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Introduction to electron microscopypy

NANOTEM Lecture Series

Characterization of materials

Arto Koistinen, M.Sc.BioMater Centre

23.11.2009

Transmission electron microscope (TEM)

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"Short history"� Louis de Broglie in the early 1920's: a theory of particles

having wave-like properties � In the 1920's: Schrödinger ja Heisenberg developed a

theory of quantum mechanics which "enabled" electrontheory of quantum mechanics, which "enabled" electron microscopy

� In 1926 H. Busch proved mathematically that electrons can be focused by a magnetic field with the similar way as light is focused in an optical lens

� Ernst Ruska developed a lens system able to magnify specimen by 16x! (Published in 1931; they used a term 'electron microscope')p )

� R. Ruedenberg (working for Siemens) applied a patent and in some references he has been mentioned as the inventor of EM.

� In1939 the first TEM was manufactured (by Siemens)

� In1986 Ruska was awarded with the Nobel Price

Transmission electron

microscope,TEM

� Ultra thin slices of specimens or very small particles are investigated.

� The principle of operation:

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Structure of TEM;TEM vs. LM (Light microscope)

� In fact, the microscopes arepretty similar!

Sample preparation for TEM

� Sample preparation is the most critical part in EM studies!!!studies!!!

� Special equipment and skillful technicial are needed

� Biological samples for TEM need…

fixation dehydration embedding cutting staining

� Notes: � sample size at final state < 1 mm� typical slice thickness about 50 nm

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Example: TEM sample preparationUNIVERSITY OF KUOPIO

BioMater Centre 110

BASIC METHOD FOR ANIMAL TISSUES, phosphate bufferPre fixation:Pre-fixation:- perfusion fixation � and/or immersion fixation �- 2 % glutaraldehyde in 0,1 M phosfate butter, pH 7,4 2-4 hRinsing:- 0,1 M phosfate buffer, pH 7,4 15 minPost fixation:- 1 % osmiumtetraoxide (OsO4) in 0,1 M phosfate buffer, pH 7,4 2 hRinsing:- 0,1 M phosfate buffer, pH 7,4 15 minDehydration:- 70 % ethanol 10 min- 90 % ethanol 10 min- 94 % ethanol 10 min- abs. ethanol 3 x 10 min- propyleneoxide 15 min

propyleneoxide 10 min- propyleneoxide 10 minInfiltration:- Mix of propyleneoxide and LX-112 1:1 2 h- LX-112 overnightEmbedding:- fresh LX-112, embedding in appropriate moldsPolymerization:- 37°C (in heat oven) 24 h- 60°C 48 h

Note: This takes 4-5 days!Still cutting (with diamond blade) and staining with heavy element salts are needed.

Some examples

TEM imagesLM images

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Sample preparation for TEM:"Hard samples"

� Ion beam milling is used

Operation of TEM; Basics of image formation

� Part of the beam electrons hit the nuclei or electrons of the atoms in specimen, i.e. they are scatteredp , y

� Scattered electrons are cropped by using apertures

� Dense sections in the specimen (i.e. stained parts) cause more scattering and are dark in the image plane

� The most important factor in image formation in TEM is scatteringg(NOTE! In light microscopy; absorbtion)

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Structure of TEM 1;Cross-section of the equipment

Structure of TEM 2;"Electron gun"

� Electron source ("gun")� Electrons are emitted from a tungsten filament (thin wire)� Electrons are emitted from a tungsten filament (thin wire)

� Also modern types of guns are developed with higher stability, longevity and brightness; LaB6 and field emission

� Electrons are accelerated with an electric field (80 kV or 200 kV, for example) towards the specimen

"Electron gun" "Properties of guns"

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Structure of TEM 3;Lens system

� Lens system� All lenses are electromagnetic lenses

� Electrons can be controlled by the� Electrons can be controlled by the magnetic field

� Firstly, electron beam is focused to the sample by condensor lenses

� Objective lens (after the sample) forms an image of the specimen

� Intermediate lenses and projector lens magnify the imageg y g

� Image recording system� Nowadays, the image is recorded by a

CCD camera (or still by using plate films)

Basics of microscopy

� Resolution (r, "resolving power")� Resolving power is the minimum distance between two spots

that can be seen as individual spots� Human eye: 0.1 mm = 100 μm = 100000 nm� Light microscopy: 0.0002 mm = 0.2 μm = 200 nm� Electron microscopy: 0.0000001 mm = 0.0001 μm = 0.1 nm

r

l ik k i

silmä

Light microscopy

Human eye

0 1 10 100 1000 10000 100000 1000000

1 10 100 1000

10,10,01

0,10,01

nm

um

mm

valomikroskooppi

läpäisyelektronimikroskooppiTransmission electron microscopy

Light microscopy

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Basics of microscopy;Resolving power

� Resolving power…� depends on the wavelength of the light� is roughly half of the wavelength

� For example; Using visible light (n. 400 – 700 nm) the resolution is about 200 nm at maximum

� "Behind the scenes":612.0

i612.0

ANr λλ ⋅

=⋅

=..sin ANn α⋅

where, = wavelength, = refractive index,= angle in the lens system,= numerical aperture

λnα

..AN

Point source

Diffraction in the slit or aperture

Formed image

Basics of microscopy; Resolving power (TEM)

� Also motion of the electrons Acceleration Wavelength (nm)include wave-like behaviour (theory by de Broglie), and the wavelength depends on the acceleration voltage:

voltage (kV) Wavelength (nm)

10 0.0122

50 0.0054

100 0.0037

1000 0.0009

"Behind the scenes":Energy of particle = Energy of quantum: λ/2 hcmcE ==de Broglie wavelength can be calculated:

Speed of electrons can also be calculated (assuming energy from acceleration = kinetic energy of the particle):

⇒

NOTE! With acceleration voltage 50 kV the speed of the electrons is about 15 % of the light speed --> theory of the relativity has to be considered

mch

=λ

meVv 2

=2

21 mveV =

h = Planck's constantm = electron massc = speed of light

e = electron chargeV = acceleration voltagev = speed of electron

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Some examples 2

Capillary Capillary (scale bar 2 μm)

Bacteria(scale bar 0.2 μm)

"Dust particles" (scale bar 50 nm)

Modern techniques: Tomography with TEM

3D3D--object => set of 2Dobject => set of 2D--projectionsprojections 2D2D--projections => 3Dprojections => 3D--reconstructionreconstruction

S. Nickell, C. Kofler, A. Leis, W. Baumeister: Nature Reviews Molecular Cell BiologyS. Nickell, C. Kofler, A. Leis, W. Baumeister: Nature Reviews Molecular Cell Biology

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Example of tomography:3D organization of organelles in cells

Different types of MLLs. A) Tomographic slice (resolution of 4nm) of 250nm section showing the concentric o g ni tion of inte n l

Murk et al. Traffic 2004; 5: 936-945

organization of internal membranes in a high-pressure frozen hDC. B) MLL in high pressure frozen B -lymphocyte containing membrane sheets and small vesicles. C, D) 3-D model of internal membranes with an onion-like organization of vacuoles present in MLL shown in A.

Scanning electron microscope, SEM

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"Short history"� Developed by M. Knoll in 1935; � Patented by M. von Ardenne in 1937

� The first commercial SEM in 1965� Cambridge Scientific Instruments: Mark I� This was a breakthrough of electron microscopy, because SEM was

found to besuitable in various applications� Note! TEM was developed earlier in the 1930's

� In the end of 1960's, elemental analysis attached (WDS)

� Thereafter, methodological and technological developmenthave improved the performancehave improved the performance

� For example; electron source stability --> better resolution, vacuum systems --> different imaging modes,information technology --> data storage and manipulation

� Nowadays, SEM if by far the most common type of electron microscopes

Basics� Surfaces and surface related structures,

topography and morphlogy of the specimensare investigated with SEMare investigated with SEM

� Basic components in the equipment:� Electron source, vacuum system, magnetic lenses

and signal detection unit� Note! Can you define SEM as a microscope?!?!

SEM, Philips XL30

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Operation of SEM;SEM vs. TV

� Electron gunLenses� Lenses

� All are condensing

� Deflector� Scanning

� Detector"P l t "� "Pulse meter"

� Visualization

Sample preparation for SEM;Basic requirements

� Samples must fulfil the basic requirements:

1 - Must fit in the specimen chamber and the holders2 - Stability;

- no evaporation of liquids is allowed- sample must remain unchainged in electron bombing--> Risk of contamination and structural changes

3 - Conductivity; charging of the sample creates givespoor results

Coatings low acceleration voltage or special euipment- Coatings, low acceleration voltage or special euipmentprevent the problem

4 - Cleanliness; - dirt on the sample may interfere the investigation--> Note: sometimes the "dirt" is being investigated

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Charging / stability

Charging of the sample Damage due to electrons

Examples

Metallic screw(untreated, SEM mode)

Polymeric implant(untreated, low vacuum mode)

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Sample preparation for SEM

� Again, sample preparation is critical in SEM studies� Special equipment and reagents are typically used� Special equipment and reagents are typically used

� Biological samples for SEM need…

fixation dehydration coating(e.g. critical point drying) (sputter coating with Au or Pt)

Physical fixation Chemical fixation(fro en and fract red)

Sample preparation for SEM; effect of fixation method

(frozen and fractured)

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Sample preparation for SEM;effect of drying method

Sample preparation for SEM:"Hard samples"

� Ion beam cutter

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Operation of SEM;Image formation

� High-energy beam electrons hit the atoms in specimen and thus, secondary electrons are scattered from the specimen and detectedspecimen and detected.

� Note! Beam electrons have energy 2- 30 kV, whereas the detectable electrons (secondary electrons) have energy only about 10-20 eV

Examples: Biological samples� Pollen: Cultured cells:

� Bacteria: Red cells:

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Operation of SEM;Beam/specimen interaction

� Due to electron bombingdifferent types of particlesdifferent types of particlesor radiation is emitted fromthe sample

� These signals can bedetected and used for characterization

� Resolution of the signals are� Resolution of the signals areproportional to the interactionvolume

� Note: For imaging, the resolution can be < 1 nm!

Imaging with SEM:Effect of acceleration voltage

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Other imaging modes with SEM: BSE

� Backscattered electrons (BSE):� BSEs are beam electrons which escape� BSEs are beam electrons which escape

from the specimen --> BSEs have higher energy than SEs� Information acquired with BSEs:

� Depth-related structural information � Info of chemical composition

BSE, 10 kV BSE, 3.5 kV

Backscattered Electron Image

Other modes of SEM:Low vacuum -mode

� Used for imaging of non-conductive samples� polymers, biological samples...p y , g p

� Relative humidity in the chamber is raised, and ionized gas molecules transfer excessive electrons to prevent charging

� Additional GSE-detector is required

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Other modes of SEM:Environmental SEM (ESEM)

� Relative humidity and temperature can becontrolledcontrolled--> solid/liquid phases--> swelling, etc.

An example: salt crystals

Modern techniques:Tomography with SEM

� Principle:

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Example of tomography with SEM

� A ceramic sphere containg bubbles. Sphere diameter 90 microns.Sphere diameter 90 microns.

Data courtesy of Dr Sherry Mayo

THANK YOU!

� For more information, please visit http://www uku fi/biomaterhttp://www.uku.fi/biomater