arto koistinen, m.sc. biomater centre 23.11 micros… · · 2017-09-17arto koistinen, m.sc....
TRANSCRIPT
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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