Center for Microscopy and Image Analysis
Introduction to
Electron Microscopy
Andres Kaech
Instrumentation
Why electron microscopy?
Resolution Limit
Wavelength/Size
Object
1 mm
MRI, CT
Radio
Human eye
100 μm
Cells
Infrared
10 μm
Red blood cells
Visible
Light microscope
Ultraviolet
1 μm
Bacteria
100 nm
Mycoplasma
n
o
r
t
c
Ele
py
o
c
ro s
c
i
m
Viruses
10 nm
Proteins
x,γ-rays
1 nm
Electron microscope
0.1 nm
Amino acids
Atoms
Ultrastructure
The types of electron microscopes
Transmission electron microscope (TEM)
Scanning electron microscope (SEM)
Hela Cells
2 µm
1 µm
The types of electron microscopes
Transmission electron microscope (TEM)
Electron beam
Specimen
Scanning electron microscope (SEM)
Electron beam
~100 nm
Specimen
Projection
Surface
2 µm
1 µm
Why electron microscopy?
Rat intestine
Light microscopy
Electron microscopy
Microvilli
1 µm
Adherence junction: ß-catenin visualized
by immunolabelling using „immunogold“
10 µm
Green…F-actin
Yellow…ß-catenin
Orange…Nuclei
Schwarz & Humbel 2007: Methods in Molecular Biology, vol. 369, Electron Microscopy: Methods and Protocols, Second Edition
Edited by: J. Kuo © Humana Press Inc., Totowa, NJ
Why electron microscopy?
Mouse intestine
Actin filaments
Glycocalix
Junction
Microvilli
1 µm
Elektronenmikroskopie ETH Zurich
Specimens courtesy of Bärbel Stecher, Institute of Microbiology, ETH Zurich
Why electron microscopy?
Mouse intestine
Membrane
(lipid bilayer)
Actin filaments
500 nm
Elektronenmikroskopie ETH Zürich
From photons to electrons
Light microscopes
Photons
Photons are substituted with electrons
Glass lenses are substituted with electromagnetic and electrostatic lenses
Electron microscope
Electrons
e-
From photons to electrons
e-
Similarities to photons:
Wave-particle duality of electrons
Optical properties
(Diffraction, chromatic abberation, spherical abberation,
astigmatism etc.)
Resolution depends on aperture and wavelength
(Diffraction limited resolution)
Abbe’s equation d = 0.61 λ/NA
NA = n ⋅ sin α
Resolution of electron microscopes
The higher the energy of the electrons, the lower the wavelength,
the higher the resolution
Acceleration voltages of electrons:
Transmission electron microscopes (TEM): 40 – 1200 kV
Scanning electron microscopes (SEM): 1 – 30 kV
Effective instrument resolution TEM: ≈ 0.1 nm
Effective instrument resolution SEM: ≈ 1 nm
However:
Resolution of biological objects is limited by specimen preparation:
Practical resolution: > 1 nm
Transmission electron microscope vs. Widefield light microscope
2 µm
Transmission electron microscope
Electron gun
Electromagnetic
lens
TEM grid
Widefield light microscope
Illumination
Condenser lens
Specimen
Lamp
Glass lens
Slide
Electromagnetic
lens
Objective lens
Glass lens
Electromagnetic
lens
Projector lens
Glass lens
Phosphorescent
screen
CCD camera
Final image
Eye
CCD camera
Scanning electron microscope vs. Confocal scanning laser microscope
1 µm
Scanning electron microscope
Confocal scanning laser microscope
Laser
Illumination
Electron gun
Photomultiplier (Detector)
Photomultiplier
Detector
Electromagnetic
lenses
Electromagnetic lens
Electromagnetic/
electrostatic lens
Lens system
“condenser”
Beam scanner
Objective
X-ray,
photomultiplier
Specimen
Glass lenses
Mirror
Glass lenses
Electron microscopes are high vacuum systems
Transmission electron microscope (TEM)
Scanning electron microscope (SEM)
High vacuum
Without vacuum:
• Electrons would collide with gas molecules
• Electron source (tungsten) would blow
Vacuum systems
Example: Transmission electron microscope
Cathode
10-7 - 10-10 mbar
IGP
Ion getter pump / Oil diffusion pump
Specimen holder
(object plane)
10-5 - 10-7 mbar
IGP
Turbo molecular pump
TMP
10-0 - 10-2 mbar
Rotary pump
Viewing screen
RP
Atmosphere: 1000 mbar
Electron source (Electron gun)
High voltage
(acceleration
voltage of e-)
Electron source (Electron gun)
Thermionic emission
(tungsten, LaB6, Schottky emitter)
Cold field emission
(quantum-mechanical tunneling)
Tungsten
Thermionic
LaB6
Schottky
Cold field emission
Material
W
LaB6
ZrO/W
W
Heating temp. (K)
2700
1800
1800
300
Normalized brightness
Required vacuum (Pa)
∆E (eV)
Ultra high
high
Chromatic aberration!
Electromagnetic lenses
Electromagnetic lens of a transmission electron microscope
Electromagnetic lenses
Magnetic field depends on current and number of windings
e-
e-
Advantage of a electromagnetic lenses:
The focal length can be changed by changing the
current:
I
No movement or exchange of the lens is required
for changing focus or magnification!
►Image is inverted and rotated
►Image rotation is corrected in microscopes
Electromagnetic lenses
Axial astigmatism of electromagnetic lenses … confusion of the image
Most relevant aberration in biological electron microscopy
x
Objective lens
“True” image
of object
Optical axis
Object:
Source of e(off-axis)
z
y
Focus
Reasons:
• Inhomegenities of the lens
• Contamination of lenses and apertures
• Charging of specimen
Electromagnetic lenses
Axial astigmatism of electromagnetic lenses … confusion of the image
Most relevant aberration in biological electron microscopy
x
“True” image
of object
Objective lens
Optical axis
Object:
Source of e(off-axis)
y
Cellulose filter
paper in SEM
Under focused image
elliptic deformation
Focus
circle of least confusion
Over focused image
elliptic deformation
Electromagnetic lenses
Axial astigmatism of electromagnetic lenses … confusion of the image
Most relevant aberration in biological electron microscopy
x
Objective lens
Corrector coils
“True” image
of object
Optical axis
Object:
Source of e(off-axis)
y
Cellulose filter
paper in SEM
Focus, corrected astigmatism
circle of confusion minimized
Electromagnetic lenses
Chromatic aberration
Due to energy difference of electrons (wavelength)
e- (98 kV)
e- (100 kV)
e- (102 kV)
Curvature and distortion of field
Spherical aberrations
Specimen holders and stages
2 µm
Transmission electron microscope
Goniometer: x, y, z, r
Specimen size:
• 3 mm in diameter!
• Ca. 100 nm in thickness
(electron transparent)
Specimen holders and stages
2 µm
Specimen on a TEM grid
3 mm
Specimen holder
Specimen holders and stages
1 µm
Scanning electron microscope
Viewing chamber = Specimen chamber
Gun
Objective lens
Specimen stub
Stub holder
Stage
Specimen stage (x, y, z, r, tilt)
Specimen size:
• 100 mm in diameter
• 2 cm in z-direction (not electron transparent)
Electron – specimen interactions
Primary electrons (E0)
Backscattered electrons (E=E0)
Elastic
(higher angle, E=E0)
Inelastic
(low angle, E=E0-∆E)
Unscattered
(E=E0)
Electron – specimen interactions
Inelastic scattering:
Primary electrons hit electrons of the
specimen atom
2
Energy is transferred from the
primary electron to the specimen
K
1
L
Emission of electrons and radiation
M
N
Electron – specimen interactions
Primary electrons
Backscattered electrons
X-rays
Secondary electrons
SEM analysis
Cathode luminescense
Heat
Auger electrons
Specimen
Interaction volume
TEM analysis
Elastically scattered electrons
Inelastically scattered electrons
Unscattered electrons
Imaging in the transmission electron microscope
2 µm
Illumination
Condenser lens
Specimen
Specimen: Electron transparent
(very thin: 100 nm)
Objective lens
Projector lens
Final image
Image: 2D projection of a volume
• CCD camera
• Phosphorescent screen
• Conventional photosensitive film
Imaging in the transmission electron microscope
2 µm
Contrast formation in TEM
™ Absorption of electrons
™ Scattering of electrons
™ Phase contrast
NOTE: All mechanisms occur at the same time (superposition)
Question: Which mechanism is most relevant for biological specimens?
Imaging in the transmission electron microscope
2 µm
Contrast formation in TEM
Biological specimen consist of light elements:
™ Absorption contrast weak
™ Scattering contrast weak
“LOW CONTRAST”
™ Phase contrast weak
Contrast enhancement required:
Treatment with heavy metals (Ur, Pb, Os)!
Heavy metals attach differently to different components
Imaging in the transmission electron microscope
2 µm
Main contrast formation in plastic embedded specimens
™ Scattering of electrons through heavy metals
Primary electron beam
…Heavy metal ions
phospholipids
Specimen
ribosome
Objective aperture
Signal
Intensity
Specimen profile
Imaging in the transmission electron microscope
2 µm
Thin section of alga stained with heavy metals (Ur, Pb)
Electron microscopy ETH Zurich
Imaging in the transmission electron microscope
2 µm
Thin section of alga without heavy metal staining
1 µm
Electron microscopy ETH Zurich
Imaging in the transmission electron microscope
2 µm
Contrast enhancement by underfocusing
Phase differences of diffracted and non-diffracted rays are increased or
decreased by changing the focus
Underfocus
Close to focus
Overfocus
Fresnel rings
Carbon film , ca. 4 nm
100 nm
Imaging in the transmission electron microscope
2 µm
Contrast enhancement by underfocusing
Thin section of a frozen-hydrated apple leaf (“unstained”)
1 µm
Electron microscopy ETH Zurich
Phase contrast only (H2O vs. biological material)
Imaging in the scanning electron microscope
1 µm
Scanning electron microscope
Illumination
Photomultiplier
Detector
• Photomultiplier
• No CCD camera
Lens system
“condenser”
Beam scanner
Objective
Specimen
Specimen: Bulk specimen
Imaging in the scanning electron microscope
1 µm
Scanning and signal detection
Scanning of the specimen
Imaging in the scanning electron microscope
1 µm
Signal and detection
Primary electrons
Backscattered electrons
X-rays
Secondary electrons
Heat
Auger electrons
Specimen
SEM analysis
Cathode luminescense
Interaction volume
TEM analysis
Elastically scattered electrons
Inelastically scattered electrons
Unscattered electrons
Imaging in the scanning electron microscope
1 µm
Signal and detection
Different properties of the different signals
► Specific detectors
► Different/specific information
Imaging in the scanning electron microscope
1 µm
Secondary electron detector
Primary electrons
+200-500V – Collector voltage
+7-12kV HV
SE
Electrons
Photons
Photomultiplier
Electrons
Imaging in the scanning electron microscope
1 µm
Contrast formation in SEM (using SE and BSE)
Different number of electrons from different spots of the specimen
Dependent on
™ location of the detector
™ topography of the specimen
™ acceleration voltage of primary electrons
™ composition of the specimen
Imaging in the scanning electron microscope
1 µm
Contrast based on SE
Virtual light source
SE detector
(inlens)
SE detector
Leg of an ant, coated with ca. 10 nm Platinum
Electron microscopy ETH Zurich
Imaging in the scanning electron microscope
1 µm
Contrast based on SE
PE
PE
SE
R
PE
PE
PE
PE
Φ
SE
SE
R
R
R
R
Specimen
R
Primary electrons
Secondary electrons
Excited volume
Imaging in the scanning electron microscope
1 µm
Contrast formation in SEM
Biological material (light elements):
Only few electrons escape from specimen
Almost no contrast, similar contrast everywhere on specimen
Unsharp image (electrons from “large” volume)
Contrast enhancement important & needed:
Localization of the signal to the surface
Coating of biological specimen with thin heavy metal layer (a few nm)
Reducing acceleration voltage
Imaging in the scanning electron microscope
1 µm
Contrast based on SE: Non-coating vs. coating with heavy metals
Uncoated
Coated with 4 nm platinum
500 nm
Freeze-fractured yeast
Electron microscopy ETH Zurich
Imaging in the scanning electron microscope
1 µm
Contrast formation
Uncoated
Coated with 4 nm platinum
Primary electron beam
Primary electron beam
Platinum