introduction to electron microscopy andres kaech ......electron microscopy andres kaech...

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