Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Optical materials characterization Julio Soares Frederick Seitz Materials Research Laboratory University of Illinois at Urbana-Champaign © 2014University of Illinois Board of Trustees. All rights reserved. Why optical characterization? 2 Why optical characterization? 2 Optics Communications, Vol 284(9), 2376-2381 (2011) Why optical characterization? 2 Why optical characterization? 2 Why optical characterization? 2 Why optical characterization? 2 Why optical characterization? 3 Why optical characterization? 4 Why optical characterization? 5 • The photoelectric effect The Nobel Prize in Physics 1921 Albert Einstein "for his services to Theoretical Physics, and especially for his discovery of the law of the photoelectric effect" The Nobel Foundation The Nobel Prize in Physics 1923 Robert A. Millikan "for his work on the elementary charge of electricity and on the photoelectric effect" The Nobel Foundation library.thinkquest.org Why optical characterization? 6 Black body radiation, which lead to quantum physics… Classical (Rayleigh-Jeans) Quantum (Planck) 0.030 0.025 r(n) 0.020 0.015 The Nobel Prize in Physics 1918 Max Planck 0.010 0.005 0.000 0 10,000 20,000 30,000 "in recognition of the services he rendered to the advancement of Physics by his discovery of energy quanta" wavenumbers (1/cm) Copyright 2001 B.M. Tissue The Nobel Foundation Why optical characterization? 7 2009 2005 Roy J. Glauber John L. Hall Theodor W. Hänsch Charles Kuen Kao Willard S. Boyle George E. Smith transmission of light in fibers for optical communication and invention of the CCD 1981 Claude Cohen-Tannoudji William D. Phillips Nicolaas Bloembergen Arthur Leonard Schawlow development of methods to cool and trap atoms with laser light 1923 1921 1964 1997 contributions to the quantum theory of optical coherence and the development of laser-based precision spectroscopy Steven Chu 1930 Robert Andrews Millikan Albert Einstein contribution to the development of laser spectroscopy Sir Chandrasekhara Venkata Raman the photoelectric effect Charles Hard Townes Nicolay Gennadiyevich Basov Aleksandr Mikhailovich Prokhorov fundamental work in the field of quantum electronics, which has led to the construction of oscillators and amplifiers based on the maser-laser principle 1953 Frits (Frederik) Zernike 1922 1918 Niels Henrik David Bohr 1966 investigation of the structure of atoms and of the radiation emanating from them Alfred Kastler discovery and development of optical methods for studying Hertzian resonances in atoms demonstration of the phase contrast method, especially for his invention of the phase contrast microscope discovery of the Raman effect 1907 Max Karl Ernst Ludwig Planck discovery of energy quanta 1908 Albert Abraham Michelson for his optical precision instruments and the spectroscopic and metrological investigations carried out with their aid Gabriel Lippmann method of reproducing colours photographically based on the phenomenon of interference The Nobel Foundation Light – matter interaction 8 Optical methods for materials characterization 9 • Interrogating the material properties with light. – Transmission/Reflection spectroscopy (identification, structure, concentration, speed, etc.) • Spectrophotometry, FTIR – Ellipsometry (dielectric function, layer thickness, carrier density, etc.) – Raman (identification, structure, phase, crystallinity, stress, drug distribution, diagnosis, etc.) – PL/PLE (electronic structure, carrier life-time, defect levels, carrier concentration, size distribution, etc.) – SFG (surface/interface chemistry, identification, orientation, etc.) – Modulation spectroscopy (electronic structure, carrier life-time, defect levels, internal electric fields, thermal properties, mechanical properties, etc.) • PR, ER, TDTR, FDTR – Photocurrent/Photovoltage (electronic structure, energy band states, etc.) • Quantum efficiency, solar cell and detector characterization profile, defect Transmission, Reflection, Absorption 10 What is measured? The transmission, and reflection of light as a function of the incident photon energy. Basic principle: The absorption, reflection and transmission of light by a material depends on its electronic, atomic, chemical and morphological structure. 15 Transmission, Reflection, Absorption 11 UV-VIS-NIR FTIR 16 Transmission, Reflection, Absorption Raman Spectroscopy 12 17 Impurities Excited states Donnor levels Virtual state Acceptor levels Vibrational states Ground state IR UV/VIS Spectrophotometry (UV-VIS-NIR) 13 Instrumentation: Transmission Specular reflectance Diffuse reflectance 18 Spectrophotometry (UV-VIS-NIR) 14 Instrumentation: 19 Fourier Transform IR spectroscopy (FTIR) 20 15 The Nobel Prize in Physics 1907 Albert A. Michelson "for his optical precision instruments and the spectroscopic and metrological investigations carried out with their aid" Instrumentation: The FTIR uses a Michelson interferometer with a moving mirror, in place of a diffraction grating or prism. The Nobel Foundation fixed mirror DL = nl => constructive interference DL = (n+1/2) l => destructive interference moving mirror Beam splitter sample detector Detector voltage IR source -10 -8 -6 -4 -2 0 2 4 6 8 10 Time Fourier Transform IR spectroscopy (FTIR) 21 15 The Nobel Prize in Physics 1907 Albert A. Michelson "for his optical precision instruments and the spectroscopic and metrological investigations carried out with their aid" Instrumentation: The FTIR uses a Michelson interferometer with a moving mirror, in place of a diffraction grating or prism. The Nobel Foundation fixed mirror DL = nl => constructive interference DL = (n+1/2) l => destructive interference moving mirror Beam splitter sample detector Detector voltage IR source -10 -8 -6 -4 -2 0 2 4 6 8 10 Time Fourier Transform IR spectroscopy (FTIR) 22 16 . I (n ) S (t )e 2int dt -10 -8 -6 -4 -2 0 2 4 6 8 10 Time Intensity Detector voltage Spectrum formation: 0.0 0.4 0.8 Frequency 1.2 Fourier Transform IR spectroscopy (FTIR) 23 17 An example of an interferogram and its corresponding FTIR spectrum. Frequency (Hz) Polystyrene Amplitude The signal is detected as intensity vs. time (interferogram). The Fourier transform of the interferogram gives the spectrum, as intensity vs. wave number. Absorbance 2.0 1.5 1.0 0.5 0.0 1000 1500 2000 2500 3000 -1 Wavenumber (cm ) 3500 4000 Spectrophotometry (UV-VIS-NIR) 18 Instrumentation: 24 Fourier Transform IR spectroscopy (FTIR) 19 FT vs. Dispersive optics spectrometers Advantages: Disadvantages: •Multiplexing (all wavelengths are measured simultaneously). •More complex system. •No chromatic aberrations or artifacts. •Higher throughput. •Improved S/N ratio. •Linearity of detectors (working closer to saturation levels). •Stray light scattering at low signal level. 25 Spectrophotometry (UV-VIS-NIR) 26 20 0.4 0.3 Abs = K l c = a l .5mM solutions 0.2 0.5 0.4 0.1 450 500 550 600 650 Wavelength (nm) Using absorption to determine Au/Hg concentration in water solutions As Au relative concentration rises, the absorption peak shifts toward shorter wavelengths, increase in intensity, and its FWHM decreases. The intensity increase follows Beer-Lambert’s law. Absorption Absorption Beer-Lambert Law 4ml Au/.1ml Hg 3ml Au/1ml Hg 2ml Au/2ml Hg 1ml Au/3ml Hg 0.3 0.2 0.1 0.0 0.1 0.2 0.3 0.4 Au concentration (mM) 0.5 Spectrophotometry (UV-VIS-NIR) l (nm) 500 400 58 600 700 800 58.0 57.5 57.0 57 56.5 T(%) 56.0 14000 𝜆𝑚 = 𝑚 𝜈 = 2𝑛𝑑 sin 𝜃 peak index / 2n (m/cm) 21 13000 56 27 peak positions linear fit 30000 20000 10000 Slope = 2.76 m 0 14000 16000 18000 20000 22000 24000 55 60000 22500 20000 17500 n (cm-1) 15000 12500 Using transmission interference fringes to determine thickness Two sets of interference fringes are present on the spectrum, corresponding to each film that composes the system. peak index / 2n (m/cm) 54 25000 n (cm-1) 50000 peak positions linear fit 40000 30000 20000 10000 Slope = 50.99 m 0 12500 13000 n (cm-1) 13500 Spectrophotometry (UV-VIS-NIR) 22 Excitations in materials •Plasmons Phys. Today, 64, 39 (2011) http://juluribk.com Plasmons are quanta of collective motion of charge-carriers in a gas with respect of an oppositely charged background. They play a significant role on transmission and 3 m reflection of light. Spectrophotometry (UV-VIS-NIR) 29 23 M X k0 G 200 nm kx 3 m M G X Optics Express 13, 5669 (2005) Plasmonic crystal Brillouin zone from the transmission spectra measured for many different angles of incidence. Fourier Transform IR spectroscopy (FTIR) 24 http://www.clockhours.com Fingerprinting: FTIR can be used to identify components in a mixture by comparison with reference spectra. Discovery of beeswax as binding agent on a 6th-century BC Chinese turquoise-inlaid bronze sword Wugan Luo, Tao Li, Changsui Wang, Fengchun Huang J. of Archaeological Sci. 39 (2012), 1227 30 Fourier Transform IR spectroscopy (FTIR) 25 Fingerprinting: FTIR can be used to identify components in a mixture by comparison with reference spectra. Complementary characterization techniques, like XRD can provide conclusive evidence for the identification. J. of Archaeological Sci. 39 (2012), 1227 31 Spectrophotometry (UV-VIS-NIR) and FTIR 26 Strengths: •Very little to no sample preparation. •Simplicity of use and data interpretation. •Short acquisition time, for most cases. •Non destructive. •Broad range of photon energies. •High sensitivity (~ 0.1 wt% typical for FTIR). 32 Spectrophotometry (UV-VIS-NIR) and FTIR 27 Limitations: • • • • Reference sample is often needed for quantitative analysis. Many contributions to the spectrum are small and can be buried in the background. Usually, unambiguous chemical identification requires the use of complementary techniques. Limited spatial resolution. Complementary techniques: Raman, Electron Energy Loss Spectroscopy (EELS), Extended X-ray Absorption Fine Structure (EXAFS), XPS, Auger, SIMS, XRD, SFG. 33 Polarization 34 28 www.bobatkins.com Guimond and Elmore - Oemagazine May 2004 Ellipsometry 29 What is measured? The changes in the polarization state of light upon reflection from a mirror like surface. 35 Ellipsometry 36 30 Basic principle: The reflected light emerges from the surface elliptically polarized, i.e. its p and s polarization components are generally different in phase and amplitude. f ~ Rp iD tan( )e ~ Rs Ellipsometry 37 31 fa a d fb b c fc ~ R p,s E E r p,s i p,s n~1 sin f1 n~2 sin f2 n~ n + ik ~ r12p , s n~2,1 cos f1 n~1, 2 cos f2 ~ n cos f + n~ cos f 2 ,1 1 1, 2 ~ rabp , s + ~ rbcp , s e 2i ~ R p,s 1+ ~ rabp , s ~ rbcp , s e 2i 2d ~ nb cos fb e i ( pr ,s pi ,s ) ~ Rp iD tan( )e ~ Rs l ~ Rp tan( ) ~ Rs r i D 2 Ellipsometry 32 38 Applications 21.9 Å Si SiO2 thickness c 2 4.31 2.19 ± 0.04 nm 14 12 10 8 6 4 2 0 Model fit Experiment D 400 500 600 700 800 Wavelength (nm) 180 160 140 120 100 80 60 D (degrees) SiO2 (degrees) Film thickness Ellipsometry 33 39 Applications Composition Surface roughness Film thickness Band gap energy Ellipsometric (l) and D(l) spectra of Cd1-xZnxS thin films deposited under the different concentration of ammonia: 0.19, 0.38, 0.56, and 0.75 M. The solid lines represent the best fit to the theoretical model. (a) and (b) were measured under 68° and 72°, respectively. Jpn. J. Appl. Phys. 49 (2010) 081202 Ellipsometry 34 40 Applications Composition Surface roughness Film thickness Band gap energy Parameters of the Cd1xZnxS thin films as a function of different ammonia concentrations obtained from SE analysis. [NH4OH] (M) Thickness (nm) Roughness (nm) ZnS (%) Band-gap (eV) 0.19 42.12 23.77 99.7 3.49 0.38 73.79 7.15 45.5 2.52 0.56 50.89 5.94 32.3 2.45 0.75 18.59 4.54 5.2 2.43 Jpn. J. Appl. Phys. 49 (2010) 081202 Ellipsometry 35 41 Applications Composition Surface roughness Film thickness Band gap energy Optical constants (dielectric function) Refractive index (n) and extinction coefficient (k) as a function of wavelength of Cd1-xZnxS thin films under different ammonia concentrations. Plots of (αhν)2 versus photon energy h for Cd1-xZnxS thin films under different ammonia concentration. Jpn. J. Appl. Phys. 49 (2010) 081202 Ellipsometry 36 Optical Hall Effect 42 Ellipsometry 37 43 Applications Electrical properties From the fit for the C-face sample: Two graphene layers with distinctly different properties: a bottom p-type channel with Ns=(5.5±0.4)x1013 cm-2 and =1521±52 cm2 V-1 s-1 a top p-type channel with Ns=(3.4±0.6)x1014 cm-2 and =18±4 cm2 V-1 s-1 From electrical dc Hall effect measurement: p-type conductivity with Ns=(3.0±0.5) x1013 cm-2 and =3407±250 cm2 V-1 s-1 Ellipsometry 38 44 Applications Electrical properties From the fit for the Si-face sample: A p-type channel with Ns=(1.2±0.3)x1012 cm-2 and =794±80 cm2 V-1 s-1 From electrical dc Hall effect measurement: p-type conductivity with Ns=(1.9±0.2) x1012 cm-2 and =891±250 cm2 V-1 s-1 The correspondence between electrical and OHE data is very good. Ellipsometry 39 45 Applications Electrical properties C-face: Top layer: m* = (0.19−0.08√B) m0 Bottom layer: m* = 0.035 m0 Si-face: m* = 0.03 m0 Ellipsometry 46 40 Stregths: – Fast. – Measures a ratio of two intensity values and a phase. • Highly accurate and reproducible (even in low light levels). • No reference sample necessary. • Not as susceptible to scatter, lamp or purge fluctuations. • Increased sensitivity, especially to ultrathin films (<10nm). – Can be used in-situ. DC Comics Ellipsometry 47 41 Limitations: – Flat and parallel surface and interfaces with measurable reflectivity. – A realistic physical model of the sample is usually required to obtain useful information. Neil Miller Complementary techniques: PL, Modulation spectroscopies, X-Ray Photoelectron Spectroscopy, Secondary Ion Mass Spectroscopy, XRD. 42 Raman spectroscopy Raman Spectroscopy 48 Sir Chandrasekhara Venkata Raman The Nobel Prize in Physics 1930 was awarded to Sir Venkata Raman "for his work on the scattering of light and for the discovery of the effect named after him". The Nobel Foundation Raman spectroscopy Raman Spectroscopy Anti-Stokes Stokes Scattered intensity 43 What is measured? The light inelastically scattered by the material. Photon energy Stokes Rayleigh scattering Anti-Stokes Raman shift Excited state Virtual states Basic principle: The impinging light couples with the lattice vibrations (phonons) of the material, and a small portion of it is inelastically scattered. The difference between the energy of the scattered light and the incident beam is the energy absorbed or released by the phonons. Vibrational states Ground state 49 Resonance Raman IR Raman spectroscopy 44 Excitations in materials •Phonons •Molecular vibrations http://www.physik.tu-berlin.de/institute/IFFP/richter/new/research/surface-phonons.shtml Phonons are the quanta of Collective lattice vibrations 45 Raman spectroscopy Raman Spectroscopy 51 51 -1 Wavenumber (cm ) 1000 2000 2500 3000 Polyetherurethane Intensity Like the FTIR, Raman spectroscopy measures the interaction of photons with the vibration modes of materials, but the physical processes behind the two techniques are fundamentally different and so are the selection rules that apply to each. 1500 FTIR Raman The two techniques are complementary, rather than equivalent. 1000 1500 2000 2500 -1 Raman shift (cm ) 3000 Raman spectroscopy Raman Spectroscopy 46 52 52 Molecular and crystalline structure characterization Raman is sensitive to the atomic structure of the material. Raman intensity (arb. units) Diamond C Nanotube Coal (Rock)-norm Graphite coal (wood) 1000 2000 3000 -1 Physics Reports, 409 (2005), 47 Raman Shift (cm ) Raman spectroscopy Raman Spectroscopy 47 53 53 Chemical composition & component identification Components distribution at micron & sub-micron scale Distribution of ingredients in a pharmaceutical tablet The AAPS Journal 2004; 6 (4), 32 Renishaw, Inc. Raman spectroscopy Raman Spectroscopy 48 Molecular and crystalline structure characterisation Presence of N vacancies yields poor crystallinity <C Substitutional C fills N vacancies improving the crystallinity >C C incorporates interstitially causing a degradation of the crystal lattice 54 54 Raman spectroscopy Raman Spectroscopy 49 Phase transition monitoring Stress measurements Mapping the Raman peak position of a micro indentation in a silicon wafer. Renishaw, Inc. 55 55 Raman spectroscopy 50 A complete Raman mapping of phase transitions in Si under indentation C. R. Das, H. C. Hsu, S. Dhara, A. K. Bhaduri, B. Raj, L. C. Chen, K. H. Chen, S. K. Albert, A. Raye and Y. Tzengc 51 Raman spectroscopy Raman Spectroscopy 57 57 Raman spectroscopy is able to clearly distinguish areas of differing numbers of layers in thin graphene sheets. Graphene monolayer, bilayer and other multiple-layer regions identified K.S. Novoselov et al., Science 306, 666 (2004). 52 Raman spectroscopy Raman Spectroscopy 58 58 Oriented CNT between two electrodes FS-MRL Raman spectroscopy Raman Spectroscopy 53 Primary Strengths: • • • • • Very little sample preparation. Structural characterization. Non destructive technique. Chemical information. Complementary to FTIR. Super Optical Powers 59 59 Raman spectroscopy Raman Spectroscopy 54 60 60 Primary Limitations: • • • Expensive apparatus (for high spectral/spatial resolution and sensitivity). Weak signal, compared to fluorescence. Limited spatial resolution. Kryptonite DC Comics Complementary techniques: FTIR, EELS, Mass spectroscopy, EXAFS, XPS, AES, SIMS, XRD, SFG. Raman spectroscopy Raman Spectroscopy 55 61 Impurities Virtual states Excited states Excitation Donnor levels Acceptor levels Vibrational states Ground state Luminescence Raman scattering Raman spectroscopy Raman Spectroscopy 55 62 Impurities Excited states Donnor levels Excitation Virtual states Acceptor levels Vibrational states Ground state Luminescence Raman scattering Luminescence 56 63 Lifetime: Phosphorescence, fluorescence Mechanism: Photoluminescence, bioluminescence, chemoluminescence, thermoluminescence, piezoluminescence, etc. Radim Schreiber Trevor Morris Photoluminescence 64 57 http://www.evidentcrimescene.com Charles Hedgcock © University of Arizona, Tucson, AZ http://kevincollinsphoto.smugmug.com http://uclagettyprogram.wordpress.com Photoluminescence 58 What is measured? The emission spectra of materials due to radiative recombination following photo-excitation. Basic principle: The impinging light promote electrons from the less energetic levels to excited levels, forming electron-hole pairs. As the electrons and holes recombine, they may release some of the energy as photons. The emitted light is called luminescence. Conduction band Valence band direct band gap indirect band gap 65 Photoluminescence 59 Excitations in materials •Excitons •Bound excitons •Excitonic complexes Peter Abbamonte et al., Proc. Nat. Acad. Sci. 105 (34) Exciton describes the bound state of an electronhole pair due their mutual Coulomb attraction Photoluminescence 60 Photoluminescence spectra of InN layers with different carrier concentrations. 1 - n = 6x1018 cm-3 (MOCVD); 2 - n = 9x1018 cm-3 (MOMBE); 3 - n = 1.1x1019 cm-3 (MOMBE); 4 - n = 4.2x1019 cm-3 (PAMBE). Solid lines show the theoretical fitting cures based on a model of interband recombination in degenerated semiconductors. As a result, the true value of InN band gap Eg~0.7 eV was established. Davydov et al. Phys. Stat. Solidi (b) 230 (2002b), R4 67 Photoluminescence 68 61 InxGa1-xN alloys. Luminescence peak positions of catodoluminescence and photoluminescence spectra vs. concentration x. The plots of luminescence peak positions can be fitted to the curve Eg(x)=3.48 - 2.70x - bx(1-x) with a bowing parameter of b=2.3 eV Ref.1 - Wetzel., Appl. Phys. Lett. 73, 73 (1998). Ref.2 - V. Yu. Davydov., Phys. Stat. Sol. (b) 230, R4 (2002). Ref.3 - O’Donnel., J. Phys .Condens. Matt. 13, 1994 (1998). Hori et al. Phys. Stat. Sol. (b) 234 (2002) 750 Photoluminescence Photoluminescence 69 62 GaAs Conduction band D + (D ,X) FE (Do,X) (Ao,X) A Valence band Photoluminescence 63 Using PL to determine the width and quality of InGaAsN/GaAs quantum wells. 3nm InGaAsN QW 5nm InGaAsN QW 9nm InGaAsN QW 70 Photoluminescence Photoluminescence 64 Strengths: • • • Very little to none sample preparation. Non destructive technique. Very informative spectrum. 71 Photoluminescence Photoluminescence 72 65 Limitations: • • • Often requires low temperature. Data analysis may be complex. Many materials luminescence weakly. Complementary techniques: Ellipsometry, Modulation spectroscopies, Spectrophotometry, Raman. Time-domain thermoreflectance 73 66 What is measured? The reflectance variation with temperature. Basic principle: The dielectric function of a material, and thus its reflectance, is a function of temperature. By modulating the material’s temperature, we can measure the variation in its reflectivity, caused by that modulation. With time resolved measurements, we can calculate the thermal conductivity of the sample. hn0 hn0 Time-domain thermoreflectance 74 67 Instrumentation: • fs Ti:Sapphire laser operating in the range 700-800 nm. • A variable delay line in the pump beam path for time resolution. • Scanning sample stage. • Cryostats. • Magnetic field. Applications of TDTR include: Spatially resolved thermal and elastic properties of materials: •Thermal effusivity (LC) and conductivity. •Mechanical properties (by determination of the speed of sound). J. Appl. Phys., Vol. 93, 793 (2003). Time-domain thermoreflectance 68 The lowest thermal conductivity so far observed for a fully dense solid. (48 mWm-1K-1) 75 Time-domain thermoreflectance 68 The lowest thermal conductivity so far observed for a fully dense solid. (48 mWm-1K-1) 76 Time-domain thermoreflectance 69 77 Time-domain thermoreflectance 69 78 Time-domain thermoreflectance 70 Strengths: – – – – Thermoconductivity over a broad range can be measured. Can measure interface thermal conductance. Spatial resolution. Measurements can be made over a wide range of temperatures. – Fast. Science 315, 342 (2007) 79 Time-domain thermoreflectance 80 71 Limitations: – Samples need coating with an Al thin film. – Alignment can be tedious. – Thermal conductivity cannot be measured for films much thinner than the thermal penetration depth. Complementary techniques: 3w method, Raman spectroscopy, Nanoindentation. Optical microscopy 72 Optical microscopy 73 "Classical" Optical Microscopy Image Formation •In the optical microscope, light from the microscope lamp passes through the condenser and then through the specimen (assuming the specimen is a light absorbing specimen). • Some of the light passes both around and through the specimen undisturbed in its path (direct light or undeviated light). •The direct or undeviated light is projected by the objective and spread evenly across the entire image plane at the diaphragm of the eyepiece. •Some of the light passing through the specimen is deviated when it encounters parts of the specimen (deviated light or diffracted light). Optical microscopy 74 Optical microscopy 75 Objective Type Spherical Aberration Chromatic Aberration Field Curvature Achromat 1 Color 2 Colors No Plan Achromat 1 Color 2 Colors Yes Fluorite 2-3 Colors 2-3 Colors No Plan Fluorite 3-4 Colors 2-4 Colors Yes Plan Apochromat 3-4 Colors 4-5 Colors Yes Optical microscopy 76 Phase contrast Bright field Dark field Polarizing Optical microscopy 77 Difference interference contrast Optical microscopy 78 Phase contrast Bright field Fibers in bright field and dark field Dark field Polarizing Optical microscopy 79 Fluorescence Optical microscopy 80 Contrast-Enhancing Techniques for Optical Microscopy Specimen Type Imaging Technique Transmitted Light Transparent Specimens Phase Objects Bacteria, Spermatozoa, Cells in Glass Containers, Protozoa, Mites, Fibers, etc. Light Scattering Objects Diatoms, Fibers, Hairs, Fresh Water Microorganisms, Radiolarians, etc. Light Refracting Specimens Colloidal Suspensions powders and minerals Liquids Amplitude Specimens Stained Tissue Naturally Colored Specimens Hair and Fibers Insects and Marine Algae Fluorescent Specimens Cells in Tissue Culture Fluorochrome-Stained Sections Smears and Spreads Birefringent Specimens Mineral Thin Sections Liquid Crystals Melted and Recrystallized Chemicals Hairs and Fibers Bones and Feathers Phase Contrast Differential Interference Contrast (DIC) Hoffman Modulation Contrast Oblique Illumination Rheinberg Illumination Darkfield Illumination Phase Contrast and DIC Phase Contrast Dispersion Staining DIC Brightfield Illumination Fluorescence Illumination Polarized Illumination http://micro.magnet.fsu.edu Optical microscopy 81 Contrast-Enhancing Techniques for Optical Microscopy Reflected Light Specular (Reflecting) Surface Thin Films, Mirrors Polished Metallurgical Samples Integrated Circuits Diffuse (Non-Reflecting) Surface Thin and Thick Films Rocks and Minerals Hairs, Fibers, and Bone Insects Amplitude Surface Features Dyed Fibers Diffuse Metallic Specimens Composite Materials Polymers Birefringent Specimens Mineral Thin Sections Hairs and Fibers Bones and Feathers Single Crystals Oriented Films Fluorescent Specimens Mounted Cells Fluorochrome-Stained Sections Smears and Spreads Brightfield Illumination Phase Contrast, DIC Darkfield Illumination Brightfield Illumination Phase Contrast, DIC Darkfield Illumination Brightfield Illumination Darkfield Illumination Polarized Illumination Fluorescence Illumination http://micro.magnet.fsu.edu Optical microscopy Near-field Scanning Optical microscopy 82 Resolution Based on the Airy discs formation Abbé derived a theoretical limit to an optical instrument resolution. Arch. Microskop. Anat. 9, 413 (1873) He defined the resolving power of an optical instrument as the distance between two point sources for which the center of one Airy disk coincides with the first zero of the other: d 0.61l NA Rayleigh criterion (light emitting particle) 𝜆 𝜆 𝑑≈ ≈ 𝑁𝐴𝑐𝑜𝑙 + 𝑁𝐴𝑜𝑏𝑗 2 𝑁𝐴 Abbé criterion (illuminated particle) 91 Optical microscopy 83 0.61 𝜆 𝑑≈ 𝑛 sin 𝜇 𝜆 𝑑≈ 2 𝑛 sin 𝜇 Rayleigh criterion (light emitting particle) Abbé criterion (illuminated particle) Confocal microscopy 84 • In confocal microscopy, due to the constrained light path provides an increased contrast, allowing for resolving objects with intensity differences of up to 200:1. • The in plane resolution, in confocal microscopy, is also slightly increased (1.5 times) while the resolution along the optical axis is high. •These improvements are obtained at the expense of the utilization of mechanisms for scanning either by moving a specimen or by readjustment of an optical system. Scanning application allows to increase field of view as compared with conventional microscopes. Confocal microscopy 85 The relation of the first ring maximum amplitude to the amplitude in the center is 2% in case of conventional point spreading function (PSF) in a focal plane while in case of a confocal microscope this relation is 0.04%. Confocal microscopy 86 Confocal microscopy 87 Anna-Katerina Hadjantonakis; Virginia E Papaioannou BMC Biotechnology 2004, 4:33 Confocal microscopy 88 Strengths: Limitations: – Optical sectioning (0.5 m). – three-dimensional images. – Improved contrast (200:1). – Better resolution (1.5x). – Field of view defined by the scanning range. – Image is scanned, resulting in slower data acquisition. – High intensity laser radiation can damage some samples. – Cost (typically 10x more than a comparable wide-field system). Beyond confocal microscopy 89 Beyond confocal microscopy 90 Nature Reviews Genetics 4, 613 (2003) Biophysical J. 75, 2015 (1998) (courtesy of Brad Amos MRC, Cambridge) Beyond confocal microscopy 91 Beyond confocal microscopy 92 Beyond confocal microscopy 93 • • • • • • • Sample is illuminated with a patterned light source. The pattern interact with structures finer than the diffraction limit; The pattern projection itself is diffraction limited. The big advantage of HR-SIM is again its ease of-use. Uses common Fixation and labeling procedures are similar to normal fluorescence microscopy,. Dye labeling must be stable enough to survive the acquisition of multiple images. Beyond confocal microscopy 94 PALM (photo-activated localization microscopy) Widefield PALM Widefield PALM Biophysical Journal 91, 4258 (2006) Nature Methods 6, 689 (2009) Nature Protocols 4, 291 (2009) Other similar echniques: STORM (Stochastic optical reconstruction microscopy) FIONA (Fluorescence Imaging with One Nanometer Accuracy) SHReC (Single Molecule High Resolution Colocalization) SPDM (Spectral Precision Distance Microscopy) And many variants. Near-field scanning optical microscopy (NSOM) Near-field Scanning Optical microscopy 95 10 4 Near-field microscopy: resolution beyond the diffraction limit! The near field microscope resolves objects much smaller than the wavelength of light by measuring at very short distances from the sample surface, through sub-wavelength apertures. Basic principle: The principle of operation of the NSOM was devised by Synge in 1928 †. •The sample is kept in the near-field regime of a sub-wavelength source. •If z<<l, the resolution is determined by the aperture, not wavelength. •The image is constructed by scanning the aperture across the sample and recording the optical response. † Philos.Mag. 6, 356 (1928). Near-field scanning optical microscopy (NSOM) Near-field Scanning Optical microscopy 96 Instrumentation: 10 5 Near-field scanning optical microscopy (NSOM) 97 10 6 Strengths: – – – – Powerful, very attractive. No sample preparation. Non destructive technique. Sub diffraction limit resolution (50 nm). CBS Broadcasting Inc. Near-field scanning optical microscopy (NSOM) 98 10 7 Requirements and limitations: – – – – – Careful alignment required. Very low throughput. Slow data acquisition. Limited to fairly flat samples (20 m). Interaction between tip and sample may make analysis difficult. Complementary techniques: AFM, SEM,TEM, Confocal microscopy. It is Important to use complementary techniques! 99 Thank you! 10 8 100 Thanks to our Platinum Sponsors: Thanks to our sponsors: © 2014 University of Illinois Board of Trustees. All rights reserved.