Optical materials characterization

Transcription

1 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.

2 2 Why optical characterization?

3 Why optical characterization? 2 Optics Communications, Vol 284(9), (2011)

4 2 Why optical characterization?

5 2 Why optical characterization?

6 2 Why optical characterization?

7 2 Why optical characterization?

8 3 Why optical characterization?

9 4 Why optical characterization?

10 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" library.thinkquest.org The Nobel Foundation

11 r(n) Why optical characterization? 6 Black body radiation, which lead to quantum physics Classical (Rayleigh-Jeans) Quantum (Planck) ,000 20,000 30,000 The Nobel Prize in Physics 1918 Max Planck "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

12 Why optical characterization? 2005 Charles Kuen Kao Willard S. Boyle George E. Smith Roy J. Glauber John L. Hall Theodor W. Hänsch 1964 transmission of light in fibers for optical communication and invention of the CCD 1981 contribution to the development of laser spectroscopy 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 Nicolaas Bloembergen Arthur Leonard Schawlow Charles Hard Townes Nicolay Gennadiyevich Basov Aleksandr Mikhailovich Prokhorov 1966 demonstration of the phase contrast method, especially for his invention of the phase contrast microscope Alfred Kastler discovery and development of optical methods for studying Hertzian resonances in atoms contributions to the quantum theory of optical coherence and the development of laser-based precision spectroscopy Steven Chu Claude Cohen-Tannoudji William D. Phillips development of methods to cool and trap atoms with laser light Robert Andrews Millikan Albert Einstein the photoelectric effect Niels Henrik David Bohr investigation of the structure of atoms and of the radiation emanating from them Albert Abraham Michelson for his optical precision instruments and the spectroscopic and metrological investigations carried out with their aid Sir Chandrasekhara Venkata Raman discovery of the Raman effect Max Karl Ernst Ludwig Planck discovery of energy quanta Gabriel Lippmann method of reproducing colours photographically based on the phenomenon of interference The Nobel Foundation

13 8 Light matter interaction

14 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 profile, defect states, etc.) Quantum efficiency, solar cell and detector characterization

15 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

16 11 Transmission, Reflection, Absorption 16 UV-VIS-NIR FTIR

17 12 Transmission, Reflection, Absorption Raman Spectroscopy Impurities 17 Excited states Donnor levels Virtual state Acceptor levels Vibrational states Ground state IR UV/VIS

18 13 Instrumentation: Spectrophotometry (UV-VIS-NIR) 18 Transmission Diffuse reflectance Specular reflectance

19 Spectrophotometry (UV-VIS-NIR) 14 Instrumentation: 19

20 Detector voltage 15 Fourier Transform IR spectroscopy (FTIR) The Nobel Prize in Physics 1907 Albert A. Michelson 20 "for his optical precision instruments and the spectroscopic and metrological investigations carried out with their aid" The Nobel Foundation fixed mirror Instrumentation: The FTIR uses a Michelson interferometer with a moving mirror, in place of a diffraction grating or prism. moving mirror IR source DL = nl => constructive interference DL = (n+1/2) l => destructive interference Beam splitter sample detector Time

21 Detector voltage 15 Fourier Transform IR spectroscopy (FTIR) The Nobel Prize in Physics 1907 Albert A. Michelson 21 "for his optical precision instruments and the spectroscopic and metrological investigations carried out with their aid" The Nobel Foundation fixed mirror Instrumentation: The FTIR uses a Michelson interferometer with a moving mirror, in place of a diffraction grating or prism. moving mirror IR source DL = nl => constructive interference DL = (n+1/2) l => destructive interference Beam splitter sample detector Time

22 Detector voltage Intensity 16 Fourier Transform IR spectroscopy (FTIR) 22 Spectrum formation:. 2in t I( n ) S( t) e dt Time Frequency

23 Absorbance Amplitude 17 Fourier Transform IR spectroscopy (FTIR) 23 An example of an interferogram and its corresponding FTIR spectrum. Frequency (Hz) The signal is detected as intensity vs. time (interferogram). Polystyrene 2.0 The Fourier transform of the interferogram gives the spectrum, as intensity vs. wave number Wavenumber (cm -1 )

24 18 Instrumentation: Spectrophotometry (UV-VIS-NIR) 24

25 19 Fourier Transform IR spectroscopy (FTIR) 25 FT vs. Dispersive optics spectrometers Advantages: Multiplexing (all wavelengths are measured simultaneously). No chromatic aberrations or artifacts. Higher throughput. Improved S/N ratio. Disadvantages: More complex system. Linearity of detectors (working closer to saturation levels). Stray light scattering at low signal level.

26 Absorption Absorption Spectrophotometry (UV-VIS-NIR) ml Au/.1ml Hg 3ml Au/1ml Hg 2ml Au/2ml Hg 1ml Au/3ml Hg Beer-Lambert Law Abs = K l c = a l 0.2.5mM solutions 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 Au concentration (mm)

27 peak index / 2n ( m/cm) T(%) peak index / 2n ( m/cm) Spectrophotometry (UV-VIS-NIR) l (nm) λm = m ν = 2nd sin θ peak positions linear fit Slope = 2.76 m n (cm -1 ) peak positions linear fit n (cm -1 ) 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 Slope = m n (cm -1 )

28 Spectrophotometry (UV-VIS-NIR) 22 Excitations in materials Plasmons Phys. Today, 64, 39 (2011) 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 reflection of light. 3 m

29 Spectrophotometry (UV-VIS-NIR) M X k 0 G 200 nm k x 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.

30 24 Fourier Transform IR spectroscopy (FTIR) 30 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

31 25 Fourier Transform IR spectroscopy (FTIR) 31 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

32 26 Spectrophotometry (UV-VIS-NIR) and FTIR 32 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).

33 Spectrophotometry (UV-VIS-NIR) and FTIR 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.

34 28 Polarization 34 Guimond and Elmore - Oemagazine May

35 29 Ellipsometry 35 What is measured? The changes in the polarization state of light upon reflection from a mirror like surface.

36 30 Ellipsometry 36 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 tan( ) ~ R i p e D ~ Rs

37 37 ) (,,,,, ~ i s p r s p i i s p r s p s p e E E R f f f f i s p bc s p ab i s p bc s p ab s p s p e r r e r r R n n n n r 2,, 2,,, 2 1,2 1 2,1 2 1,2 1 2,1, 12 ~ ~ 1 ~ ~ ~ cos ~ cos ~ cos ~ cos ~ ~ ik n n + ~ a b c d f b D D i r s p s p i R R R R e ~ ~ ) tan( ~ ~ ) tan( f a f c b n b d f l cos ~ sin ~ sin ~ f f n n Ellipsometry 31

38 32 Ellipsometry Applications 38 Film thickness SiO 2 Si SiO 2 thickness c Å 2.19 ± 0.04 nm (degrees) Model fit Experiment D D (degrees) Wavelength (nm)

39 33 Composition Surface roughness Film thickness Band gap energy Ellipsometry Applications 39 Ellipsometric (l) and D(l) spectra of Cd 1-x Zn x S 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)

40 34 Composition Surface roughness Film thickness Band gap energy Ellipsometry Applications 40 [NH4OH] (M) Parameters of the Cd1xZnxS thin films as a function of different ammonia concentrations obtained from SE analysis. Thickness (nm) Roughness (nm) ZnS (%) Band-gap (ev) Jpn. J. Appl. Phys. 49 (2010)

41 35 Composition Surface roughness Film thickness Band gap energy Optical constants (dielectric function) Ellipsometry Applications 41 Refractive index (n) and extinction coefficient (k) as a function of wavelength of Cd 1-x Zn x S thin films under different ammonia concentrations. Plots of (αhν) 2 versus photon energy h for Cd 1-x Zn x S thin films under different ammonia concentration. Jpn. J. Appl. Phys. 49 (2010)

42 36 Ellipsometry Optical Hall Effect 42

43 37 Ellipsometry Applications 43 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)x10 13 cm -2 and =1521±52 cm 2 V -1 s -1 a top p-type channel with Ns=(3.4±0.6)x10 14 cm -2 and =18±4 cm 2 V -1 s -1 From electrical dc Hall effect measurement: p-type conductivity with N s =(3.0±0.5) x10 13 cm -2 and =3407±250 cm 2 V -1 s -1

44 38 Ellipsometry Applications 44 Electrical properties From the fit for the Si-face sample: A p-type channel with Ns=(1.2±0.3)x10 12 cm -2 and =794±80 cm 2 V -1 s -1 From electrical dc Hall effect measurement: p-type conductivity with N s =(1.9±0.2) x10 12 cm -2 and =891±250 cm 2 V -1 s -1 The correspondence between electrical and OHE data is very good.

45 39 Ellipsometry Applications 45 Electrical properties C-face: Top layer: Bottom layer: m* = ( B) m 0 m* = m 0 Si-face: m* = 0.03 m 0

46 Ellipsometry 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

47 Ellipsometry 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.

48 42 Raman spectroscopy Raman Spectroscopy Sir Chandrasekhara Venkata Raman 48 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

49 Anti-Stokes Rayleigh scattering Stokes Scattered intensity Stokes Anti-Stokes 43 Raman spectroscopy Raman Spectroscopy 49 What is measured? The light inelastically scattered by the material. Basic principle: Excited state Raman shift Photon energy Virtual states 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 Resonance Raman IR

50 Raman spectroscopy 44 Excitations in materials Phonons Molecular vibrations Phonons are the quanta of Collective lattice vibrations

51 Intensity 45 Raman spectroscopy Raman Spectroscopy Wavenumber (cm -1 ) 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. Polyetherurethane FTIR Raman The two techniques are complementary, rather than equivalent Raman shift (cm -1 )

52 Raman intensity (arb. units) 46 Raman spectroscopy Raman Spectroscopy Molecular and crystalline structure characterization Raman is sensitive to the atomic structure of the material. Diamond C Nanotube Coal (Rock)-norm Graphite coal (wood) Physics Reports, 409 (2005), 47 Raman Shift (cm -1 )

53 47 Raman spectroscopy Raman Spectroscopy 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.

54 48 Raman spectroscopy Raman Spectroscopy 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

55 49 Raman spectroscopy Raman Spectroscopy Phase transition monitoring Stress measurements Mapping the Raman peak position of a micro indentation in a silicon wafer. Renishaw, Inc.

56 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

57 51 Raman spectroscopy Raman Spectroscopy 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).

58 52 Raman spectroscopy Raman Spectroscopy Oriented CNT between two electrodes FS-MRL

59 53 Raman spectroscopy Raman Spectroscopy Primary Strengths: Very little sample preparation. Structural characterization. Non destructive technique. Chemical information. Complementary to FTIR. Super Optical Powers

60 54 Raman spectroscopy Raman Spectroscopy Primary Limitations: Expensive apparatus (for high spectral/spatial resolution and sensitivity). Weak signal, compared to fluorescence. Limited spatial resolution. Complementary techniques: Kryptonite FTIR, EELS, Mass spectroscopy, EXAFS, XPS, AES, SIMS, XRD, SFG. DC Comics

61 Excitation 55 Impurities Raman spectroscopy Raman Spectroscopy 61 Virtual states Excited states Donnor levels Acceptor levels Vibrational states Ground state Luminescence Raman scattering

62 Excitation 55 Impurities Raman spectroscopy Raman Spectroscopy 62 Excited states Donnor levels Virtual states Acceptor levels Vibrational states Ground state Luminescence Raman scattering

63 Luminescence Lifetime: Phosphorescence, fluorescence Mechanism: Photoluminescence, bioluminescence, chemoluminescence, thermoluminescence, piezoluminescence, etc. Radim Schreiber Trevor Morris

64 57 Photoluminescence 64 Charles Hedgcock University of Arizona, Tucson, AZ

65 58 Photoluminescence 65 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

66 Photoluminescence 59 Excitations in materials Excitons Bound excitons Excitonic complexes Exciton describes the bound state of an electronhole pair due their mutual Coulomb attraction Peter Abbamonte et al., Proc. Nat. Acad. Sci. 105 (34)

67 60 Photoluminescence 67 Photoluminescence spectra of InN layers with different carrier concentrations. 1 - n = 6x10 18 cm -3 (MOCVD); 2 - n = 9x10 18 cm -3 (MOMBE); 3 - n = 1.1x10 19 cm -3 (MOMBE); 4 - n = 4.2x10 19 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 E g ~0.7 ev was established. Davydov et al. Phys. Stat. Solidi (b) 230 (2002b), R4

68 61 Photoluminescence 68 In x Ga 1-x N alloys. Luminescence peak positions of catodoluminescence and photoluminescence spectra vs. concentration x. The plots of luminescence peak positions can be fitted to the curve E g (x)= x - 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

69 62 Photoluminescence 69 GaAs Conduction band D (D +,X) FE (D o,x) Valence band A (A o,x)

70 63 Photoluminescence 70 Using PL to determine the width and quality of InGaAsN/GaAs quantum wells. 3nm InGaAsN QW 5nm InGaAsN QW 9nm InGaAsN QW

71 Photoluminescence Strengths: Very little to none sample preparation. Non destructive technique. Very informative spectrum.

72 Photoluminescence Limitations: Often requires low temperature. Data analysis may be complex. Many materials luminescence weakly. Complementary techniques: Ellipsometry, Modulation spectroscopies, Spectrophotometry, Raman.

73 Time-domain thermoreflectance 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

74 Time-domain thermoreflectance Instrumentation: fs Ti:Sapphire laser operating in the range 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: J. Appl. Phys., Vol. 93, 793 (2003). Thermal effusivity (LC) and conductivity. Mechanical properties (by determination of the speed of sound).

75 68 Time-domain thermoreflectance 75 The lowest thermal conductivity so far observed for a fully dense solid. (48 mwm -1 K -1 )

76 68 Time-domain thermoreflectance 76 The lowest thermal conductivity so far observed for a fully dense solid. (48 mwm -1 K -1 )

77 69 Time-domain thermoreflectance 77

78 69 Time-domain thermoreflectance 78

79 Time-domain thermoreflectance 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)

80 Time-domain thermoreflectance 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.

81 72 Optical microscopy

82 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).

83 74 Optical microscopy

84 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

85 76 Optical microscopy Phase contrast Bright field Dark field Polarizing

86 Optical microscopy 77 Difference interference contrast

87 78 Optical microscopy Phase contrast Bright field Dark field Polarizing Fibers in bright field and dark field

88 Optical microscopy 79 Fluorescence

89 80 Specimen Type Optical microscopy Contrast-Enhancing Techniques for Optical Microscopy 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 Transmitted Light Imaging Technique 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

90 81 Optical microscopy Contrast-Enhancing Techniques for Optical Microscopy 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 Reflected Light Brightfield Illumination Phase Contrast, DIC Darkfield Illumination Brightfield Illumination Phase Contrast, DIC Darkfield Illumination Brightfield Illumination Darkfield Illumination Polarized Illumination Fluorescence Illumination

91 82 Near-field Scanning Optical microscopy Optical microscopy 91 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 d 0.61l NA λ λ NA col + NA obj 2 NA Rayleigh criterion (light emitting particle) Abbé criterion (illuminated particle)

92 Optical microscopy 83 d d 0.61 λ n sin μ λ 2 n sin μ Rayleigh criterion (light emitting particle) Abbé criterion (illuminated particle)

93 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.

94 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%.

95 86 Confocal microscopy

96 Confocal microscopy 87 Anna-Katerina Hadjantonakis; Virginia E Papaioannou BMC Biotechnology 2004, 4:33

97 Confocal microscopy 88 Strengths: Optical sectioning (0.5 m). three-dimensional images. Improved contrast (200:1). Better resolution (1.5x). Field of view defined by the scanning range. Limitations: 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).

98 89 Beyond confocal microscopy

99 Beyond confocal microscopy 90 Nature Reviews Genetics 4, 613 (2003) (courtesy of Brad Amos MRC, Cambridge) Biophysical J. 75, 2015 (1998)

100 91 Beyond confocal microscopy

101 92 Beyond confocal microscopy

102 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.

103 Beyond confocal microscopy 94 PALM (photo-activated localization microscopy) Widefield PALM Widefield PALM Biophysical Journal 91, 4258 (2006) Nature Methods 6, 689 (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. Nature Protocols 4, 291 (2009)

104 95 Near-field scanning Scanning optical Optical microscopy microscopy (NSOM) 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 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).

105 96 Near-field scanning Scanning optical Optical microscopy microscopy (NSOM) 10 5 Instrumentation:

106 97 Near-field scanning optical microscopy (NSOM) 10 6 Strengths: Powerful, very attractive. No sample preparation. Non destructive technique. Sub diffraction limit resolution (50 nm). CBS Broadcasting Inc.

107 98 Near-field scanning optical microscopy (NSOM) 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.

108 99 It is Important to use complementary techniques! 10 8 Thank you!

109 100 Thanks to our Platinum Sponsors: Thanks to our sponsors: 2014 University of Illinois Board of Trustees. All rights reserved.