Microscopy techniques for biomaterials. Engenharia Biomédica. Patrícia Almeida Carvalho

Microscopy techniques for biomaterials Engenharia Biomédica Patrícia Almeida Carvalho 1

2 Why microscopy? http://www.cellsalive.com/howbig.htm

3 Why microscopy?

Resolution of an optical system Diffraction at an aperture - Rayleigh criterion http://micro.magnet.fsu.edu/primer The Rayleigh criterion for the resolution of an optical system states that two points will be resolvable if the maximum of the intensity of the Airy ring from one of them coincides with the first minimum intensity of the Airy ring of the other. This implies that the resolution, ρ (strictly resolving power) is given by: ρ = 0.6λ/η /ηsinα where λ is the wavelength, η the refractive index and α is the semi-angle at the specimen. 4

Introduction to lenses http://micro.magnet.fsu.edu/primer 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: f object = f image 1/a + 1/b = 1/f M = b/a 5

Image Formation http://micro.magnet.fsu.edu/primer 6

Compound Microscope http://micro.magnet.fsu.edu/primer 7

Compound Microscope Transmission (Diascopic) http://micro.magnet.fsu.edu/primer Transmission (Diascopic)

Compound Microscope Transmission (Diascopic) http://micro.magnet.fsu.edu/primer Reflection (Episcopic)

Fluorescence Microscopy http://micro.magnet.fsu.edu/primer 10

Fluorescence Microscopy http://micro.magnet.fsu.edu/primer 11

Fluorescence Microscopy http://micro.magnet.fsu.edu/primer 12

Confocal microscopy http://micro.magnet.fsu.edu/primer The Instrument (Laser Scanning confocal microscope) Confocal or conjugate planes ρ = 0.6λ/ηsinα ρ = 0.41λ/ηsinα Reflection mode: surface topography 13

Confocal microscopy http://micro.magnet.fsu.edu/primer 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. 14

Confocal microscopy http://micro.magnet.fsu.edu/primer 15

Confocal microscopy http://micro.magnet.fsu.edu/primer Tomography 16

17 Microscope working principles

Image Types JEOL images 18

Light vs electrons ρ = 0.61λ/ηsinα 19

20 Electromagnetic Lenses

21 Electromagnetic Lenses

22 Electromagnetic Lenses

23 Electromagnetic Lenses

24 Electromagnetic Lenses

Electromagnetic Lenses Electromagnetic lenses 25

Scanning Electron Microscopy working principles 26

Scanning Electron Microscopy The Microscope Hitachi S 2400

Scanning Electron Microscopy Depth of field - Light vs electrons (JEOL) Optical Microscope Image SEM Image of Same Object 28

Scanning Electron Microscopy Woven polyester-fiber cloth House dust mite

Scanning Electron Microscopy The instrument Electron gun Gun alignment coils Condenser lens Secondary electron detector Deflection coils Objective lens Backscatter electron detector Specimen goniometer stage Oil diffusion vacuum pump Specimen chamber 30

Scanning Electron Microscopy Tungsten Hairpin Filament LaB 6 Pointed Rod Electron Gun - Cathodes Tungsten field-emission cathode 31

Scanning Electron Microscopy Electron Gun - Thermionic vs Field Emission 32

Scanning Electron Microscopy The condenser lens, often composed of a series of condensers, is primarily responsible for the production of the small probe diameter. With the demagnification of the probe, there is an unavoidable loss in current from the probe. Condenser and Objective (lenses) The purpose of the objective lens is to obtain the smallest diameter beam at the surface of the sample. It achieves this operating in the very same manner as the condenser lens. However, it received the name "objective" in analogy to the focusing done by the objective lens in the light microscope. 33

Scanning Electron Microscopy Scanning Coils Deflection Coils The scanning coils serve many functions, all related to the placement of the electron beam on the sample: -raster scanning, coupled to the image CRT. This is the mechanism for image formation and magnification. -fine positioning of the raster on the sample, by applying a DC voltage to the coils (electronic imageshift). -selected area electron channeling patterns, by rocking the beam about a point on the specimen. -x-ray analysis of a single feature or spot, by shutting off the rastering and using the scanning coils to position the beam on the sample. 34

Scanning Electron Microscopy Beam-specimen interactions (signal types) 35

Scanning Electron Microscopy Everhart-Thornley Electron Detector 36

Scanning Electron Microscopy Topographic contrast (where do the shades come from?) The effect of surface topography on the escape of secondary electrons from within the sample 37

Scanning Electron Microscopy Backscattered electron detector 38

Scanning Electron Microscopy Secondary vs Backscattered electron images (JEOL) Topographic contrast (SE mode) Atomic number contrast (BSE mode) 39

Scanning Electron Microscopy BSE images 40

41 Energy Dispersive Spectroscopy

Energy Dispersive Spectroscopy Background Deconvolution Corrections ZAF Z atomic number A Absorption F Fluorescence 42

Energy Dispersive Spectroscopy Analyses Types Qualitative Semi-quantitative (standardless) Quantitative Semi-quantitative (standardless) Element Atomic % Weight % Al 2.588 1.203 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 43

Energy Dispersive Spectroscopy X-Ray maps 44

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 (porous, biological samples ) - inability to show internal detail - inability to obtain highest resolution - conductive layer 45

Environmental SEM Beam-Gas Interactions 46

Environmental SEM Environmental Secondary Detector The environmental Secondary Detector uses gas ionization to amplify the secondary electron signal. In non-conductive samples, positive ions are attracted to the sample surface where negative charges from the beam tend to accumulate. The positive ions effectively suppress charge artifacts!!! What are the implications for ceramic, polymer and biological samples observation? 47

Environmental SEM PLA Pressure Limiting Aperture 48

49 Environmental SEM

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. 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. 50

Transmission electron microscopy The Instrument A TEM usually operates at 10-6 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. 51

Transmission electron microscopy Ray Diagram The optical system of the TEM: The objective lens simultaneously generates the diffraction pattern and the first intermediate image. Note that the ray paths are identical until the intermediate lens, where the field strengths are changed, depending on the desired operation mode. The field strengths can be changed by setting the focal lengths (the distance from the lens to the ray crossover). A higher field strength (shorter focal length) is used for imaging, whereas a weaker field strength (longer focal length) is used for diffraction. 52

Transmission electron microscopy What results from a higher field strength for a fixed final image plane? 53

Transmission electron microscopy Imaging Techniques Bright field Bright-field imaging is used for examination of most microstructural imaging. In order to examine samples in bright-field, the objective aperture must be inserted. The objective aperture is a metal plate with holes of various sizes machined into it. The aperture is inserted into the back focal plane, the same plane at which the diffraction pattern is formed. The back focal plane is located just below the sample and objective lens. When the aperture is inserted, it only allows the electrons in the transmitted beam to pass and contribute to the resulting bright-field image. 54

Transmission electron microscopy Imaging Techniques Dark field Dark-field images occur when the objective aperture is positioned off-axis from the transmitted beam in order to allow only a diffracted beam to pass. In order to minimize the effects of lens aberrations, the diffracted beam is deflected along the optic axis 55

Transmission electron microscopy Bright Field Images When an electron beam strikes a sample, some of the electrons pass directly through while others may undergo slight inelastic scattering from the transmitted beam. Contrast in an image 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 as a bright field image. Bright field images are commonly used to examine micro-structural related features. BF image of a twinned crystal in strong contrast. Crystalline defects shown in a BF image 56

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 allowed to pass through the objective aperture a dark field image is obtained. In order to reduce spherical aberration and astigmatism and to improve overall image resolution, the diffracted beam will be deflected such that it lies parallel the optic axis of the microscope. This type of image is said to be a centered dark field image. Dark field images are particularly useful in examining micro-structural detail in a single crystalline phases. DF image of a twinned crystal in strong contrast. Crystalline defects shown in a DF image 57

Transmission electron microscopy Bright Field Images (J.S.J. Vastenhout, Microsc Microanal 8 Suppl. 2, 2002) Stained with OsO 4 and RuO 4 vapors 58

Transmission electron microscopy (more examples) acrylonitril-butadiene-styrene copolymer (ABS) - (cellular rubber particles) (LEICA) Schierholz JU, Hellmann GP. In situ graft copolymerization: salami morphologies in PMMA/EP blends: part I. Polymer 2003;44:2005 13. 59

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) 60

Transmission electron microscopy High-Resolution An atomic resolution image is formed by the "phase contrast" technique, which exploits the differences in phase among the various electron beams scattered by the sample in order to produce contrast. A large objective lens aperture allows the transmitted beam and at least four diffracted beams to form an image. 61

Transmission electron microscopy (more examples) HRETM 62

Transmission electron microscopy Electron tomography Imaging technique combines many images taken from different perspectives to create a three dimensional model of the sample. Data collection involves collecting images while tilting the specimen around a single axis. Software is used to combine the images into a 3-D representation. Nephrin at a porous slit diaphragm (Sidec Technologies ) 100 nm 5 nm 63

Transmission electron microscopy Diffraction Techniques Spot Patterns (Selected area diffraction SAD) are created when electrons are diffracted in a single crystal region of a given specimen. The center spot corresponds to the transmitted electron beam. Other spots are diffracted portions of the initial electron beam. Spot Patterns can be used for unknown phase identification and identification of crystal structure and orientation. The location of the spots are again governed by Bragg's law. SAD is a technique to reduce both the area and intensity of the beam contributing to a diffraction pattern by the insertion of an aperture into the image plane of the objective lens. This produces a virtual diaphragm in the plane of the specimen. Diffraction pattern from a single crystal of silicon in the <111> orientation. 64

Transmission electron microscopy Diffraction Techniques Kikuchi Patterns created when a selected area diffraction pattern is taken from a thick region of the thin foil. 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 radii diffraction cones. One cone will be more intense than the general background and produce a bright line. This is the excess line. The second cone will be less intense and produce a dark line (see arrows). This is the deficit line. Kikuchi Patterns allow for very accurate determination of crystal orientation and are an important factor in establishing the proper diffracting conditions for generating high-contrast images. Kikuchi Pattern from a thick sample of silicon. The six-fold symmetrical distribution in this pattern is a good indicator that the sample is very close to the <111> orientation. 65

Transmission electron microscopy Diffraction Techniques Convergent-Beam Electron Diffraction CBED Patterns allow examination of diffraction patterns from specimen volumes less than 0.5 mm in diameter. In order to generate a diffraction pattern from such a small area, an electron beam must be focused. The resulting diffraction pattern will be a series of disks rather than sharp spots due to highly convergent electron probe. The diameter of these disks is then proportional to the beam convergence angle. CBED patterns are routinely used to do crystallographic analysis of small volumes, such as fine precipitates in thin foil specimens. CBED pattern taken from a stainless steel single crystal viewing along the [111] zone axis. 66

Transmission electron microscopy Diffraction Techniques Ring Patterns created when electron diffraction occurs simultaneously from many grains with different orientations relative to the incident electron beam. Analogous to x-ray powder diffraction, ring patterns, can be used to identify unknown phases or characterize the crystallography of a material. The radii and spacing of the rings are governed by: Rd = λl where d is the interplanar spacing, R is the ring radius, and L is known as the camera constant. Ring diffraction patterns from polycrystalline gold indexed to show which atomic planes (hkl) are contributing to the ring. 67

Transmission electron microscopy Structural and chemical diversity of sialoliths Calcification Hydroxyapatite 500 nm

Transmission electron microscopy Structural and chemical diversity of sialoliths Calcification 100 nm 250 nm [0001] - [1010] Hydroxyapatite

Transmission electron microscopy (more examples) Bright-Field TEM 70

Transmission electron microscopy (more examples) Ring Diffraction patterns Crystalline with planar defects Partially amorphous 71

Transmission electron microscopy (more examples) Structure identification?? Simulations 72

Transmission electron microscopy (more examples) Structure identification Simulations?? 73

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. For penetration of a 200 kv electron beam, a typical metal, ceramic, or semiconductor specimen must be less than 100 nm thick. However, the specimen is required to represent the unaltered bulk material in terms of structure, chemistry, and content of defects. If one also wishes this specimen to have large amounts of electrontransparent 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. Some specimens such as thin films may be examined directly with very little specimen preparation. 74

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. For penetration of a 200 kv electron beam, a typical metal, ceramic, or semiconductor specimen must be less than 100 nm thick. However, the specimen is required to represent the unaltered bulk material in terms of structure, chemistry, and content of defects. If one also wishes this specimen to have large amounts of electrontransparent 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. Some specimens such as thin films may be examined directly with very little specimen preparation. 75

Transmission electron microscopy Sample Preparation Bulk/Interfaces - ceramics or metals Powder Samples prepared by suspension in a liquid. The suspension is dropped onto a mounting grid disperses the powder across it. The solvent, which is unreactive with the sample material evaporates and leaves the powder ready for investigation. Methanol and Acetone are two such solvents commonly used 76 (Cryo)Ultra-microtomy used to be a standard technique in biology, but is now a more general and especially important for polymers. Preparation by Ultra-microtomy means cutting thin pieces of a specimen with the aid of a diamond knife. These shaves are already thin enough to be electron-transparent. It is often used when the material is too hard, too soft (polymer, biological samples) or when the damage of the sample may occur with other final thinning methods.

Transmission electron microscopy Mechanical + Ion-Thinning Sample Preparation Takes between 1 and 30 h and involving the following steps: The bulk material is reduced to a disc of 3 mm diameter by sanding, cutting, electro-erosion, crushing or repeated cleaving. The preparation of a transparent area on the edge of a specimen by polishing it at a slight (less than 5 ) angle is called wedge polishing and is a very common method. Dimpling is another common preparation technique. The sample is pre-thinned to 30-50 µm and a bowl-shaped dimple is polished in the center. One way of achieving the electron-transparent thickness of 5 µm is ion-milling at a low angle (10 to 15 ) to create a transparent area with a centered hole in the specimen. 77

Transmission electron microscopy Ultra-cryomicrotomy 78

Transmission electron microscopy Advantages Highest spatial resolution (atomic scale resolution (sub Å) Quantitative identification of structural defects (determination of the Burgers vector Disadvantages TEM is an expensive and destructive technique Some materials are sensitive to electron beam radiation, resulting in a loss of crystallinity and mass Sample preparation is time consuming Sample dimension is small (3 mm diameter, less than ~100 nm thick for transparency) 79

Atomic force microscopy Working principles 80

Atomic force microscopy Operation modes Contact Tapping (intermitent, noncontact) 81

Si 3 N 4 Si 3 N 4 Atomic force microscopy Probes: cantilever and tip Si Si 82

Atomic force microscopy Contact mode (repulsive mode) F = nn-µn Topography F = k. z - Constant height - Constant load 83

Atomic force microscopy Force spectroscopy Repulsive Pull-out force Adhesive 84

Atomic force microscopy Tapping mode f ~ f R A = 20-100 nm 85

Atomic force microscopy TEM Examples Cork cell walls (contact) A B 86

Atomic force microscopy Examples PVD TiN coatings on stainless steel (tapping) 87

Atomic force microscopy 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 in dynamic mode) 88

89 Atomic force microscopy

Atomic force microscopy Lateral Force Microscopy (LFM) Cantilever torsion is proportional to height and to friction 90

Atomic force microscopy 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. V + - MFM EFM 91

Atomic force microscopy MFM example Recording tape CrO 2 Topographic Image Magnetic Force Image 92

Atomic force microscopy EFM example - two-component LB-film (NT-MDT) Topography Electric field distribution 93

94 Atomic force microscopy

95 Near-Field Scanning Optical Microscopy (NSOM or SNOM) http://micro.magnet.fsu.edu/primer

Near-Field Scanning Optical Microscopy (NSOM or SNOM) http://www.ntmdt.com/ Shear Force Microscopy DNA Scan size: 1.25 µm x1.25 µm 96

Near-Field Scanning Optical Microscopy (NSOM or SNOM) http://www.ntmdt.com/ Transmission mode 97

Near-Field Scanning Optical Microscopy (NSOM or SNOM) http://www.ntmdt.com/ Reflection mode 98

Near-Field Scanning Optical Microscopy (NSOM or SNOM) http://www.ntmdt.com/ Fluorescence mode 99

Microscopy techniques for biomaterials Discussion 100 Engenharia Biomédica