Transmission Electron
Microscopy
Henning Stahlberg
Purified (Membrane) Protein
Single
Particles
Nuclear Magn.
Resonance
3D model
(small
proteins)
2D
Crystallization
Electron
Microscopy
3D model
Resolution:
12 Å
3D
Crystallization
Atomic Force
Microscopy
3D model
Resolution:
x,y: 2.5 Å
z: 3.0 Å
2D surface
Resolution:
x,y: 5 Å
z: 1 Å
X-Ray
Diffraction
3D model
Resolution:
2Å
Dimensions in Life Sciences
light microscopes
electron microscopes
scanning probe microscopes
nuclear magnetic resonance
X-ray diffraction
Wave-Particle Dualism
!
Waves
<===>
Particles
momentum p=h/!
wavelength: !
frequency: "
velocity: v=!#"
mass: m = m 0 / 1- (v / c)2
(e.g., wavefront, diffraction, ...)
(e.g., trajectories, scattering, ...)
velocity: v
momentum p=m*v
Information Carriers
Transmission Microscopes
Photons
Infrared
Visible
Ultraviolet
0.1 - 1 eV
1 - 3 eV
3 - 500 eV
10 - 1 µm
1 - 0.3 µm
0.3 - 0.01 µ m
Electrons
1 kV
10 kV
100 kV
1000 kV
0.4 Å
0.12 Å
0.037 Å
0.009 Å
The Transmission Electron Microscope
-100’000 Volt !
Sample
Vacuum !
Viewing
Screen
8
Electron Sources
Heated filament
Field emission gun
Electrons are focused by magnetic fields
The magnetic electron lens
Flying Electrons require Vacuum
Interaction Electrons-Specimen
Electrons are scattered, not absorbed by the atoms of the sample.
Transmission Electron Microscopes
300 kV
FEG
The Champion
300 kV
Helium Cold Stage
Energy Filter
4k CCD Camera
Transmission Electron Microscopes
300 kV
FEG
The Champion
200 kV
200 kV
120 kV
The Crashtest Dummy
LN2 Cold Stage
4k CCD Camera
The Reliable
Transmission Electron
Microscope
Specimen Preparation
eElectron
source
Electron
beam
Eye
Specimen
Vacuum
Viewing
screen
Microscopy Grid
Carbon-Film
Specimen Preparation Methods
Negative Stain
Shadowing
Replica
Labeling
Cryo EM
Transmission Electron
Microscope
Specimen Preparation
e-
Proteins
Negative Stain
Carbon-Film
LON
Neg. Stain
TEM
Continous
C-film
Room
Temperature
Negative Stain
GroEL:
Protein Folding Tool
10 nm
Negative Stain
0°-/45°-tilt pair of negatively stained, unfixed
'plug-spoke' complexes, i.e., a distinct nuclear
pore complex (NPC) component, obtained
after detergent treatment (0.1% Triton X-100)
of spread Xenopus laevis oocyte nuclear
envelopes (NEs).
Negative Stain
Electron micrographs of negatively stained Nudaurelia capensis b icosahedral virus (NbV)
Virions viewed close to their icosahedral 2-fold (2) or 3-fold (3) axes are labeled.
Specimen Preparation Methods
Negative Stain
Shadowing
Replica
Labeling
Cryo EM
Specimen Preparation
e-
Holey Carbon Film
Carbon-Film
Microscopy Grid
Transmission Electron
Microscope
Specimen Preparation
eElectron
source
Proteins
Cold: -180°C !!!
Buffer
Solution
Negative
Stain
Carbon-Film
Cryo EM : Sample Preparation
Blott the drop
away with
blotting-paper
Copper-grid with
holey carbon film
Put a drop onto it
Plunge the grid
into liquid ethane
(T = -180°C)
LON
CryoTEM
Holey C-film
-180°C
Neg. Stain
vs.
Cryo-TEM
Electron micrographs of
negatively stained (top)
and frozen-hydrated (bottom)
Nudaurelia capensis
b icosahedral virus (NbV),
Cryo-Electron Microscopy
Infection of bacteria
(here liposomes with FhuA)
by the Bacteriophage T5
Animation by Rossmann group (Purdue),
based on data by Peter Leiman et al. (now EPFL)
Lambert et al. Mol Microbiol 1998
Specimen Preparation Methods
Negative Stain
Shadowing
Replica
Labeling
Cryo EM
Cryo
TEM
Continous
C-film
-180°C
da Vinci
100 %
AFM
90 %
TEM neg.stain
60 %
70 %
80 %
TEM cryo holey C-film
50 %
30 %
40 %
TEM cryo closed C-film
20 %
10 %
0%
The Transmission Electron Microscope
-100’000 Volt !
Sample
Vacuum !
Viewing Screen
Fourier Theorem
Real Space
Fourier Space
g(x) = h(x) x f(x)
Image = PointSpreadFunction
convoluted with
Structure
FT(Image) =
ContrastTransferFunction
multiplied with
FT(Structure)
These two things are IDENTICAL !!!
Transfer Function
Transfer Function
Transfer Function of Microscopes
Otto Scherzer
(Mar. 9, 1909 - Nov. 15, 1982)
Imaging of atoms, optimum phase contrast, spherical correction
Electron Sample Interaction
e
e
e
e
energy
(light, etc.)
e
e
+
e
sample
atom
wide angles
unscattered
primary electrons
e
X-rays
+
e
small angles
e
e
e
secondary
electrons
inelastically scattered
primary electrons
elastically scattered
primary electrons
Electron Sample Interaction
Auger electrons
E < 10 eV
electron beam
E0
backscattered electrons
E0 - E
secondary electrons
E < 20 ... 50 eV
X-ray radiation
positive ions
visible light
thin sample
elastically scattered electrons
E0
inelastically scattered electrons
E0 - E
unscattered electrons
E0
Contrast
electron beam
thin sample
scattering angle
objective lens
scattered
beam
(phase shift = W)
unscattered
beam
Interference gives contrast
screen
Scherzer Formula
sin($(u)): phase contrast transfer function
cos($(u)): amplitude contrast transfer function
u: scattering vector (!scattering angle)
W: wave aberation
!: electron wavelength
%z: defocus
Cs: spherical aberation constant
CTF
CTF(u) = { A * cos($(u)) - sqrt(1-A2 ) * sin($(u)) } * E(u)
sin($(u)): phase contrast transfer function
cos($(u)): amplitude contrast transfer function
u: scattering vector (!scattering angle)
A: Amplitude contrast fraction. (neg. stain: use 0.07)
The CTF with Envelope Function
%Cs
%z
1
1
!z = -200 mm
-1
Cs = 1 mm
Cs = 2 mm
Cs = 3 mm
!z = -60 nm
1
2
3
4
-1
5
Resolution [1/nm]
!z = -50 mm
!z = -400 mm
Cs = 1.6 mm
1
2
CTF(u) = sin($(u)) * E(u)
3
4
5
Resolution [1/nm]
Otto Scherzer
Lecture on Thermodynamics, 1958
Thon rings = Zeros of the CTF
Real Space:
Point Spread Function
Object X PSF = Image
Fourier Space:
Contrast Transfer Function
FT(Object) • CTF = FT(Image)
FT(PSF) ! CTF
PSF ! FT(CTF)
Can we correct the CTF ?
Otto Scherzer
Lecture on Thermodynamics, 1958