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Microscopy techniques for biomaterials
Engenharia Biomédica
Patrícia Almeida Carvalho
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Why microscopy? http://www.cellsalive.com/howbig.htm
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Why microscopy?
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Resolution of an optical system
Diffraction at an aperture - Rayleigh criterion
The Rayleigh criterion for the resolution of an optical system states that two points willbe resolvable if the maximum of the intensity of the Airy ring from one of themcoincides with the first minimum intensity of the Airy ring of the other. This implies thatthe resolution, ρ (strictly resolving power) is given by:
ρρρρ = 0.6λλλλ/η/η/η/ηsinαααα
http://micro.magnet.fsu.edu/primer
ρρρρ = 0.6λλλλ/η/η/η/ηsinαααα
where λ λ λ λ is the wavelength, ηηηη the refractive index and αααα is the semi-angle at the specimen.
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Introduction to lenses
� Ray diagrams (geometrical optics):1. The optical axis contains the object focal point and the image focal point.2. Rays going through the lens optical center (principal rays) are not deflected. 3. Parallel rays diverge from and converge to the focal points.4. For identical optical media on both sides: fobject = fimage
http://micro.magnet.fsu.edu/primer
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1/a + 1/b = 1/f
M = b/a
Image Formation http://micro.magnet.fsu.edu/primer
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Compound Microscope http://micro.magnet.fsu.edu/primer
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Compound Microscope http://micro.magnet.fsu.edu/primer
Transmission (Diascopic)Transmission (Diascopic)
Transmission (Diascopic)
Compound Microscope http://micro.magnet.fsu.edu/primer
Reflection (Episcopic)
Fluorescence Microscopy http://micro.magnet.fsu.edu/primer
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Fluorescence Microscopy http://micro.magnet.fsu.edu/primer
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Fluorescence Microscopy http://micro.magnet.fsu.edu/primer
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Confocal microscopy
The Instrument (Laser Scanning confocal microscope)
http://micro.magnet.fsu.edu/primer
Confocal or conjugate planes
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ρρρρ = 0.6λλλλ/ηηηηsinαααα
ρρρρ = 0.41λλλλ/ηηηηsinαααα
Reflection mode: surface topography
Confocal microscopy http://micro.magnet.fsu.edu/primer
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Samples are stained with fluorochromes that stain specific phases or organelles (labeling).
Corresponding images are collected in the red, green, and blue channels and recombined.
Confocal microscopy http://micro.magnet.fsu.edu/primer
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Confocal microscopy http://micro.magnet.fsu.edu/primer
Tomography
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Microscope working principles
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Image Types JEOL images
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Light vs electrons
ρρρρ = 0.61λλλλ/ηηηηsinαααα
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Electromagnetic Lenses
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Electromagnetic Lenses
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Electromagnetic Lenses
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Electromagnetic Lenses
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Electromagnetic Lenses
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Electromagnetic Lenses
Electromagnetic lenses
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Scanning Electron Microscopy
working principles
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The Microscope Hitachi S 2400
Scanning Electron Microscopy
Depth of field - Light vs electrons (JEOL)
Scanning Electron Microscopy
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Optical Microscope Image SEM Image of Same Object
Scanning Electron Microscopy
Woven polyester-fiber clothWoven polyester-fiber cloth
House dust mite
Electron gun
Gun alignment coils
Condenser lens
Objective lens
Deflection coils
Secondary
The instrument
Scanning Electron Microscopy
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Backscatter electron detector
Specimen chamber
Specimen goniometer stage
Oil diffusion vacuum pump
Secondary electron detector
Electron Gun - CathodesTungsten Hairpin Filament
LaB6 Pointed Rod
Tungsten field-emission cathode
Scanning Electron Microscopy
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Electron Gun - Thermionic vs Field Emission
Scanning Electron Microscopy
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The condenser lens, often composedof a series of condensers, is primarilyresponsible for the production of thesmall probe diameter. With thedemagnification of the probe, there isan unavoidable loss in current fromthe probe.
Condenser and Objective (lenses)
Scanning Electron Microscopy
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The purpose of the objective lens isto obtain the smallest diameter beamat the surface of the sample. Itachieves this operating in the verysame manner as the condenser lens.However, it received the name"objective" in analogy to the focusingdone by the objective lens in the lightmicroscope.
Scanning Coils – Deflection Coils
The scanning coils serve many functions, all related tothe placement of the electron beam on the sample:
-raster scanning, coupled to the image CRT. This is themechanism for image formation and magnification.
Scanning Electron Microscopy
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-fine positioning of the raster on the sample, by applyinga DC voltage to the coils (electronic imageshift).
-selected area electron channeling patterns, by rockingthe beam about a point on the specimen.
-x-ray analysis of a single feature or spot, by shutting offthe rastering and using the scanning coils to position thebeam on the sample.
Beam-specimen interactions (signal types)
Scanning Electron Microscopy
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Everhart-Thornley Electron Detector
Scanning Electron Microscopy
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Scanning Electron Microscopy
Topographic contrast
(where do the shades come from?)
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The effect of surface topography on the escape ofsecondary electrons from within the sample
Backscattered electron detector
Scanning Electron Microscopy
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Secondary vs Backscattered electron images (JEOL)
Scanning Electron Microscopy
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Topographic contrast (SE mode) Atomic number contrast (BSE mode)
BSE images
Scanning Electron Microscopy
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Energy Dispersive Spectroscopy
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Energy Dispersive Spectroscopy
Background
Deconvolution
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CorrectionsZAF
Z – atomic number
A – Absorption
F – Fluorescence
Energy Dispersive Spectroscopy
Element Atomic % Weight %
Al 2.588 1.203
Si 4.247 2.056Qualitative
Analyses Types
Semi-quantitative (standardless)
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Si 4.247 2.056
Ti 0.365 0.301
Cr 21.793 19.529
Mn 0.229 0.216
Fe 3.931 3.783
Ni 58.337 59.013
Nb 3.261 5.221
Mo 5.249 8.677
Semi-quantitative (standardless)
Quantitative
Energy Dispersive Spectroscopy
X-Ray maps
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Advantages vs Disadvantages of SEM
� Advantages- high depth of field- direct observation of the external form of real objects at high magnifications- wide range of magnifications (below 50 x to over 100 000x)
� Disadvantages of SEM- high vacuum environment of the specimen- high vacuum environment of the specimen(porous, biological samples…)
- inability to show internal detail- inability to obtain highest resolution- conductive layer…
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Environmental SEM
Beam-Gas Interactions
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Environmental SEM
Environmental Secondary Detector
The environmental SecondaryDetector uses gas ionization toamplify the secondary electron signal.In non-conductive samples, positive
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In non-conductive samples, positiveions are attracted to the samplesurface where negative charges fromthe beam tend to accumulate. Thepositive ions effectively suppresscharge artifacts!!!
What are the implications for ceramic, polymer and biological samples observation?
Environmental SEM
PLA – Pressure Limiting Aperture
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Environmental SEM
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Transmission electron microscopy
� TEM is an analytical tool that allows detailed investigation of the morphology, structure, and local chemistry of metals, ceramics, polymers, biological materials and minerals. It also enables the investigation of crystal structures, crystallographic orientations through electron diffraction, as well as second phase, precipitates and contaminants distribution by x-ray and electron-energy analysis.analysis.
� Magnifications of up to 500,000x and detail resolution below 1 nm are achieved routinely. Quantitative and qualitative elemental analysis can be provided from features smaller than 30 nm. For crystals with interplanar spacing greater than 0.12 nm, crystal structure, symmetry and orientation can be determined. Structural identification of defects, including stacking faults and dislocations is possible.
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Transmission electron microscopy
A TEM usually operates at 10-6
The Instrument
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Torr in the column and 10-7 Torr
in the electron gun chamber.
These are the important parts of
a TEM and their general
interaction.
Transmission electron microscopy
The optical system of the TEM: Theobjective lens simultaneously generates thediffraction pattern and the first intermediateimage. Note that the ray paths are identicaluntil the intermediate lens, where the field
Ray Diagram
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until the intermediate lens, where the fieldstrengths are changed, depending on thedesired operation mode. The field strengthscan be changed by setting the focal lengths(the distance from the lens to the raycrossover). A higher field strength (shorterfocal length) is used for imaging, whereas aweaker field strength (longer focal length) isused for diffraction.
Transmission electron microscopy
What results from a higher field strength for a fixed final image plane?
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Transmission electron microscopy
Imaging Techniques – Bright field
Bright-field imaging is used forexamination of most microstructuralimaging. In order to examine samples inbright-field, the objective aperture mustbe inserted. The objective aperture is ametal plate with holes of various sizes
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metal plate with holes of various sizesmachined into it. The aperture is insertedinto the back focal plane, the same planeat which the diffraction pattern isformed. The back focal plane is locatedjust below the sample and objective lens.When the aperture is inserted, it onlyallows the electrons in the transmittedbeam to pass and contribute to theresulting bright-field image.
Transmission electron microscopy
Imaging Techniques – Dark field
• Dark-field images occur when the objective aperture is positioned off-axis from thetransmitted beam in order to allow only a diffracted beam to pass. In order to minimize theeffects of lens aberrations, the diffracted beam is deflected along the optic axis
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Transmission electron microscopy
Bright Field Images
When an electron beam strikes a sample, some of the electrons pass directly through whileothers may undergo slight inelastic scattering from the transmitted beam. Contrast in animage is created by differences in scattering. By inserting an aperture in the back focal plane,an image can be produced with these transmitted electrons. The resulting image is known asa bright field image. Bright field images are commonly used to examine micro-structuralrelated features.
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Crystalline defects shown in a BF imageBF image of a twinned crystal in strong contrast.
Transmission electron microscopy
Dark Field Images
If a sample is crystalline, many of the electrons will undergo elastic scattering from the various (hkl)planes. This scattering produces many diffracted beams. If any of these diffracted beams is allowedto pass through the objective aperture a dark field image is obtained. In order to reduce sphericalaberration and astigmatism and to improve overall image resolution, the diffracted beam will bedeflected such that it lies parallel the optic axis of the microscope. This type of image is said to be acentered dark field image. Dark field images are particularly useful in examining micro-structuraldetail in a single crystalline phases.
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Crystalline defects shown in a DF imageDF image of a twinned crystal in strong contrast.
Transmission electron microscopy
Bright Field Images (J.S.J. Vastenhout, Microsc Microanal 8 Suppl. 2, 2002)
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Stained with OsO4 and RuO4 vapors
Transmission electron microscopy (more examples)
acrylonitril-butadiene-styrene copolymer (ABS) - (cellular rubber particles) (LEICA)
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Schierholz JU, Hellmann GP. In situ graft copolymerization: salami morphologies in PMMA/EP blends: part I. Polymer 2003;44:2005–13.
Transmission electron microscopy (more examples)
Copolymer stained with iodine vapor. The insert shows the FFT of the image (Akora, Briber, Kofinas, Polymer 47 (2006) 2018)
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Transmission electron microscopy
High-Resolution
An atomic resolution imageis formed by the "phasecontrast" technique, whichexploits the differences inphase among the variouselectron beams scattered by
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electron beams scattered bythe sample in order toproduce contrast. A largeobjective lens apertureallows the transmitted beamand at least four diffractedbeams to form an image.
Transmission electron microscopy (more examples)
HRETM
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Transmission electron microscopy
Electron tomography
Imaging technique combines manyimages taken from differentperspectives to create a threedimensional model of the sample.Data collection involves collectingimages while tilting the specimen
Nephrin at a porous slit diaphragm
(Sidec Technologies )
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images while tilting the specimenaround a single axis. Software isused to combine the images into a3-D representation.
100 nm 5 nm
Transmission electron microscopy
are created when electrons are diffracted in a single crystalregion of a given specimen. The center spot corresponds tothe transmitted electron beam. Other spots are diffractedportions of the initial electron beam. Spot Patterns can beused for unknown phase identification and identification ofcrystal structure and orientation. The location of the spotsare again governed by Bragg's law.
Diffraction Techniques – Spot Patterns (Selected area diffraction – SAD)
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are again governed by Bragg's law.
Diffraction pattern from a single crystal of silicon in the <111> orientation.
SAD is a technique to reduceboth the area and intensity ofthe beam contributing to adiffraction pattern by theinsertion of an aperture into theimage plane of the objectivelens. This produces a virtualdiaphragm in the plane of thespecimen.
Transmission electron microscopy
Diffraction Techniques – Kikuchi Patterns
created when a selected area diffraction pattern is taken from a thick region of the thinfoil. Some electrons will be elastically scattered by Bragg diffraction from the various(hkl) atomic planes. Two cones of diffracted electrons are created by each set of (hkl)planes. The Kikuchi lines are actually arcs of intersection from these large radiidiffraction cones. One cone will be more intense than the general background andproduce a bright line. This is the excess line. The second cone will be less intense and
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produce a bright line. This is the excess line. The second cone will be less intense andproduce a dark line (see arrows). This is the deficit line.
Kikuchi Pattern from a thick sample of silicon. The six-foldsymmetrical distribution in this pattern is a good indicator that thesample is very close to the <111> orientation.
Kikuchi Patterns allow for very accurate determination ofcrystal orientation and are an important factor inestablishing the proper diffracting conditions for generatinghigh-contrast images.
Transmission electron microscopy
Diffraction Techniques – Convergent-Beam Electron Diffraction
CBED Patterns allow examination of diffraction patterns from specimen volumes lessthan 0.5 mm in diameter. In order to generate a diffraction pattern from such a smallarea, an electron beam must be focused. The resulting diffraction pattern will be aseries of disks rather than sharp spots due to highly convergent electron probe. Thediameter of these disks is then proportional to the beam convergence angle.
CBED patterns are routinely used to do crystallographic analysis of small volumes, such
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CBED pattern taken from a stainless steel single crystal viewingalong the [111] zone axis.
CBED patterns are routinely used to do crystallographic analysis of small volumes, suchas fine precipitates in thin foil specimens.
Transmission electron microscopy
Diffraction Techniques – Ring Patterns
created when electron diffraction occurs simultaneously from many grains withdifferent orientations relative to the incident electron beam. Analogous to x-ray powderdiffraction, ring patterns, can be used to identify unknown phases or characterize thecrystallography of a material. The radii and spacing of the rings are governed by:
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Ring diffraction patterns from polycrystalline gold indexed toshow which atomic planes (hkl) are contributing to the ring.
Rd = λL
where d is the interplanar spacing, R is the ring radius, and Lis known as the camera constant.
Structural and chemical diversity of sialoliths
Calcification
Hydroxyapatite
Transmission electron microscopy
500 nm
Hydroxyapatite
100 nm
Transmission electron microscopy
Structural and chemical diversity of sialoliths
Calcification
Hydroxyapatite
[0001] [1010]-
100 nm
250 nm
Transmission electron microscopy (more examples)
Bright-Field TEM
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Transmission electron microscopy (more examples)
Ring Diffraction patterns
Crystalline with planar defects Partially amorphous
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Transmission electron microscopy (more examples)
Structure identification
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Simulations
Transmission electron microscopy (more examples)
Structure identification
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Simulations
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Transmission electron microscopy
Sample Requisites
Specimens for examination by TEM must meet the following requirements:
� Thin enough for electrons to penetrate without excessive energy loss.� Be representative of the structure and composition.
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For penetration of a 200 kV electron beam, a typical metal, ceramic, or semiconductorspecimen must be less than 100 nm thick. However, the specimen is required torepresent the unaltered bulk material in terms of structure, chemistry, and content ofdefects. If one also wishes this specimen to have large amounts of electron-transparent thin area, be flat and unbent, and be strong enough to be easily handled,the task of making such a specimen from an arbitrary material is very difficult. Somespecimens such as thin films may be examined directly with very little specimenpreparation.
Transmission electron microscopy
Sample Requisites
Specimens for examination by TEM must meet the following requirements:
� Thin enough for electrons to penetrate without excessive energy loss.� Be representative of the structure and composition.
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For penetration of a 200 kV electron beam, a typical metal, ceramic, or semiconductorspecimen must be less than 100 nm thick. However, the specimen is required torepresent the unaltered bulk material in terms of structure, chemistry, and content ofdefects. If one also wishes this specimen to have large amounts of electron-transparent thin area, be flat and unbent, and be strong enough to be easily handled,the task of making such a specimen from an arbitrary material is very difficult. Somespecimens such as thin films may be examined directly with very little specimenpreparation.
Transmission electron microscopy
Sample Preparation
Powder Samplesprepared by suspension in a liquid. The suspensionis dropped onto a mounting grid disperses thepowder across it. The solvent, which is unreactivewith the sample material evaporates and leaves thepowder ready for investigation. Methanol and
Bulk/Interfaces - ceramics or metals
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powder ready for investigation. Methanol andAcetone are two such solvents commonly used
(Cryo)Ultra-microtomyused to be a standard technique in biology, but isnow a more general and especially important forpolymers. Preparation by Ultra-microtomy meanscutting thin pieces of a specimen with the aid of adiamond knife. These shaves are already thinenough to be electron-transparent. It is often usedwhen the material is too hard, too soft (polymer,biological samples) or when the damage of thesample may occur with other final thinning methods.
Transmission electron microscopy
Takes between 1 and 30 h and involving the following steps:
• The bulk material is reduced to a disc of 3 mm diameterby sanding, cutting, electro-erosion, crushing or repeatedcleaving.
• The preparation of a transparent area on the edge of aspecimen by polishing it at a slight (less than 5°) angle is
Mechanical + Ion-Thinning Sample Preparation
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specimen by polishing it at a slight (less than 5°) angle iscalled wedge polishing and is a very common method.
• Dimpling is another common preparation technique. Thesample is pre-thinned to 30-50 µm and a bowl-shapeddimple is polished in the center.
• One way of achieving the electron-transparent thicknessof 5 µm is ion-milling at a low angle (10° to 15°) to createa transparent area with a centered hole in the specimen.
Transmission electron microscopy
Ultra-cryomicrotomy
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Transmission electron microscopy
Advantages
� Highest spatial resolution (atomic scale resolution (sub Å)� Quantitative identification of structural defects (determination of theBurgers vector
Disadvantages
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Disadvantages
� TEM is an expensive and destructive technique� Some materials are sensitive to electron beam radiation, resulting in a loss ofcrystallinity and mass
� Sample preparation is time consuming� Sample dimension is small (3 mm diameter, less than ~100 nm thick fortransparency)
Atomic force microscopy
Working principles
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� Contact
Atomic force microscopy
Operation modes
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� Tapping (intermitent, non-contact)
Probes: cantilever and tip
Si
Si
Atomic force microscopy
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Si3N4Si3N4
Contact mode (repulsive mode)
Topography
Atomic force microscopy
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F = nN-µN
zkF ∆−= .
Topography
- Constant load- Constant height
Force spectroscopy
Atomic force microscopy
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Repulsive
Adhesive
Pull-out force
Tapping mode
Atomic force microscopy
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f ~ fR
A = 20-100 nm
Atomic force microscopy
Examples – Cork cell walls (contact)
TEM
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A
B
Atomic force microscopy
Examples – PVD TiN coatings on stainless steel (tapping)
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Contact vs tapping
� Hard samples (contact/taping)� Soft samples(tapping)� Tip damage (tapping)� Adsorption layers – e.g. water (contact)� High scanning speed (contact – slow feedback
Atomic force microscopy
in dynamic mode)
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Atomic force microscopy
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Lateral Force Microscopy (LFM)
Cantilever torsion is proportional to height and to friction
Atomic force microscopy
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Magnetic/ Electric Force Microscopy (MFM and EFM)
• Step 1 – In a first scan topography is measured under tapping mode
• Step 2 – In a second scan phase, amplitude or frequency changes of the cantilever caused by magnetic or electric interactions are measured.
Atomic force microscopy
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V+
-
MFM EFM
MFM example – Recording tape CrO2
Atomic force microscopy
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Topographic Image Magnetic Force Image
EFM example - two-component LB-film (NT-MDT)
Atomic force microscopy
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Topography Electric field distribution
Atomic force microscopy
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Near-Field Scanning Optical Microscopy (NSOM or SNOM) http://micro.magnet.fsu.edu/primer
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Near-Field Scanning Optical Microscopy (NSOM or SNOM) http://www.ntmdt.com/
Shear Force Microscopy
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DNA Scan size:1.25 µm x1.25 µm
Near-Field Scanning Optical Microscopy (NSOM or SNOM) http://www.ntmdt.com/
Transmission mode
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Near-Field Scanning Optical Microscopy (NSOM or SNOM) http://www.ntmdt.com/
Reflection mode
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Near-Field Scanning Optical Microscopy (NSOM or SNOM) http://www.ntmdt.com/
Fluorescence mode
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Microscopy techniques for biomaterials
DiscussionDiscussion
Engenharia Biomédica100