Lecture3 Optical Microscopy Introduction Lens formula Image formation

Lecture-3 Optical Microscopy • Introduction • Lens formula, Image formation and Magnification • Resolution and lens defects • Basic components and their functions • Common modes of analysis • Specialized Microscopy Techniques • Typical examples of applications http: //www. youtube. com/watch? v=P 2 te. E 17 z. T 4 I&list=PLKst. G-8 VPWKz. Oe 4 Tkv. A 7 F 6 q. Ml. G 2 HH 8 me. X at~0: 46 -1: 33

Review Problems on Optical Microscopy 1. Compare the focal lengths of two glass converging lenses, one with a larger curvature angle and the other with a smaller curvature angle. 2. List the parameters that affect the resolution of optical microscopes. 3. A student finds that some details on the specimen cannot be resolved even after the resolution of the microscope was improved by using the oil immersion objective. The student thinks that the details can be resolved by enlarging a photograph taken with the microscope at maximum magnification. Do you agree? Justify your answer. http: //www. doitpoms. ac. uk/tlplib/optical-microscopy/questions. php

http: //micro. magnet. fsu. edu/primer/java/microscopy/immersion/index. html Resolution of a Microscope (lateral) The smallest distance between two specimen points that can still be distinguished as two separate entities dmin = 0. 61 l/NA NA=nsin( ) l – illumination wavelength (light) NA – numerical aperture -one half of the objective angular aperture n-imaging medium refractive index dmin ~ 0. 3 m for a midspectrum l of 0. 55 m https: //www. youtube. com/watch? v=n 2 asdnc. MYMo at~5: 35 -6: 00

Numerical Aperture (NA) NA=1 - NA = n(sin ) theoretical maximum numerical aperture of a lens operating with air as the imaging medium n: refractive index of the imaging medium between the front lens of objective and specimen cover glass Objective lens Angular aperture ( 72 degrees) One half of A-A Specimen cover glass NA of an objective is a measure of its ability to gather light and resolve fine specimen detail at a fixed object distance. https: //en. wikipedia. org/wiki/Angular_aperture http: //micro. magnet. fsu. edu/primer/java/microscopy/immersion/index. html

Numerical Aperture NA = n(sin ) Imaging Medium Air n=1. 0 Immersion oil n=1. 515 http: //www. youtube. com/watch? v=RSKB 0 J 1 s. Rn. U oil immersion objective use in microscope at~0: 33

Axial resolution – Depth of Field Ranges m) (F (F m) Depth of focus (f mm) NA f F 0. 13 15. 5 0. 4 3. 8 5. 8. 95 80. 0 0. 19 The distance above and below The axial range through which geometric image plane within an object can be focused without which the image is in focus any appreciable change in image sharpness M NA f F F is determined by NA. http: //www. matter. org. uk/tem/depth_of_field. htm http: //www. youtube. com/watch? v=Fv. C 2 WLUq. Eug at~3: 40

Depth of Focus The distance above and below geometric image plane within which the image is in focus. Depth of focus (f mm) CCD camera

Axial resolution – Depth of Field The axial range through which an object can be focused without any appreciable change in image sharpness. Depth of focus (f mm) NA f F 0. 13 15. 5 0. 4 3. 8 5. 8. 95 80. 0 0. 19 25 m Small F Large F

Basic components and their functions http: //www. youtube. com/watch? v=RKA 8_mif 6 -E Microscope Review (simple, clear) http: //www. youtube. com/watch? v=b 2 PCJ 5 s-iyk Microscope working in animation (How to use a microscope) http: //www. youtube. com/watch? annotation_id=annotation_100990&featur e=iv&src_vid=L 6 d 3 z. D 2 Lt. SI&v=nt. Pju. UMd. Xbg (I) http: //www. youtube. com/watch? v=VQt. MHj 3 va. Lg (II) Parts and Function of a Microscope (details) http: //www. youtube. com/watch? v=X-w 98 KA 8 Uq. U&feature=related How to use a microscope (specimen preparation at~1: 55 -2: 30) http: //www. youtube. com/watch? v=b. GBg. ABLEV 4 g How to care for and operate a microscope

Basic components and their functions (1) Eyepiece (ocular lens) (2) Revolving nose piece (to hold multiple objective lenses) (3) Objective lenses (4) And (5) Focus knobs (4) Coarse adjustment (5) Fine adjustment (6) Stage (to hold the specimen) (7) Light source (lamp) (8) Condenser lens and diaphragm (9) Mechanical stage (move the specimen on two horizontal axes for positioning the specimen)

Functions of the Major Parts of a Optical Microscope § § § Lamp and Condenser: project a parallel beam of light onto the sample for illumination Sample stage with X-Y movement: sample is placed on the stage and different part of the sample can be viewed due to the X-Y movement capability Focusing knobs: since the distance between objective and eyepiece is fixed, focusing is achieved by moving the sample relative to the objective lens

Light Sources

Condenser Light from the microscope light source Condenser gathers light and concentrates it into a cone of light that illuminates the specimen with uniform intensity over the entire viewfield http: //www. youtube. com/watch? annotation_id=annotation_100990&feature=iv&src_vid=L 6 d 3 z. D 2 Lt. SI&v=nt. Pju. UMd. Xbg ~8: 08 to 9: 40 http: //micro. magnet. fsu. edu/primer/java/kohler/contrast/index. html

Specimen Stage http: //micro. magnet. fsu. edu/primer/flash/stage/index. html

Functions of the Major Parts of a Optical Microscope § Objective: does the main part of § § magnification and resolves the fine details on the samples (mo ~ 10 – 100) Eyepiece: forms a further magnified virtual image which can be observed directly with eyes (me ~ 10) Beam splitter and camera: allow a permanent record of the real image from the objective be made on film (for modern research microscope)

camera Beam splitter Reflected light Olympus BX 51 Research Microscope Cutaway Diagram Transmitted light http: //micro. magnet. fsu. edu/primer/java/microassembly/index. html

Objective Lens dmin = 0. 61 l/NA Objective specifications Anatomy of an objective rical ture DIC-differential interference contrast Objectives are the most important components of a light microscope: image formation, magnification, the quality of images and the resolution of the microscope http: //www. youtube. com/watch? v=P 0 Z 4 H 2 O_Stg Objectives to~5: 26 http: //www. youtube. com/watch? v=H 8 PQ 9 RMUo. A 8 Grades of objectives to~2: 30 & 3: 25 -4: 50 https: //www. youtube. com/watch? v=Fw. Bjpi 8 ck 1 Y Alignment of OM

Defects in Lens § § Curvature of Field - When visible light is focused through a curved lens, the image plane produced by the lens will be curved The image appears sharp and crisp either in the center or on the edges of the viewfield but not both http: //micro. magnet. fsu. edu/primer/java/aberrations/curvatureoffield/index. html

Defects in Lens § Chromatic Aberration § § Axial - Blue light is refracted to the greatest extent followed by green and red light, a phenomenon commonly referred to as dispersion Lateral - chromatic difference of magnification: the blue image of a detail was slightly larger than the green image or the red image in white light, thus causing color ringing of specimen details at the outer regions of the field of view A converging lens can be combined with a weaker diverging lens, so that the chromatic aberrations cancel for certain wavelengths: The combination – achromatic doublet weaker diverging lens http: //www. youtube. com/watch? v=y. H 7 rb. Ru 7 Av 8&list=PL 02 D 1 D 436 A 44 B 521 A chromatic aberration http: //www. youtube. com/watch? v=H 8 PQ 9 RMUo. A 8 at~3: 30 -4: 30

Eyepiece Lens (Diaphragm) M=(L/fo)(25/fe) Eyepieces (Oculars) work in combination with microscope objectives to further magnify the intermediate image http: //micro. magnet. fsu. edu/primer/anatomy/oculars. html http: //www. birdwatching. com/optics/diopter_set. html

Lecture-3 Optical Microscopy • Introduction • Lens formula, Image formation and Magnification • Resolution and lens defects • Basic components and their functions • Common modes of analysis • Specialized Microscopy Techniques • Typical examples of applications

Common Modes of Analysis Depending on the nature of samples, different illumination methods must be used • Transmitted OM - transparent specimens thin section of rocks, minerals and single crystals • Reflected OM - opaque specimens most metals, ceramics, semiconductors Specialized Microscopy Techniques • Polarized LM - specimens with anisotropic optical character Characteristics of materials can be determined morphology (shape and size), phase distribution (amorphous or crystalline), transparency or opacity, color, refractive indices, dispersion of refractive indices, crystal system, birefringence, degree of crystallinity, polymorphism and etc.

Anatomy of a modern OM http: //www. youtube. com/watch? v=7 -Tlyd 7 pi. SM Trans OM to~1: 37 Refle OM from 1: 38 -end Illumination System Reflected OM Transmitted OM Illumination System http: //www. youtube. com/watch? v=zq 13 e 36 cs 3 s at~0: 20 -1: 40 Field diaphragm

camera Beam splitter Olympus BX 51 Research Microscope Cutaway Diagram http: //micro. magnet. fsu. edu/primer/java/microassembly/index. html

Common Modes of Analysis Depending on the nature of samples, different illumination methods must be used • Transmitted OM - transparent specimens thin section of rocks, minerals and single crystals • Reflected OM - opaque specimens most metals, ceramics, semiconductors Specialized Microscopy Techniques • Polarized LM - specimens with anisotropic optical character Characteristics of materials can be determined morphology (shape and size), phase distribution (amorphous or crystalline), transparency or opacity, color, refractive indices, dispersion of refractive indices, crystal system, birefringence, degree of crystallinity, polymorphism and etc.

Polarized Light Microscopy Polarized light microscope is designed to observe specimens that are visible primarily due to their optically anisotropic character (birefringent). The microscope must be equipped with both a polarizer, positioned in the light path somewhere before the specimen, and an analyzer (a second polarizer), placed in the optical pathway between the objective rear aperture and the observation tubes or camera port. birefringent - doubly refracting

http: //www. youtube. com/watch? v=rbx 3 K 1 x. Bx. VU polarized light Polarization of Light When the electric field vectors of light are restricted to a single plane by filtration, then the light is said to be polarized with respect to the direction of propagation and all waves vibrate in the same plane. http: //www. youtube. com/watch? v=l. Z-_i 82 s 16 E&feature=endscreen&NR=1 to~3: 30 min http: //www. youtube. com/watch? v=E 9 qpbt 0 v 5 Hw http: //micro. magnet. fsu. edu/primer/java/polarizedlight/filters/index. html

Birefringence is optical property of a material having a refractive index that depends on the polarization and propagation direction of light. Isotropic anisotropic Ca. CO 3 Anisotropic Double Refraction (Birefringence) http: //www. youtube. com/watch? v=Wdr. YRJfi. Uv 0

Anisotropic Optical Character Cubic (Birefringence) a Crystals are classified as being either isotropic or anisotropic depending upon their optical behavior and whether or not their crystallographic axes are equivalent. All isotropic crystals have equivalent axes that interact with light in a similar manner, regardless of the crystal orientation with respect to incident light waves. Light entering an isotropic crystal is refracted at a constant angle and passes through the crystal at a single velocity without being polarized by interaction with the electronic components of the crystalline lattice. tetragonal c Anisotropic crystals have crystallographically distinct axes and a interact with light in a manner that is dependent upon the orientation of the crystalline lattice with respect to the incident light. When light enters the optical axis (c) of anisotropic crystals, it acts in a manner similar to interaction with isotropic crystals and passes through at a single velocity. However, when light enters a non-equivalent axis (a), it is refracted into two rays each polarized with the vibration directions oriented at right angles to one another, and traveling at different velocities. This phenomenon is termed "double" or "bi" refraction and is seen to a greater or lesser degree in all anisotropic crystals. http: //micro. magnet. fsu. edu/primer/java/polarizedlight/crystal/index. html

Polarized Light Microscopy Polarized light microscope is designed to observe specimens that are visible primarily due to their optically anisotropic character (birefringent). The microscope must be equipped with both a polarizer, positioned in the light path somewhere before the specimen, and an analyzer (a second polarizer), placed in the optical pathway between the objective rear aperture and the observation tubes or camera port. birefringent - doubly refracting

Polarized Optical Microscopy (POM) Reflected POM Transmitted POM (a)Surface features of a microprocessor integrated circuit (b)Apollo 14 Moon rock http: //micro. magnet. fsu. edu/primer/virtual/polarizing/index. html

Common Modes of Analysis Depending on the nature of samples, different illumination methods must be used • Transmitted OM - transparent specimens thin section of rocks, minerals and single crystals • Reflected OM - opaque specimens most metals, ceramics, semiconductors Specialized Microscopy Techniques • Polarized LM - specimens with anisotropic optical http: //www. youtube. com/watch? v=ul. NZ 3 u 7_J 5 I to ~1: 05 character Characteristics of materials can be determined morphology (shape and size), phase distribution (amorphous or crystalline), transparency or opacity, color, refractive indices, dispersion of refractive indices, crystal system, birefringence, degree of crystallinity, polymorphism and etc. http: //www. youtube. com/watch? v=Iw 734 z 1 e 6 w. Q to~1: 30

Lecture-3 Optical Microscopy • Introduction • Lens formula, Image formation and Magnification • Resolution and lens defects • Basic components and their functions • Common modes of analysis • Specialized Microscopy Techniques • Typical examples of applications

Specialized OM Techniques • Enhancement of Contrast Darkfield Microscopy Phase contrast microscopy Differential interference contrast microscopy Fluorescence microscopy-medical & organic materials • Scanning confocal optical microscopy (relatively new) Three-Dimensional Optical Microscopy inspect and measure submicrometer features in semiconductors and other materials • Hot- and cold-stage microscopy melting, freezing points and eutectics, polymorphs, twin and domain dynamics, phase transformations • In situ microscopy E-field, stress, etc. • Special environmental stages-vacuum or gases

http: //micro. magnet. fsu. edu/primer/techniques/contrast. html Contrast is defined as the difference in light intensity between the specimen and the adjacent background relative to the overall background intensity. Image contrast, C is defined by Sspecimen-Sbackgroud C= Sspecimen S = SA Sspecimen (Smax) and Sbackgroud (Smin) are intensities measured from specimen and background, e. g. , A and B, in the scanned area. Cminimum ~ 2% for human eye to distinguish differences between the specimen (image) and its background. http: //www. youtube. com/watch? v=SVK 4 Ok. UK 0 Yw at~1: 47 -3: 04

Contrast in Optical Microscope https: //www. youtube. com/watch? v=L 3 Ssx. IUm 0 As at~2: 17 -3: 46 Interaction of light with matter Contrast produced in the specimen by the absorption of light (directly related to the chemical composition of the absorber) and the predominant source of contrast in the ordinary optical microscope, brightness, reflectance, birefringence, light scattering, diffraction, fluorescence, or color variations have been the classical means of imaging specimens in brightfield microscopy. Enhancement of contrast by darkfield microscopy Darkfield microscopy is a specialized illumination technique that capitalizes on oblique illumination to enhance contrast in specimens that are not imaged well under normal brightfield illumination conditions. http: //micro. magnet. fsu. edu/primer/virtualzoo/index. html http: //www. youtube. com/watch? v=P 2 te. E 17 z. T 4 I&list=PLKst. G-8 VPWKz. Oe 4 Tkv. A 7 F 6 q. Ml. G 2 HH 8 me. X at~1: 33 -2: 21

http: //www. youtube. com/watch? v=d 6 jsn. LIs. Nw. I at~3: 40 -5: 20 § § § Angle of Illumination Bright filed illumination – The normal method of illumination, light comes from above (for reflected OM) Oblique illumination – light is not projected along the optical axis of the objective lens; better contrast for detail features Dark field illumination – The light is projected onto specimen surface through a special mirror block and attachment in the objective – the most effective way to improve contrast. Light stop Imax Imin Imax-Imin C= Imax C-contrast http: //www. youtube. com/watch? v=7 V 3 ny. RGeha 4 Dark field microscopy http: //micro. magnet. fsu. edu/primer/techniques/darkfieldreflect. html

Condenser Oblique hollow cone of light Cone of light Reflected light Light stop Bright field illumination Dark field illumination Condenser gathers light and concentrates it into a cone of light that illuminates the specimen with uniform intensity over the entire viewfield.

http: //micro. magnet. fsu. edu/primer/java/darkfield/cardioid/index. html http: //micro. magnet. fsu. edu/primer/techniques/darkfieldreflect. html reflected DF Transmitted Dark Field Illumination Oblique rays specimen Reflected beam I Parallel beam distance I distance http: //www. youtube. com/watch? v=I 4 ZQm-CAg. L 8 at~5: 24 -8: 14

Contrast Enhancement OM images of the green alga Micrasterias

Specialized OM Techniques • Enhancement of Contrast Darkfield Microscopy Phase contrast microscopy Differential interference contrast microscopy Fluorescence microscopy-medical & organic materials • Scanning confocal optical microscopy (relatively new) Three-Dimensional Optical Microscopy inspect and measure submicrometer features in semiconductors and other materials • Hot- and cold-stage microscopy melting, freezing points and eutectics, polymorphs, twin and domain dynamics, phase transformations • In situ microscopy E-field, stress, etc. • Special environmental stages-vacuum or gases

http: //www. microscopyu. com/articles/phasecontrast/phasemicroscopy. html Phase Contrast Microscopy Phase contrast microscopy is a contrast-enhancing optical technique that can be utilized to produce high-contrast images of transparent specimens, such as living cells, thin tissue slices, lithographic patterns, fibers, latex dispersions, glass fragments, and subcellular particles (including nuclei and other organelles). http: //www. youtube. com/watch? v=I 4 ZQm-CAg. L 8 at~0: 50 -5: 20 http: //www. youtube. com/watch? v=Wvy. Cg 1 uz. G 5 c

Crystals Growth by Differential Interference contrast microscopy (DIC) Growth spiral on cadmium iodide crystals growing From water solution (1025 x). http: //www. youtube. com/watch? v=P 2 te. E 17 z. T 4 I at~23: 05 -30: 50 http: //micro. magnet. fsu. edu/primer/techniques/dichome. html Fluorescence microscopy - medical & organic materials http: //www. youtube. com/watch? v=i. Pr. Z 84 k. HH 2 U at~1: 50 -3: 15 http: //micro. magnet. fsu. edu/primer/techniques/fluorescence/fluorhome. html

http: //micro. magnet. fsu. edu/primer/techniques/confocal/index. html Scanning Confocal Optical Microscopy Confocal microscopy is an optical imaging technique used to increase optical resolution and contrast of a micrograph by adding a spatial pinhole placed at the confocal plane of the lens to eliminate out-of-focus light. Scanning confocal optical microscopy (SCOM) is a technique for obtaining highresolution optical images with depth selectivity. (a laser beam is used) The key feature of confocal microscopy is its ability to acquire in-focus images from selected depths, a process known as optical sectioning. Images are acquired point-by-point and reconstructed with a computer, allowing three-dimensional reconstructions of topologically complex objects. http: //www. youtube. com/watch? v=mrjg. Ny. KX 8 -w Why confocal? to~1: 05 http: //www. youtube. com/watch? v=pu. T 1 cc. MWKy. Q at~0: 40 -1: 36 & 2: 40 -2: 56 http: //www. youtube. com/watch? v=Axrst 4 T__YY scanning

http: //www. olympusconfocal. com/theory/confocalintro. html Introduction Scanning Confocal Optical Microscopy Three-Dimensional Optical Microscopy w Critical dimension measurements in semiconductor metrology Cross-sectional image with line scan at PR/Si interface of a sample containing 0. 6 m-wide lines and 1. 0 m-thick photoresist on silicon. The bottom width, w, determining the area of the circuit that is protected from further processing, can be measured accurately by using SCOP. Measurement of the patterned photoresist is important because it allows the process engineer to simultaneously monitor for defects, misalignment, or other artifacts that may affect the manufacturing line. http: //www. youtube. com/watch? v=olu. JW 7 u. K 7 rw&index=12&list=PL 200 E 1 A 86911 B 0422 to~2. 44 coral under confocal http: //micro. magnet. fsu. edu/primer/virtual/confocal/index. html interactive tutorial

Lecture-3 Optical Microscopy • Introduction • Lens formula, Image formation and Magnification • Resolution and lens defects • Basic components and their functions • Common modes of analysis • Specialized Microscopy Techniques • Typical examples of applications

Typical Examples of OM Applications

Grain Size Examination 1200 C/30 min Thermal Etching a 1200 C/2 h b 20 m A grain boundary intersecting a polished surface is not in equilibrium (a). At elevated temperatures (b), surface diffusion forms a grain-boundary groove in order to balance the surface tension forces.

Grain Size Examination Objective Lens x 100 Reflected OM

Grain Growth - Reflected OM 5 m Polycrystalline Ca. F 2 illustrating normal grain growth. Better grain size distribution. 30 m Large grains in polycrystalline spinel (Mg. Al 2 O 4) growing by secondary recrystallization from a fine-grained matrix

Liquid Phase Sintering – Reflective OM Amorphous phase 40 m Microstructure of Mg. O-2% kaolin body resulting from reactive-liquid phase sintering.

Image of Magnetic Domains Magnetic domains and walls on a (110)-oriented garnet crystal (Transmitted LM with oblique illumination). The domains structure is illustrated in (b).

Phase Identification by Reflected Polarized Optical Microscopy YBa 2 Cu 307 -x superconductor material: (a) tetragonal phase and (b) orthorhombic phase with multiple twinning (arrowed) (100 x).

Specialized OM Techniques • Enhancement of Contrast Darkfield Microscopy Phase contrast microscopy Differential interference contrast microscopy Fluorescence microscopy-medical & organic materials • Scanning confocal optical microscopy (relatively new) Three-Dimensional Optical Microscopy inspect and measure submicrometer features in semiconductors and other materials • Hot- and cold-stage microscopy melting, freezing points and eutectics, polymorphs, twin and domain dynamics, phase transformations • In situ microscopy E-field, stress, etc. • Special environmental stages-vacuum or gases http: //www. nature. com/nmeth/journal/v 12/n 6/full/nmeth. 3400. html

Hot-stage POM of Phase Transformations in Pb(Mg 1/3 Nb 2/3)O 3 -Pb. Ti. O 3 Crystals n T(o. C) (a) and (b) at 20 o. C, strongly birefringent domains with extinction directions along <100>cubic, indicating a tetragonal symmetry; (c) at 240 o. C, phase transition from the tetragonal into cubic phase with increasing isotropic areas at the expense of vanishing strip domains.

E-field Induced Phase Transition in Pb(Zn 1/3 Nb 2/3)O 3 -Pb. Ti. O 3 Crystals a Schematic diagram for in situ domain observations. b c Single domain Domain structures of PZN-PT crystals as a function of E-field; (a)E=20 k. V/cm, (b) e=23. 5 k. V/cm (c) E=27 k. V/cm Rhombohedral at E=0 and Tetragonal was induced at E>20 k. V/cm

Review - Optical Microscopy • Use visible light as illumination source • Has a resolution of ~o. 2 m • Range of samples characterized - almost unlimited for solids and liquid crystals • Usually nondestructive; sample preparation may involve material removal • Main use – direct visual observation; preliminary observation for final characterization with applications in geology, medicine, materials research and engineering, industries, and etc. • Cost - $15, 000 -$390, 000 or more

Characteristics of Materials Can be determined By OM: Morphology (shape and size), phase distribution (amorphous or crystalline), transparency or opacity, color, refractive indices, dispersion of refractive indices, crystal system, birefringence, degree of crystallinity, polymorphism and etc.

Limits of Optical Microscopy • Small depth of field <15. 5 m Rough surface • Low resolution ~0. 2 m • Shape of specimen Thin section or polished surface Cover glass specimen Glass slide resin 20 m • Lack of compositional and crystallographic information

Optical Microscopy vs Scanning Electron Microscopy radiolarian 25 m OM Small depth of field Low resolution SEM Large depth of field High resolution http: //www. mse. iastate. edu/microscopy/ Radiolarian – marine protozoan

Scanning Electron Microscopy (SEM) • What is SEM? • Working principles of SEM • Major components and their functions • Electron beam - specimen interactions • Interaction volume and escape volume • Magnification, resolution, depth of field and image contrast • Energy Dispersive X-ray Spectroscopy (EDS) • Wavelength Dispersive X-ray Spectroscopy (WDS) • Orientation Imaging Microscopy (OIM) • X-ray Fluorescence (XRF) http: //www. mse. iastate. edu/microscopy http: //science. howstuffworks. com/scanning-electron-microscope. htm/printable