Introduction to electron microscopy py 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 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 are pretty similar!

Sample preparation for TEM Sample preparation is the most critical part in EM 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 preparation UNIVERSITY OF KUOPIO BioMater Centre BASIC METHOD FOR ANIMAL TISSUES, phosphate buffer Pre fixation: Pre-fixation: - perfusion fixation and/or immersion fixation - 2 % glutaraldehyde in 0,1 M phosfate butter, pH 7,4 Rinsing: - 0,1 M phosfate buffer, pH 7,4 Post fixation: - 1 % osmiumtetraoxide (OsO4) in 0,1 M phosfate buffer, pH 7,4 Rinsing: - 0,1 M phosfate buffer, pH 7,4 Dehydration: - 70 % ethanol - 90 % ethanol - 94 % ethanol - abs. ethanol - propyleneoxide - propyleneoxide Infiltration: - Mix of propyleneoxide and LX-112 1:1 - LX-112 Embedding: - fresh LX-112, embedding in appropriate molds Polymerization: - 37°C (in heat oven) - 60°C

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2-4 h 15 min 2h 15 min 10 min 10 min 10 min 3 x 10 min 15 min 10 min 2h overnight

24 h 48 h

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

Some examples

LM images

TEM 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, p , i.e. theyy are scattered 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 scattering g (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) 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 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 g y the image 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")

r

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 Human eye silmä Light microscopy valomikroskooppi l ik k i Transmission electron microscopy läpäisyelektronimikroskooppi

0

1

10

100

0,01

0,1

1000 1

10000

100000

10

100

0,01

0,1

1000000 nm 1000 1

um mm

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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": r=

Formed image

Point source

0.612 ⋅ λ 0.612 ⋅ λ = n ⋅ sin i α N . A.

where, λ

= wavelength, = refractive index, α = angle in the lens system, N.A. = numerical aperture

n

Diffraction in the slit or aperture

Basics of microscopy;

Resolving power (TEM)

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

Acceleration voltage (kV)

Wavelength (nm)

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0.0122

50

0.0054

100

0.0037

1000

0.0009

"Behind the scenes": Energy of particle = Energy of quantum: … de Broglie wavelength can be calculated: …

…

Speed of electrons can also be calculated

E = mc 2 = hc / λ h h = Planck's constant λ= m = electron mass mc c = speed of light

(assuming energy from acceleration = kinetic energy of the particle):

eV = …

1 2 mv 2

⇒

v=

2eV m

e = electron charge V = acceleration voltage v = speed of electron

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

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Some examples 2

Capillary

(scale bar 2 μm)

Bacteria

(scale bar 0.2 μm)

"Dust particles" (scale bar 50 nm)

Modern techniques: Tomography with TEM

3D--object => set of 2D 3D 2D--projections

2D--projections => 3D 2D 3D--reconstruction

S. 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

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

Different types of MLLs. A) Tomographic slice (resolution of 4nm) of 250nm section showing the concentric o g ni tion of inte organization internal n l 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 development have 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 specimens are 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 gun Lenses All are condensing

Deflector

Scanning

Detector

"P l meter" "Pulse t "

Visualization

Sample preparation for SEM; Basic requirements

Samples must fulfil the basic requirements: 1 - Must fit in the specimen chamber and the holders 2 - 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 gives poor results

- Coatings, Coatings low acceleration voltage or special euipment prevent 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 Biological samples for SEM need…

fixation

dehydration

coating

(e.g. critical point drying)

(sputter coating with Au or Pt)

Sample preparation for SEM; effect of fixation method Physical fixation (fro en and fractured) (frozen fract red)

Chemical fixation

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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 detected. 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 bombing different types of particles or radiation is emitted from the sample These signals can be detected and used for characterization

Resolution of the signals are proportional to the interaction volume 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 from the specimen --> BSEs have higher energy than SEs

Information acquired with BSEs: Depth-related structural information Info of chemical composition

Backscattered Electron Image BSE, 10 kV

BSE, 3.5 kV

Other modes of SEM: Low vacuum -mode

Used for imaging of non-conductive samples polymers, p y , biological g samples... 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 be controlled --> 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.

Data courtesy of Dr Sherry Mayo

THANK YOU! For more information, please visit http://www uku fi/biomater http://www.uku.fi/biomater

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