • The vacuum of the microscope - DEHYDRATION!

    • ~9 X 10-8 Torr or 1 X 10-10 atm

  • Look at the lengths we go to avoid this!

    • We dehydrate it with solvents

    • We flash freeze it and then freeze dry it

    • We embed it in heavy metals

    • We vitrify it (Flash frozen in amorphous ice)

What limits resolution1
What Limits Resolution?

  • The vacuum of the microscope - DEHYDRATION!


What limits resolution2
What Limits Resolution?

  • The vacuum of the microscope - Dehydration

  • Lack of contrast - Biological samples just don’t do a very good job at scattering electrons

  • Radiation damage - Biological samples just don’t like getting hit by the electron beam

Radiation damage primary and secondary effects
Radiation Damage - Primary and Secondary Effects

  • Primary Effects

    • Temperature independent

    • Occurs in the first 10-14 seconds

    • Excitation of orbital electrons of the sample forms ions and radicals

  • Secondary Effects

    • Secondary effects are what cause damage in the sample

    • Secondary effects are temperature dependent

    • Chemical and physical changes (breaking C-H and C-C bonds)

    • Mass loss (can be as great as 50%)

    • Charging effects

    • Contamination

      • Residual hydrocarbons in the vacuum chamber can break into fragments that become deposited on specimens.

      • Water vapor from insertion of the sample and from photographic film

    • Loss of order in crystalline specimens

Radiation damage loss of order in crystalline specimens
Radiation Damage - Loss of Order in Crystalline Specimens

Changes in the electron diffraction pattern of frozen-hydrated catalase crystals resulting from radiation damage

<1 e-/Å2

2.5 e-/Å2

2.8 Å

5.0 e-/Å2

11 e-/Å2

Example Dosage: The minimum dosage necessary to see an image on the screen at 20kX magnification is ~4e-/Å2/sec.

8.5 Å

Taylor and Glaeser (1976) J. Ultrastruc. Res. 55:448

Radiation damage frozen hydrated simian virus 40 dose series

10 e-/Å2

20 e-/Å2

30 e-/Å2

40 e-/Å2

Radiation DamageFrozen-hydrated Simian Virus 40 Dose Series


  • Metal shadowing

  • Negative/positive staining

  • Embedding in sugars and tannic acid

  • Cryo-electron microscopy

  • Cryo-negative staining

  • Cryo-tomography

Techniques metal shadowing
Techniques - Metal Shadowing

Wischnitzer (1970) Introduction to electron microscopy, 2nd ed.

Techniques metal shadowing1
Techniques - Metal Shadowing

  • Problems

    • Only see the surface

    • Standard techniques are low resolution, evaporated metals tend to be granular

    • Metal decoration

Actin + S1, Courtesy of John Heuser

Techniques negative positive staining
Techniques - Negative/Positive Staining

  • “Negative stain” is a misnomer. Most of the stain fills in sample depressions thereby preventing sample collapse. It does not stain the sample.

  • Sample appears “white” and the electron-dense stain is “black”.

  • Helps to reduce dehydration and radiation damage effects.

  • Attainable resolution is ~ 15-25 Å (~10 Å - GroEL De Carlo et al. 2008). 8 Å for crystals.

  • Positive staining occurs when ions of the stain react with the molecule.

Hayat & Miller (1990)Negative Staining

Techniques negative positive staining1
Techniques - Negative/Positive Staining

Examples of Commonly Used Negative Stains

  • Others:

  • Methylamine tungstate

  • Silver nitrate

  • Aurothioglucose

  • Sodium tetraborate

  • Cadmium iodide

Techniques negative positive staining2
Techniques - Negative/Positive Staining

Choosing the Proper Stain

  • High density

    • 3.8 - 5.7 gm/cc versus 1.37 gm/cc for protein

  • High solubility

  • High melting and boiling points

    • Stability in the beam

  • Stain needs to have a fine granularity (0.4 to 1.5 nm)

  • No chemical reaction with the specimen

  • Choice of stain may depend on the pH and salt concentration needs of the sample

Techniques negative positive staining3

Carbon-filmed grid

Techniques - Negative/Positive Staining

Procedure - Setup



Filter paperwedges

1% UA stain in water

Grid Box

Techniques negative positive staining4
Techniques - Negative/Positive Staining

Procedure - Hydrophilic Carbon Surface

Hydrophobic surface

Hydrophilic surface

Techniques negative positive staining5
Techniques - Negative/Positive Staining

Procedure - Glow Discharging the Grids

Techniques negative positive staining6
Techniques - Negative/Positive Staining

Procedure - Apply Sample to Grid

Techniques negative positive staining7
Techniques - Negative/Positive Staining

Procedure - Wash with dH2O

Techniques negative positive staining9
Techniques - Negative/Positive Staining

Procedure - Blot with Filter Paper

Techniques negative positive staining10
Techniques - Negative/Positive Staining


Bacteriophage T4

Maize Streak Virus

Techniques negative positive staining11
Techniques - Negative/Positive Staining


  • Unpredictable and uneven staining

  • High contrast images only the surface

  • Sample flattening

  • The electron beam can redistribute the stain

  • Different stains give different views

Parmecium bursariaChlorella virus

Uranyl acetate




Techniques negative positive staining12
Techniques - Negative/Positive Staining


“The question of what is artifact and what is not is a persistent one in electron microscopy, especially when micrographs depict what are essentially newer unexplored structures.“

Dr. Keith Porter, 1979.

From Heuser (2002)

Techniques embedding in sugars or tannic acid
Techniques - Embedding inSugars or Tannic acid

  • Making the preparation is similar to that of negative staining.

  • Specimen is supported by a matrix of concentrated sugar to maintain the need for hydration.

  • Often used with crystalline samples in order to keep them flat on the grid

  • Very beam sensitive, often need to keep sample at liquid nitrogen temperature

  • Very low contrast, sugar scatters electrons as well as protein (contrast matching)

Techniques cryo electron microscopy cryoem
Techniques - Cryo-Electron Microscopy (CryoEM)

  • Specimens are frozen in non-crystalline (vitreous ice). Freezing must be done in less than 10-4 sec.

  • Specimens are “frozen-hydrated”. This overcomes the problem of putting a hydrated sample in a vacuum.

  • Specimens are observed with “native contrast” - no staining, no fixatives.

  • Samples must be maintained below ~-140o C in the microscope. (below the vitreous to crystalline ice phase transition)

  • Maintains the native structure of the molecule to atomic resolution.

Bacteriophage  29

Techniques cryoem
Techniques - CryoEM

Holey Carbon Support Film

Techniques cryoem1
Techniques - CryoEM

Sample Preparation Equipment

Techniques cryoem2
Techniques - CryoEM

Sample Preparation Equipment

Guillotine Plunger

EM tweezers

LN2 dewar

Foot switch

Techniques cryoem3
Techniques - CryoEM

Sample Preparation Equipment

Guillotine Plunger


EM forceps

EM grid


LN2 dewar

Techniques cryoem4
Techniques - CryoEM

Addition of Sample and Blotting

Techniques cryoem5
Techniques - CryoEM

Addition of Sample and Blotting

Filter paper

Filter paper

EM grid


Techniques cryoem6

LN2 (-196 °C)

EM grid


Techniques - CryoEM

Plunging the Grid into Ethane and Transfer to Nitrogen

Techniques cryoem7
Techniques - CryoEM

Transferring to the Grid Storage Box

Techniques cryoem8
Techniques - CryoEM

Automated Freezing with the FEI Vitrobot

Environmental chamber


Techniques cryoem9
Techniques - CryoEM

Using the Vitrobot

Techniques cryoem10

EM tweezers

Sampleon grid

Filter paper disks

Techniques - CryoEM

Using the Vitrobot

Techniques cryoem11
Techniques - CryoEM

Using the Vitrobot

Techniques cryoem12
Techniques - CryoEM

Using the Vitrobot

Techniques cryoem13
Techniques - CryoEM

Cryo-transfer Workstation and Holder


Transfer Area


Nitrogen Dewar


Techniques cryoem14
Techniques - CryoEM

Transferring the Grid into the Cryo-holder

Grid Box




Techniques cryoem15
Techniques - CryoEM

Transferring the Grid into the Cryo-holder

Grid Box




Techniques cryoem16
Techniques - CryoEM

Transfer of the Grid to the Cryo-holder

Techniques cryoem17
Techniques - CryoEM

Cryo-holder in Microscope


Cryo negative staining
Cryo-Negative Staining

Sampleon grid

Saturated AmmoniumMolybdate

Place grid on drop

for 30 sec.

Blot with filter

paper. Allow to dry a

few seconds


Adapted from Adrian et al. (1998) Micron 29:145-160

Cryo negative staining1
Cryo-Negative Staining

  • Produces high contrast images

  • Maintains about 30% water by volume

  • Possibly somewhat higher potential resolution than standard negative stain technique

  • No addition of noise from a carbon support film

  • Little or no sample flattening

  • Some sample-stain interaction with sensitive samples (sample dissociation)

  • Structure information dominated by the stain envelope


Tomato bushy stunt virus



Cryo-negative stained

Adrian et al. (1998) Micron 29:145-160

Cryo tomography


  • Plunge freezing reduces artifacts, visualizes the sample in its native, hydrated state.

  • Provides resolution between 40 and 70 Å.

  • Images have very low contrast.

  • Samples are extremely beam sensitive.

  • Limited to small cells or isolated organelles. Multiple electron scattering through large objects reduces resolution.



Bridging the Gap between Cellular and Macromolecular Microscopy

Section from a 3D cryo-tomographic reconstructionof a Caulobacter crescentus cell

Jensen and Briegel (2007) COSB 17(2): 260-267.

Low dose microscopy
Low Dose Microscopy

  • Liquid nitrogen temperature reduces the effects of the electron beam but the sample is still extremely beam sensitive.

  • It is not possible to view the sample without destroying it - in effect - you are shooting blind.

  • Exposure and magnification parameters are set for a Search position, two Focus positions, and an Exposure position.

Low dose microscopy1
Low Dose Microscopy




8 X 10 cmmicrograph

Focus pointburn holes

Demonstration and lab procedures
Demonstration and Lab Procedures

  • Tim Baker’s courses

  • Molecular Microscopy: Winter Quarter

  • Structural Virology: Spring Quarter



  • Adrian, M., J. Dubochet, S. D. Fuller, and R. Harris (1998). Cryo-negative staining. Micron 29(2/3), 145-160.

  • Baker, T. S., and R. Henderson (2001). Electron cryomicroscopy. In "International Tables for Crystallography" (Rossmann, M. G., and E. Arnold, Eds.), Vol. F, pp. 451-463, 473-479. Dordrecht:Kluwer Academic Publishers, The Netherlands.

  • Baker, T. S., N. H. Olson, and S. D. Fuller (1999). Adding the third dimension to virus life cycles: Three-dimensional reconstruction of icosahedral viruses from cryo-electron micrographs. Microbiol. Mol. Biol. Rev. 63(4), 862-922.

  • Beck, M., V. Lucic, F. Forster, W. Baumeister, and O. Medalia (2007). Snapshots of nuclear pore complexes in action captured by cryo-electron tomography. Nature 449(7162), 611-615.

  • Briegel, A., D. P. Dias, Z. Li, R. B. Jensen, A. S. Frangakis, and G. J. Jensen (2006). Multiple large filament bundles observed in Caulobacter crescentus by electron cryotomography. Mol. Microbiol. 62(1), 5-14.

  • De Carlo, S., N. Boisset, and A. Hoenger (2008). High-resolution single-particle 3D analysis on GroEL prepared by cryo-negative staining. Micron 39(7), 934-943.

  • Dubochet, J., M. Adrian, J. J. Chang, J. C. Homo, J. Lepault, A. W. McDowall, and P. Schultz (1988). Cryo-electron microscopy of vitrified specimens. Q. Rev. Biophys. 21(2), 129-228.

  • Fujiyoshi, Y. (1998). The structural study of membrane proteins by electron crystallography. Adv. Biophys. 35, 25-80.

  • Gonen, T., Y. Cheng, P. Sliz, Y. Hiroaki, Y. Fujiyoshi, S. C. Harrison, and T. Walz (2005). Lipid-protein interactions in double-layered two-dimensional AQP0 crystals. Nature 438(7068), 633-638.

  • Hayat, M. A., and S. E. Miller (1990). "Negative Staining." McGraw Hill Publishing, New York.


  • Henderson, R. (2004). Realizing the Potential of Electron Cryomicroscopy. Q. Rev. Biophys. 37(1), 3-13.

  • Heuser, J. (2002). Whatever happened to the 'microtrabecular concept'? Biol. Cell 94(9), 561-596.

  • Jensen, G. J., and A. Briegel (2007). How electron cryotomography is opening a new window onto prokaryotic ultrastructure. COSB 17(2), 260-267.

  • Leis, A., B. Rockel, L. Andrees, and W. Baumeister (2009). Visualizing cells at the nanoscale. TIBS 34(2), 60-70.

  • Liang, Y., J. Jakana, X.-K. Yu, J.-Q. Zhang, W. Chiu, and Z. H. Zhou (2003). High-resolution 3D reconstruction of cytoplasmic polyhedrosis virus. Microsc. Microanal. 9(Suppl. 2), 1366-1367.

  • LeBarron, J., R. A. Grassucci, T. R. Shaikh, W. Baxter, J. Sengupta, and J. Frank (2008). Exploration of Parameters in Cryo-EM Leading to an Improved Density Map of the E. coli Ribosome. J. Struct. Biol. 164(1):24-32.

  • Ludtke, S. J., M. L. Baker, D.-H. Chen, J.-L. Song, D. T. Chuang, and W. Chiu (2008). De Novo Backbone Trace of GroEL from Single Particle Electron Cryomicroscopy. Struct. 16(3), 441-448.

  • Matadeen, R., A. Patwardhan, B. Gowen, E. V. Orlova, T. Pape, M. Cuff, F. Mueller, R. Brimacombe, and M. van Heel (1999). The Escherichia coli large ribosomal subunit at 7.5 Å resolution. Struct. 7(12), 1575-1583.

  • Mitsuoka, K., T. Hirai, K. Murata, A. Miyazawa, A. Kidera, Y. Kimura, and Y. Fujiyoshi (1999). The Structure of Bacteriorhodopsin at 3.0 angstroms Resolution Based on Electron Crystallography:Implication of the Charge Distribution. J. .Mol. Biol. 286, 861-882.


  • Sachse, C., J. Z. Chen, P.-D. Coureux, M. E. Stroupe, M. Fandrich, and N. Grigorieff (2007). High-resolution electron microscopy of helical specimens: A fresh look at tobacco mosaic virus. J. Mol. Biol. 371(3), 812-835.

  • Taylor, K. A., and R. M. Glaeser (1976). Electron microscopy of frozen hydrated biological specimens. J. Ultrastruct. Res. 55, 448-456.

  • Wischnitzer, S. (1970). In "Introduction to Electron Microscopy". PergamonPress, N. Y.

  • Wolosewick, J.J., Porter, K.R., 1979. Microtrabecular lattice of the cytoplasmic ground substance. Artifact or reality. J. Cell Biol. 82 (1),114–139.

  • Yonekura, K., S. Maki-Yonekura, and K. Namba (2005). Building the Atomic Model for the Bacterial flagellar filament by electron cryomicroscopy and image analysis. Struct. 13(3), 407-412.

  • Zhang, X., E. Settembre, C. Xu, P. R. Dormitzer, R. Bellamy, S. C. Harrison, and N. Grigorieff (2008). Near-atomic resolution using electron cryomicroscopy and single-particle reconstruction. PNAS 105(6), 1867-1872.

  • Zhu, P., J. Liu, J. Bess, E. Chertova, J. D. Lifson, H. Grisé, G. A. Ofek, K. A. Taylor, and K. H. Roux (2006). Distribution and three-dimensional structure of AIDS virus envelope spikes. Nature 441(7095), 847-852.