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?

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
Bacteriorhodopsin
~58 Å, ~26 kDa
Theoretical Biophysics Group
Beckman Institute
University of Illinois at
Urbana-Champaign
100 Å
70S E. coli ribosome
~250 Å
Courtesy J. Frank
Paramecium bursaria
Chlorella Virus 1
Caulobacter
crescentus
~1900 Å, ~1 GDa
~0.6 X 2 µm
Timothy S. Baker Group
UCSD
Briegel, et al. (2006)
1000 Å
1 µm
Types of Samples Studied
by Macromolecular Microscopy
Repeating Assemblies
70S E. coli
ribosome
Single particles
with little or no
symmetry
Hepatitis B
virus core
Single particles
with
icosahedral or
other
symmetries
Actin-myosin
filament
Helical
symmetry
Light-harvesting
2D crystal
Two-dimensional
crystals
Baker and Henderson (2001)
Types of Samples Studied
by Macromolecular Microscopy
Cells, Organelles, Pleomorphic Viruses, etc.
Baker and
Henderson (2001)
Nuclear Pore Complex
Beck, et al. (2007)
Human
Immunodeficiency
Virus - 1
Zhu, et al. (2006)
The Never-Ending Quest for Higher Resolution in EM
Points of Resolution in Structural Information of Proteins
Resolution
(Ångstroms)
Observable Structural Features
20.0
Overall shape of molecule
7.0
Helices of rods of strong
intensity
3.5
The main chain with some
ambiguities
3.0
The side chains partly resolved
2.5
Side chains well resolved; Atoms
located to about +/- 0.04
1.5
Atoms located to about +/- 0.01
1.0
Anisotropic structure of atomic
potential
Fujiyoshi (1998) Adv. Biophys 35:25-80
The Never-Ending Quest for Higher Resolution in EM
Limits of Resolution of Various Imaging Technologies
& NMR
Currently achieved resolution
Prediction for resolution
improvement
Leis, et al. (2009)
The Never-Ending Quest for Higher Resolution in EM
Resolution of Selected Solved Structures
Resolution
(Ångstroms)
Structure, Authors, Date
1.9
Aquaporin - Gonen et al. (2005)
3.8
Rotavirus - Zhang et al. (2008)
4.0
GroEL - Ludtke et al. (2008)
5.0
Tobacco mosaic virus - Sachse et al.
(2007)
6.7
E. coli ribosome complex - LeBarron et
al. (2008)
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?

The vacuum of the microscope - DEHYDRATION!
-8 Torr or 1 X 10-10 atm
 ~9 X 10
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 Resolution?

The vacuum of the microscope - DEHYDRATION!

Lack of contrast - BIOLOGICAL SAMPLES JUST DON’T
DO A VERY GOOD JOB AT SCATTERING ELECTRONS!
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

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
<1 e-/Å2
2.5 e-/Å2
2.8 Å
5.0 e-/Å2
11 e-/Å2
8.5 Å
Changes in the
electron diffraction
pattern of frozenhydrated catalase
crystals resulting
from radiation
damage
Example Dosage:
The minimum dosage
necessary to see an
image on the screen
at 20kX
magnification is ~4e/Å2/sec.
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
Techniques

Metal shadowing

Negative/positive staining

Embedding in sugars and tannic acid

Cryo-electron microscopy

Cryo-negative staining

Cryo-tomography
Techniques - Metal Shadowing
Wischnitzer (1970) Introduction to electron
microscopy, 2nd ed.
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

“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 Staining
Examples of Commonly Used Negative Stains
Negative Stain
Chemical
Formula
pH for use
Others:
•Methylamine tungstate
Cationic stains
Uranyl acetate
UO2[CH3COO]2
2 – 4.5
Uranyl formate
UO2(CHO2)2.H2O
3.5 - 5.0
•Silver nitrate
•Aurothioglucose
•Sodium tetraborate
Anionic stains
Ammonium
molybdate
[NH4]6Mo7O26
5-8
Sodium phosphotungstate
Na3PO412WO3
5-8
Sodium
silicotungstate
Na3SiO212WO3
5-8
•Cadmium iodide
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 Staining
Procedure - Setup
dH2O
Sample
Filter paper
wedges
Carbon-filmed grid
1% UA stain
in water
Grid Box
Techniques - Negative/Positive Staining
Procedure - Hydrophilic Carbon Surface
Hydrophobic surface
Hydrophilic surface
Techniques - Negative/Positive Staining
Procedure - Glow Discharging the Grids
Techniques - Negative/Positive Staining
Procedure - Apply Sample to Grid
Techniques - Negative/Positive Staining
Procedure - Wash with dH2O
Techniques - Negative/Positive Staining
Procedure - Apply Stain
Techniques - Negative/Positive Staining
Procedure - Blot with Filter Paper
Techniques - Negative/Positive Staining
Results
Bacteriophage T4
Maize Streak Virus
Techniques - Negative/Positive Staining
Problems

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
bursaria
Chlorella virus
Uranyl
acetate
stained
Cryoelectron
microscopy
Techniques - Negative/Positive Staining
Artifacts
From Heuser (2002)
“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.
Techniques - Embedding in
Sugars 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)

Specimens are frozen in noncrystalline (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
Holey Carbon Support Film
Techniques - CryoEM
Sample Preparation Equipment
Techniques - CryoEM
Sample Preparation Equipment
Guillotine Plunger
EM tweezers
LN2 dewar
Foot switch
Techniques - CryoEM
Sample Preparation Equipment
Guillotine Plunger
EM forceps
EM grid
LN2
LN2 dewar
ethane
Techniques - CryoEM
Addition of Sample and Blotting
Techniques - CryoEM
Addition of Sample and Blotting
Filter paper
Filter paper
EM grid
Sample
Techniques - CryoEM
Plunging the Grid into Ethane and Transfer to Nitrogen
LN2 (-196 °C)
EM grid
ethane
Techniques - CryoEM
Transferring to the Grid Storage Box
Techniques - CryoEM
Automated Freezing with the FEI Vitrobot
Environmental chamber
Computer-controlled
Techniques - CryoEM
Using the Vitrobot
Techniques - CryoEM
Using the Vitrobot
EM tweezers
Sample
on grid
Filter paper disks
Techniques - CryoEM
Using the Vitrobot
Techniques - CryoEM
Using the Vitrobot
Techniques - CryoEM
Cryo-transfer Workstation and Holder
Transfer Area
Cover
Nitrogen Dewar
Workstation
Cryo-Shield
Techniques - CryoEM
Transferring the Grid into the Cryo-holder
Grid Box
Cryo-Shield
Grid
Clipring
Techniques - CryoEM
Transferring the Grid into the Cryo-holder
Grid Box
Cryo-Shield
Grid
Clipring
Techniques - CryoEM
Transfer of the Grid to the Cryo-holder
Techniques - CryoEM
Cryo-holder in Microscope
Holder
Cryo-Negative Staining
Sample
on grid
Saturated
Ammonium
Molybdate
Place grid on drop
for 30 sec.
Blot with filter
paper. Allow to dry a
few seconds
Plunge
freeze
Adapted from Adrian et al. (1998) Micron 29:145-160
Cryo-Negative Staining
Advantages





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
Tomato bushy stunt virus
Disadvantages


Some sample-stain interaction with
sensitive samples (sample
dissociation)
Structure information dominated by
the stain envelope
Frozen-hydrated
Cryo-negative stained
Adrian et al. (1998) Micron 29:145-160
Cryo-Tomography
Bridging the Gap between Cellular and Macromolecular Microscopy
Advantages
Plunge freezing reduces
artifacts, visualizes the sample
in its native, hydrated state.
 Provides resolution between 40
and 70 Å.

Disadvantages



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.
Section from a 3D cryo-tomographic reconstruction
of a Caulobacter crescentus cell
Jensen and Briegel (2007) COSB 17(2): 260-267.
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 Microscopy
1K
2K
4K CCD
8 X 10 cm
micrograph
Focus point
burn holes
Demonstration and Lab Procedures
http://cryoem.ucsd.edu/
Tim Baker’s courses
1. Molecular Microscopy: Winter Quarter
2. Structural Virology: Spring Quarter
References

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.
References

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), 13661367.

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), 441448.

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.
References

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.