introduction to electron microscopy andres kaech ......electron microscopy andres kaech...
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Introduction to
Electron Microscopy
Andres Kaech
Instrumentation and Image Formation
Center for Microscopy and Image Analysis
The types of electron microscopes
Scanning electron microscope (SEM)Transmission electron microscope (TEM)
Scanning electron microscope (SEM)Transmission electron microscope (TEM)
The types of electron microscopes
Electron beam
Specimen ~100 nm
Electron beam
Specimen
Projection Surface
1 µm1 µm
Hela Cells
Examples TEM
Mouse cerebellum
500 nm
Mitochondrium
Nucleus
GolgiRibosomes
SynapseDendrite
Microtubule
Examples TEM
Mouse cerebellum
100 nm
Mitochondrium
Nucleus
Golgi
Lipid bilayer
H/K -ATPase in cimetidine-treated resting gastric parietal
cells (rabbit).
Immunolabelling: Localization of proteins
Sawaguchi et al. 2004, Journal of Histochemistry & Cytochemistry
100 nm
Examples TEM
Examples SEM
Mouse kidney
500 µm
Mouse kidney (glomerulus)
10 µm
Examples SEM
Pseudomonas aeruginosa
500 nm
Examples SEM
Wave-particle duality
Resolution depends on aperture and wavelength
(Diffraction limited resolution)
Optical properties
(Diffraction, chromatic abberation, spherical abberation,
astigmatism etc.)
Abbe’s equation d = 0.61 λ/NA sin nNA
e-
Properties of electrons
Very similar to photons:
Resolution of biological objects limited by specimen preparation:
Practical resolution: > 1 nm
TEM: 40 – 300 kV
Effective instrument resolution TEM: 0.5 nm (120 kV)
Effective instrument resolution SEM: 1 nm
Resolution of electron microscopes
The higher the energy of the electrons, the lower the wavelength,
the higher the resolution
SEM: 0.5 – 30 kV
Widefield light microscopeTransmission electron microscope
Condenser lens
Objective lens
Projector lens
Specimen
Illumination
Final image
Transmission electron microscope vs. Widefield light microscope
Confocal laser scanning microscopeScanning electron microscope
Beam scanner
Detector
Lens system
Lens system
Specimen
Illumination
Scanning electron microscope vs. Confocal laser scanning microscope1 µm1 µm
Example: Transmission electron microscope
Cathode
Specimen holder
Viewing screen
TMP
RP
IGP
IGP
Ion getter pump
Turbo molecular pump
Oil diffusion pump
Rotary pump
Atmosphere: 1000 mbar
10-5 - 10-7 mbar
10-0 - 10-2 mbar
10-7 - 10-10 mbar
Electron microscopes are high vacuum systems
High voltage
(eg. 120 kV)
Electron source
(e.g. tungsten)
Electromagnetic lenses
Electromagnetic lens of a transmission electron microscope
The focal length can be changed by changing the current:
No movement or exchange of the lens is required for focusing or changing magnification!
Chromatic aberration
Electromagnetic lenses
Spherical aberrationsDue to energy difference of electrons (wavelength)
e- (98 kV)
e- (100 kV)
e- (102 kV)
Curvature and distortion of field
Axial astigmatism - confusion of the image
Specimen holder
Specimen on a TEM grid
Specimen holders and stages - TEM
3 mm3 mm
Specimen holders and stages - TEM
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 - SEM
Scanning electron microscope
Specimen stage (x, y, z, r, tilt)
Objective lens
Stage
Specimen stub
Stub holder
Specimen size:
• 100 mm in diameter
• 2 cm in z-direction (not electron transparent)
Electron – specimen interactions
Inelastic
(low angle, E=E0-ΔE)
Unscattered
(E=E0)
Primary electrons (E0)
Backscattered electrons (E=E0)
Elastic
(higher angle, E=E0)
Electron – specimen interactions
Primary electrons
Unscattered electrons
Inelastically scattered electrons
Elastically scattered electrons
Secondary electronsBackscattered electrons
Auger electronsHeat
Cathode luminescenseX-rays
Specimen Interaction volume
SEM analysis
TEM analysis
Contrast formation in TEM
Imaging in the transmission electron microscope
Brighter and darker “areas” in image dependent on sample composition
Absorption contrast
Diffraction contrast
Phase contrast
Contrast formation in TEM
Biological specimen consist of light elements:
Contrast enhancement required:
Treatment with heavy metals (Ur, Pb, Os)!
“LOW CONTRAST”
Heavy metals attach differently to different components
Imaging in the transmission electron microscope
Brighter and darker “areas” in image dependent on sample composition
Absorption contrast weak
Diffraction contrast weak
Phase contrast weak
No heavy metals:
Plastic embedded specimen
Specimen profile
Objective aperture
phospholipids ribosome
Signal
Intensity
Primary electron beam
Imaging in the transmission electron microscope
With heavy metal “stain”: Scattering of electrons
Specimen profile
Objective aperture
phospholipids ribosome
…Heavy metal ions
Signal
Intensity
Primary electron beam
Imaging in the transmission electron microscope
Plastic embedded specimen
Thin section of alga stained with heavy metals (Ur, Pb)
Imaging in the transmission electron microscope
Thin section of alga without heavy metal staining
1 µm
Imaging in the transmission electron microscope
Close to focus
100 nm
Fresnel rings
OverfocusUnderfocus
Carbon film , ca. 4 nm
Contrast enhancement by underfocusing
Phase differences of diffracted and non-diffracted rays are increased or
decreased by changing the focus
Imaging in the transmission electron microscope
Contrast for frozen-hydrated specimens: Underfocusing
Thin section of a frozen-hydrated apple leaf (“unstained”)
1 µm
Phase contrast only “between” H2O and biological material
Electron microscopy ETH Zurich
Imaging in the transmission electron microscope
The CCD camera for electron microscopy
Outside the microscope
Inside the microscope
(vacuum)
• Electrons need to be converted to photons (scintillator)
• CCD has to be protected from electron bombardment
Imaging in the transmission electron microscope
• Nowadays direct electron CCD available, no scintillator
required (very expensive)
Primary electrons
Unscattered electrons
Inelastically scattered electrons
Elastically scattered electrons
Secondary electronsBackscattered electrons
Auger electronsHeat
Cathode luminescenseX-rays
Specimen Interaction volume
SEM analysis
TEM analysis
Imaging in the scanning electron microscope1 µm1 µm
Scanning and signal detection
…Primary electron beam
secondary electrons
Imaging in the scanning electron microscope1 µm1 µm
Secondary electron detector
+7-12kV HVPhotomultiplier
+200-500V – Collector voltage
Photons ElectronsElectrons
Primary electrons
SE
Imaging in the scanning electron microscope1 µm1 µm
Contrast formation in SEM using secondary electrons (SE)
Different number of electrons from different spots of the specimen
composition of the specimen
topography of the specimen
acceleration voltage of primary electrons
location of the detector
Dependent on
Imaging in the scanning electron microscope1 µm1 µm
Primary electron beam
Contrast formation of biological objects
Platinum
Primary electron beam
Uncoated Coated with 4 nm platinum
Imaging in the scanning electron microscope1 µm1 µm
Only few electrons escape from specimen
Signal from a large volume (“unsharp, noisy” image)
Localization of the signal to the surface
Freeze-fractured yeast
500 nm
Uncoated Coated with 4 nm platinum
Electron microscopy ETH Zurich
Imaging in the scanning electron microscope1 µm1 µm
Contrast formation of biological objects
PE Primary electrons
SE Secondary electrons
R Excited volume
Contrast based on SE - topography
SE
PE
R
RSE
F
Specimen
Imaging in the scanning electron microscope1 µm1 µm
Contrast based on SE (Detector position)
Imaging in the scanning electron microscope1 µm1 µm
Mouse kidney (glomerulus)
10 µm
Contrast based on SE – detector position Virtual light source
Imaging in the scanning electron microscope1 µm1 µm