Introduction to Electron Microscopy - · PDF fileIntroduction to Electron Microscopy ... Cold field emission ... • The signal is displayed on the computer screen at constant pixel

Introduction to

Electron Microscopy

University of ZurichCenter for Microscopy and Image Analysis

Instrumentation

Courtesy: Andres Kaech

Atoms

1 mm

100 m

10 m

1 m

100 nm

10 nm

1 nm

0.1 nm

Cells

Red blood cells

Bacteria

Mycoplasma

Viruses

Proteins

Amino acids

Radio

Infrared

Visible

Ultraviolet

x,-rays

Human eye

Electron microscope

Light microscope

Resolution Limit Wavelength/Size Object

MRI, CT

Why electron microscopy?

Ultrastructure

Electron microscopy


10 µm

Rat intestine

Green…F-actinYellow…ß-cateninOrange…Nuclei

Light microscopy Electron microscopy

Microvilli

Adherence junction: ß-catenin visualizedby immunolabelling using „immunogold“

1 µm

Schwarz & Humbel 2007: Methods in Molecular Biology, vol. 369, Electron Microscopy: Methods and Protocols, Second EditionEdited by: J. Kuo © Humana Press Inc., Totowa, NJ

Elektronenmikroskopie ETH Zurich

1 1 µµmm

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


Mouse intestine

Microvilli

GlycocalixJunction

Actin filaments

Elektronenmikroskopie ETH Zürich

500 nm500 nm


Mouse intestine

Membrane(lipid bilayer)

Actin filaments

Extracted, CPD, Pt, SEM FD, Pt, SEM

Cytosceleton afterextraction

Single filaments and their fine structure are

visible (here SEM)


Up to macromolecular level

Time resolutionThe overall design of an electron microscope

is

similar to that of a light microscope.

From photons to electrons


Light microscopes

Photons are substituted with electrons

Glass lenses are substituted with electromagnetic and electrostatic lenses

Electron microscope

Photons

Electrons

e-

Wave-particle duality of electrons

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-


Similarities to photons:

λ = wavelengthh = Planck's constant (6.6 X 10-27)m = mass of the particlev = velocity of the particle

DeBroglie relation:

The higher the energy of the electrons, the lower the wavelength, the higher the resolution

d (100 kV) = 0.24 nm

For electron microscopes: n ≈ 1 and n*sinα ≈ α

vmh

nmV23.1

Abbe’s equation

sin61.0

nd sin nNA

d = resolution in nmα = half opening angle of objective (in radians)V = accelerating voltage

Resolution EM: nmV

d

753.0

V = accelerating voltage

Electron pathes through potential field

Resolution of electron microscopes

α ≈ 0.01 radians ≈ 0.6 grad

Resolution of biological objects is limited by specimen preparation: Practical resolution: > 1 nm

Acceleration voltages of electrons:

Transmission electron microscopes (TEM): 40 – 1200 kVScanning electron microscopes (SEM): 1 – 30 kV

However:

Effective instrument resolution TEM: 0.1 nm

Effective instrument resolution SEM: 1 nm

Resolution of electron microscopes

The types of electron microscopes

Confocal laser scanning microscope

Light is substituted with electrons

Glass lenses are substituted with electromagnetic and electrostatic lenses

Transmission electron microscope

Wide field microscopy

Scanning electron microscope

Photons

Electrons

Photons

Electrons

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


Electron gun

Phosphorescent screen

CCD camera

Electromagnetic lens



TEM grid

Widefield light microscopeTransmission electron microscope

Condenser lens

Objective lens

Projector lens

Specimen

Illumination

Final image

Lamp

Eye

CCD camera

Glass lens

Glass lens

Glass lens

Slide


versus

Photomultiplier (Detector) Photomultiplier

Electron gun

Electromagnetic lenses


Electromagnetic/electrostatic lens

Confocal scanning laser microscopeScanning electron microscope

Beam scanner

Laser

Glass lenses

Mirror

Glass lenses

X-ray, photomultiplier

Detector

Lens system“condenser”

Objective

Specimen

Illumination


versus

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

High vacuum

Electrons would collide with gas molecules

Electron source (tungsten) would blow

Without vacuum:


Components of electron microscopes

Electron source (Electron gun)

Light microscope: tungsten filament(bright field)

Electron microscope: tungsten filament(common form)

Filament is heated

Electrons are emitted from the tip

F…FilamentW…Wehnelt electrodeC…Ceramic high voltage insulatorRb…Autobias resistorIe…Electron emission current

Thermionic emission (tungsten, LaB6, Schottky emitter)


Very fine tungsten tip

No heating required (room temperature)

Cold field emission (quantum-mechanical tunneling)

ThermionicTungsten LaB6 Schottky Cold 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


High voltage



Electromagnetic lens of a transmission electron microscope


Magnetic field depends on current and number of windings

Note: Force is perpendicular to the plain defined by B and v

v…speed of electronB…magnetic fieldF…resulting force

Electrons are deviated in a magnetic field

I

Image rotation is corrected in modern microscopes


Image rotation:

Axial astigmatism of electromagnetic lenses … confusion of the image


Most relevant aberration in biological electron microscopy (in particular SEM)

Under focussed imageelliptic deformation

Over focussed imageelliptic deformation

Focuscircle of least confusion

Reasons:

• Contamination of lenses and apertures

• Inhomegenities of the lens

• Charging of specimen

Correction of astigmatism with corrector coils


Focus, corrected astigmatismcircle of confusion minimized

Chromatic aberration


Spherical aberrationsDue to energy difference of electrons (wavelength)

e- (98 kV)

e- (100 kV)

e- (102 kV)

Curvature and distortion of field

Vacuum systems


Filament chamberUltra high vacuum: < 10-9 mbar

Specimen chamberHigh vacuum: ~ 10-7 mbar

Viewing chamberHigh vacuum: ~ 10-5 mbar

Vacuum systems


Ion getter pump / Oil diffusion pump

Turbo molecular pump

Rotary pump

Atmosphere: 1000 mbar

10-5 - 10-7 mbar

10-0 - 10-2 mbar

10-7 - 10-10 mbar

Vacuum systems

Turbo molecular pump

Rotary pump

Atmosphere: 1000 mbar

10-5 - 10-7 mbar

10-0 - 10-2 mbar

10-7 - 10-10 mbar

Ion getter pump / Oil diffusion pump

Vacuum systems

Properties of vacuum systems

Vacuum systems have to be kept clean:

• No volatile components (fatt, oil, water)

• Air-lock for transfer of specimen into vacuum

• Vent with dry nitrogen gas

• High vacuum systems always require a sequence of different vacuum pumps

• Differential vacuum is maintained by small openings between “chambers” and location of the pumps

• Pumping efficiency depends on the gas

Specimen holders and stages


Goniometer: x, y, z, rSpecimen size:

• 3 mm in diameter!

• Ca. 100 nm in thickness(electron transparent)

Specimen holder

Specimen on a TEM grid


3 mm3 mm



Viewing chamber = Specimen chamber

Gun

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)


• Stages and goniometer must be extremely stable and precise!

• Any drift will cause unsharp images, in particular at high magnifications

NOTE:

Electron - specimen interactions

Electron – specimen interactions

Inelastic(low angle, E=E0-∆E)

Unscattered(E=E0)

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

Elastic(higher angle, E=E0)


Primary electrons hit electrons of the specimen atom

Emission of electrons and radiation

Inelastic scattering:

Energy is transferred from the primary electron to the specimen

K

LM

N

1

2

K

LM

N


PrimaryPrimary electronselectrons

UnscatteredUnscattered electronselectrons

ElasticallyElastically scatteredscattered electronselectronsInelastically scattered electrons

SecondarySecondary electronselectronsBackscatteredBackscattered electronselectrons

AugerAuger electronselectronsHeat

Cathode luminescenseX-rays

Specimen Interaction volume

SEM analysis

TEM analysis

TEM REM


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


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

Contrast formation in TEM

Absorption of electrons

NOTE: All mechanisms occur at the same time (superposition)

Question: Which mechanism is most relevant for biological specimens?

Scattering of electrons

Diffraction and phase contrast



Specimen low highdensity

Signal Intensity

Specimen profile

Heat (beam damage)


Absorption of electrons Scattering of electrons Diffraction and phase contrast


Specimen low high density

Specimen profile

Objective aperture


Signal Intensity



Specimen

Specimen profile

Objective lensProjective lenses

Objective lensProjective lenses


Image plain

Diffracted ray

Non-diffracted ray

Signal Intensity



Biological specimen consist of light elements:

Absorption weak

Scattering weak

Diffraction and phase weak

Contrast enhancement required:

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

“NO CONTRAST”

Heavy metals attach differently to different components


Main contrast formation in plastic embedded specimens

Scattering of electrons through heavy metals

Specimen

Specimen profile

Objective aperture

phospholipids ribosome


…Heavy metal ions

Signal Intensity

Absorption of electrons

Diffraction and phase contrast

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


Thin section of alga without heavy metal staining


1 µm

Imaging in the scanning electron microscope


Photomultiplier


Specimen: Bulk specimen

• Photomultiplier• No CCD camera

Beam scanner

Detector

Lens system“condenser”

Objective

Specimen

Illumination


Scanning and signal detection

Scanning of the specimen



…Primary electron beam

…Secondary electrons

The focused electron beam is moved from one pixel to another. At everypixel, the beam stays for a defined time and generates a signal (e.g. secondary electrons) which are detected, amplified and displayed on a computer screen.



The scan generator synchronizes the scanning of the specimen withthe display of the detected, amplified signal.


Achieving higher magnifications:• A smaller area is scanned with the same number of pixels.• The scanned pixels are smaller• The signal is displayed on the computer screen at constant pixel size

Magnifying in scanning electron microscopes

Low mag. High mag.

768 px

1024 px

768 px

1024 px

Object


Signal and detection

R…interaction volume

R


R dependent on density of material (Z) and acceleration voltage of PE (0.1 - 30 kV)

R decreases with increasing Z R increases with increasing acceleration

voltage

R

λ independent on acceleration voltage(but not the number of emitted electrons!)

λ decreases with increasing Z (density)

λ

λ C: 10 – 100 nm λ Cr: 2 – 3 nm λ Pt: 1 – 2 nm

R

Contrast based on SE

Energy of SE independent of acceleration voltage of PE


Contrast based on SE R ≤ λ: Little SE contrast = f (Detectorgeometry)

Pseudo 3-dimensional image based on position of SE detector

SE detector(inlens)

SE detector


Contrast based on SE

Leg of an ant, coated with ca. 10 nm Platinum

Virtual light source

SE Escape depth 10-100 nm

Large excitation volumeLow BSE coefficient

SE mainly from metal coat!

R > λ: SE contrast = f(Φ)

THIN (1-4 nm) metal layer, e.g. Pt

SE Escape depth 1-3 nm

Small excitation volumeHigh BSE coefficient

BSE

SE II

SE IBSE

PE


Contrast based on SE: Coating for high resolution SE imaging

Φ

Freeze-fractured yeast

500 nm


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

Uncoated Coated with 4 nm platinum


R dependent on density of material (Z) and acceleration voltage of PE (0.1 - 30 kV)

Contrast based on BSE

• Useful if specimen is coated with heavy metals

R

Biological material: “No” contrast

BUT:

• Less sensitive to charging (higher energy)• Less topographic contrast• More material contrast

BSE vs. SE

SE signal at 2 kV BSE signal at 30 kV

Fractured plant cell containing metal inclusions in chloroplasts


Contrast SE vs. BSE

Topography Material

SE signal at 20 kV SE signal at 1.7 kV

Yeast freeze-dried, coated with chromium


Contrast SE

Little topography(Signal based on SE II induced by BSE!)

Good topography(Signal based on SE I from surface layer)

Documents

Introduction to Electron Microscopy - · PDF fileIntroduction to Electron Microscopy ... Cold field emission ... • The signal is displayed on the computer screen at constant pixel