introduction to 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)

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Scanning electron microscope (SEM)Transmission electron microscope (TEM)

The types of electron microscopes

Electron beam

Specimen ~100 nm

Electron beam

Specimen

Projection Surface

Hela Cells

Elektronenmikroskopie ETH Zurich

1 µm

Specimens courtesy of Bärbel Stecher, Institute of Microbiology, ETH Zurich

Examples TEM

Mouse intestine

Microvilli

GlycocalixJunction

Actin filaments

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Elektronenmikroskopie ETH Zürich

500 nm

Mouse intestine

Membrane(lipid bilayer)

Actin filaments

Examples TEM

H/K -ATPase in cimetidine-treated resting gastric parietalcells (rabbit).

Immunolabelling: Localization of proteins

Sawaguchi et al. 2004, Journal of Histochemistry & Cytochemistry

100 nm

Examples TEM

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Examples SEM

Mouse kidney

500 µm

Mouse kidney (glomerulus)

10 µm

Examples SEM

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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:

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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

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Confocal laser scanning microscopeScanning electron microscope

Beam scanner

Detector

Lens system

Lens system

Specimen

Illumination

Scanning electron microscope vs. Confocal laser scanning microscope

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

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High voltage(eg. 120 kV)

Electron source

(e.g. tungsten)

Thermionic emission(tungsten, LaB6, Schottky emitter)

Electron source (Electron gun)

Cold field emission(quantum-mechanical tunneling)

Thermionic

Tungsten LaB6 SchottkyCold field emission

Material

Heating temp. (K)

Normalized brightness

Required vacuum (Pa)

∆E (eV)

W

2700

LaB6

1800

ZrO/W

1800

W

300

Chromatic aberration!

Ultra highhigh

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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


Spherical aberrationsDue to energy difference of electrons (wavelength)

e- (98 kV)

e- (100 kV)

e- (102 kV)

Curvature and distortion of field

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Axial astigmatism - confusion of the image


Under focused imageelliptic deformation

Over focused imageelliptic deformation

Focuscircle of least confusion

Cellulose filter paper imaged in SEM

Focus, corrected astigmatismcircle of confusion minimized

With astigmatism

Withouth astigmatism

Under focused imageconcentric unsharp

Over focused imageconcentric unsharp


Reasons:

• Inhomegenities of the lens

• Contamination of lenses and apertures

• Charging of specimen

Object: Source of e-

(off-axis)

Objective lens

Optical axis

“True” image of object

z

x

y

Focus

Corrector coils

Axial astigmatism - confusion of the image

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Specimen holder

Specimen on a TEM grid

Specimen holders and stages

3 mm3 mm

Transmission electron microscope


Transmission electron microscope

Goniometer: x, y, z, r

Specimen size:

• 3 mm in diameter

• 100 nm in thickness

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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-∆E)

Unscattered(E=E0)

Primary electrons (E0)Backscattered electrons (E=E0)

Elastic(higher angle, E=E0)

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Emission of electrons and radiation

Inelastic scattering:

Energy is transferred from the primary electron to the specimen

K

L

M

N

1

2

K

L

M

N


• Inner-shell ionisationElectron „hole“ is filled by an electron of an outer shell: Surplus energy is eitheremitted as characteristic x-ray or transferred to another electron, which is emitted(Auger electron)

• Bremsstrahlung (continuum x-rays)Deceleration of electrons in the Coulomb field of the nucleus⇒ Emission of X-ray carrying the surplus energy ∆E⇒ Uncharacteristic X-rays

• Secondary electrons (SE)Loosely bound electrons (e.g., in the conduction band) can easily be ejected⇒ low energy (< 50 eV)

• PhononsLattice vibrations (heat) (⇒ beam damage)

• PlasmonsOscillations of loosely bound electrons in metals

• Cathode luminescence

Inelastic scattering:

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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 transmission electron microscope

Specimen: Electron transparent(very thin: 100 nm)

Image: 2D projection of a volume• CCD camera• Phosphorescent screen• Conventional photosensitive film

Condenser lens

Objective lens

Projector lens

Specimen

Illumination

Final image

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Contrast formation in TEM

NOTE: Mechanisms occur at the same time (superposition)


Absorption contrast

Scattering/phase contrast

Contrast formation in TEM

Biological specimen consist of light elements:

Absorption contrast weak

Scattering/phase contrast weak

Contrast enhancement required:

Treatment with heavy metals (Ur, Pb, Os)!

“LOW CONTRAST”

Heavy metals attach differently to different components


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Main contrast formation in plastic embedded specimens

Scattering of electrons through heavy metals

Specimen

Image plane

phospholipids ribosome

…Heavy metal ions


Objective aperture(back focal plane)

Primary electron beam

Brightness

Objective lens

Objective lens

Thin section of alga stained with heavy metals (Ur, Pb)


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Thin section of alga without heavy metal staining

1 µm


Underfocus OverfocusFocusMinimum contrast

100 nm

Contrast enhancement by underfocusing


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Close to focus

100 nm

Fresnel rings

OverfocusUnderfocus

Carbon film , ca. 4 nm


Phase differences of diffracted and non-diffracted rays are increased or decreased by changing the focus



Thin section of a frozen-hydrated apple leaf (“unstained”)

1 µm

Phase contrast only between H2O and biological material

Electron microscopy ETH Zurich


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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


• 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 microscope

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Photomultiplier

Scanning electron microscope

Specimen: Bulk specimen

• Photomultiplier• No CCD camera

Beam scanner

Detector

Lens system“condenser”

Objective

Specimen

Illumination

Scanning and signal detection

…Primary electron beam

secondary electrons


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Scanning and signal detection

Scanning of the specimen


Signal and detection

Different properties of the different signals

►Specific detectors

►Different/specific information


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Secondary electron detector

+7-12kV HVPhotomultiplier

+200-500V – Collector voltage

Photons ElectronsElectrons

Primary electrons

SE


Secondary electron detector


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Contrast formation in SEM using SE

Different number of electrons from different spots of the specimen

topography of the specimen

location of the detector

acceleration voltage of primary electrons

composition of the specimen

Dependent on


PE Primary electronsSE Secondary electronsR Excited volume

Contrast based on SE - topography

SE

PE

R

RSE

Specimen


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Contrast based on SE (Detector position)

Pseudo 3-dimensional image based on position of SE detector


Wing of butterfly

Contrast based on SE



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Mouse kidney (glomerulus)

10 µm

Contrast based on SE – detector position Virtual light source


Biological material (light elements):

Only few electrons escape from specimen

Almost no contrast, similar contrast everywhere on specimen

Contrast enhancement

Localization of the signal to the surface

Coating of biological specimen with thin heavy metal layer (a few nm)

Reducing acceleration voltage

Unsharp image

Contrast formation in SEM


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Contrast formation

Platinum


Uncoated Coated with 4 nm platinum


Freeze-fractured yeast

500 nm

Contrast based on SE: Non-coating vs. coating with heavy metals

Uncoated Coated with 4 nm platinum



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Low mag. High mag.

768 px

1024 px

768 px

1024 px

Object


Focusing and magnification in electron microscopy

Focusing and magnification in light microscopy

TEM:

Focusing:

Change current in magnetic lenses for focusing (objective lens)

Move holder in z

Magnification:

Change current in magnetic lenses (projective lenses) and combine several projective lenses.

SEM:

Focusing:

Change current in magnetic lenses for focusing (objective lens)

Move stage in z

Magnification:

Change scanning field (scan a smaller or larger area with the same number of pixels), pixel size changes.

Widefield light microscopy:

Focusing:

Moving objective or

stage in z

Magnification:

Changing the whole objective.

Confocal scanning laser microscopy:

Focusing:

Moving objective or

stage in z

Magnification:

Change scanning field (scan a smaller or larger area with the same number of pixels), pixel size changes.

Changing the whole objective


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Artefacts - Charging

Yeast, freeze-fractured Wax tubes on lotus leaf


Artefacts – Beam damage

Yeast, freeze-fractured


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Element analysis using X-ray “contrast”

Detector

Specimen

Illumination

SE detector (inlens)

SE detector

BSE detector

X-ray detector


Element analysis using X-ray “contrast”

Specific regions, single pixels or the whole image can be mapped using x-rays.

Energy

Intensity


introduction to electron microscopy andres kaech

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