Lecture-3 Optical Microscopy

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 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.

Resolution of a Microscope (lateral)
Resolution of a Microscope (lateral) The smallest distance between two specimen points that can still be distinguished as two separate entities dmin = 0.61l/NA NA=nsin() l – illumination wavelength (light) NA – numerical aperture -one half of the objective angular aperture n-imaging medium refractive index Resolution is the ability to distinguish between two separate points. dmin ~ 0.3m for a midspectrum l of 0.55m at~5:35-6:00

Numerical Aperture (NA)
NA=1 - theoretical maximum numerical aperture of a lens operating with air as the imaging medium NA = n(sin ) 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 One of the most important factors in determining the resolution of an objective is the angular aperture, which has a practical upper limit of about 72 degrees (with a sine value of 0.95). NA of an objective is a measure of its ability to gather light and resolve fine specimen detail at a fixed object distance.

Numerical Aperture NA = n(sin ) Imaging Medium Air n=1.0
Immersion oil n=1.515 -Numerical Aperture Light Cones oil immersion objective use in microscope at~0:33

Axial resolution – Depth of Field
Depth of focus (f mm) Depth of Field Ranges (F m) (F mm) NA f F Depth of focus is important in photomicrography. The distance above and below geometric image plane within which the image is in focus The axial range through which an object can be focused without any appreciable change in image sharpness M NA f F F is determined by NA. 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 Depth of focus is important in photomicrography.

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 25m Depth of focus is important in photomicrography. Small F Large F

Basic components and their functions
Microscope Review (simple, clear) Microscope working in animation (How to use a microscope) (I) (II) Parts and Function of a Microscope (details) How to use a microscope (specimen preparation at~1:55-2:30) How to care for and operate a microscope review of microscope parts and their functions

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 Alignment and Field Diaphragm Opening Size -Condenser Light Cones -Condenser Aperture Diaphragm - Control Of Specimen Contrast 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 ~8:08 to 9:40

Specimen Stage

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)

Olympus BX51 Research Microscope Cutaway Diagram
camera Beam splitter Reflected light Olympus BX51 Research Microscope Cutaway Diagram Transmitted light

Objective Lens dmin = 0.61l/NA Anatomy of an objective
Objective specifications rical ture - Dry and oil immersion objective use in microscope 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 Objectives to~5:26 Grades of objectives to~2:30 & 3:25-4:50 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

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 Light is not monochromatic. Light of different wavelengths is brought to focus at different distances from the center of the lens. This occurs because the refractive index of a transparent isotropic material is greater for light of shorter wavelength than for light of longer wavelength.-dispersion. 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 chromatic aberration at~3:30-4:30

Eyepiece Lens M=(L/fo)(25/fe)
(Diaphragm) Diopter – optics. A unit of measure of the refractive power of a lens, having the dimension of the reciprocal of length and a unit equal to the reciprocal of one meter. M=(L/fo)(25/fe) Eyepieces (Oculars) work in combination with microscope objectives to further magnify the intermediate image

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 Illumination System Reflected OM Transmitted OM
Trans OM to~1:37 Refle OM from 1:38-end Illumination System Reflected OM Transmitted light microscopy is the general term used for any type of microscopy where the light is transmitted from a source on the opposite side of the specimen from the objective. Usually the light is passed through a condenser to focus it on the specimen to get very high illumination. After the light passes through the specimen, the image of the specimen goes through the objective lens and to the oculars where the enlarged image is viewed. Reflected light microscopy is often referred to as incident light or metallurgical microscopy, and is the method of choice for imaging specimens that remain opaque even when ground to a thickness of 30 microns. Transmitted OM Illumination System at~0:20-1:40 Field diaphragm

Olympus BX51 Research Microscope Cutaway Diagram
camera Beam splitter Olympus BX51 Research Microscope Cutaway Diagram

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 Polarized light microscopy is a useful method to generate contrast in birefringent specimens and to determine qualitative and quantitative aspects of crystallographic axes present in various materials.

http://www.youtube.com/watch?v=rbx3K1xBxVU polarized light
Polarization of Light When the electric field vectors of light are restricted to a single plane by filtration, then the the light is said to be polarized with respect to the direction of propagation and all waves vibrate in the same plane. to~3:30min

Birefringence anisotropic Isotropic CaCO3
Birefringence is optical property of a material having a refractive index that depends on the polarization and propagation direction of light. Isotropic anisotropic CaCO3 Double Refraction (Birefringence) Anisotropic

Anisotropic Optical Character
(Birefringence) Cubic 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 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. a

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 light microscopy is a useful method to generate contrast in birefringent specimens and to determine qualitative and quantitative aspects of crystallographic axes present in various materials.

Polarized Optical Microscopy (POM)
Reflected POM Transmitted POM Surface features of a microprocessor integrated circuit Apollo 14 Moon rock

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. to ~1:05 Published on Feb 20, 2011 Crystal growth set to music Microscopic by Gas at Video of re-crystallizing Para-dichlorobenzene (Mothballs) and DiMethyl-sulfoxide (DMSO) The crystals are melted and then allowed to cool and recrystallize under the polarizing microscope to~1:30

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 at~3:50-4:30 Fluorescence

Contrast 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 S C = = Sspecimen 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. at~1:47-3:04

Contrast in Optical Microscope
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. When imaging specimens in the optical microscope, differences in intensity and/or color create image contrast, which allows individual features and details of the specimen to become visible. at~1:33-2:21

http://www.youtube.com/watch?v=d6jsnLIsNwI 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 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 transmitted optical microscope. Different phases or variations in thickness absorb light to differing extends which causes them to differ in brightness in specimens of uniform thickness in the former and in specimens with different thickness in the latter. Furthermore, in some cases selective absorption of a particular wavelength or wavelengths of light occurs causing the phases to appear coloured.), brightness, reflectance (light from the surfaces that are perpendicular to the incident beam is reflected back into the objective, while light from tilted areas of the surfaces or grain boundaries is reflected away from the objective, so that they appear dark. Conversely with DF illumination, light reflected from grain surfaces which are almost perpendicular to the optical axis of the microscope is reflected away from the objective, while light from grain boundaries and tilted surfaces is reflected into it. Mainly for opaque specimens.), birefringence, light scattering, diffraction, fluorescence, or color variations has been the classical means of imaging specimens in brightfield microscopy. Imax-Imin Imax C= Imin Imax C-contrast Dark field microscopy

Condenser Bright field illumination Dark field illumination
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.

Transmitted Dark Field Illumination
reflected DF Transmitted Dark Field Illumination Oblique rays specimen reflected and transmitted dark field microscope No zeroth order light reaches the specimen, only light that has been diffracted, refracted and reflected from the surface of the specimen is able to enter the front lens of the objective to form an image. In the absence of a specimen, the viewfield appears totally back, because no light is reflected or diffracted into the objective. Reflected beam I Parallel beam I distance distance at~5:24-8:14 DF and BF images

Contrast Enhancement OM images of the green alga Micrasterias

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 at~3:50-4:30 Fluorescence

Phase Contrast Microscopy
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). at~0:50-5:20

Crystals Growth by Differential Interference contrast microscopy (DIC)
Growth spiral on cadmium iodide crystals growing From water solution (1025x). Fluorescence Microscopy Published on Jan 2, 2013 Fluorescence is a physical phenomenon in which a compound absorbs light and re-emits this as light of a usually higher wavelength. Since the wavelengths of the excitation light source and the emitted fluorescence can be separated very well, we can detect fluorescence with very high sensitivity, making it possible to visualize even single molecules. Many different fluorescent probes for cellular components have been developed, including genetically encoded probes like the Green Fluorescent Protein (GFP). For these reasons, fluorescence microscopy is a very powerful tool in Cell Biology research. at~23:05-30:50 Fluorescence microscopy - medical & organic materials at~1:50-3:15

Scanning Confocal Optical Microscopy
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 high-resolution 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. Confocal microscopy offers several advantages over conventional optical microscopy, including controllable depth of field, the elimination of image degrading out-of-focus information, and the ability to collect serial optical sections from thick specimens. The key to the confocal approach is the use of spatial filtering to eliminate out-of-focus light or flare in specimens that are thicker than the plane of focus. Why confocal? to~1:05 at~0:40-1:36 & 2:40-2:56 scanning

Scanning Confocal Optical Microscopy
Introduction Scanning Confocal Optical Microscopy Three-Dimensional Optical Microscopy Critical dimension measurements in semiconductor metrology w 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. Confocal microscopy offers several advantages over conventional optical microscopy, including controllable depth of field, the elimination of image degrading out-of-focus information, and the ability to collect serial optical sections from thick specimens. The key to the confocal approach is the use of spatial filtering to eliminate out-of-focus light or flare in specimens that are thicker than the plane of focus. to~2.44 coral under confocal interactive tutorial

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 Uploaded on Sep 8, 2014 Using a neutron beam, chemists and engineers at The Ohio State University were able to track the flow of lithium atoms into and out of an electrode in real time as a battery charged and discharged. This visualization shows the level of charge in the electrode rising and falling as the battery charges and discharges. The study could one day help explain why rechargeable batteries lose capacity over time, or sometimes even catch fire.

Typical Examples of OM Applications

Grain Size Examination
1200C/30min Thermal Etching a 1200C/2h 20m b 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 x100 Reflected OM

Grain Growth - Reflected OM
5mm 30mm Polycrystalline CaF2 illustrating normal grain growth. Better grain size distribution. Large grains in polycrystalline spinel (MgAl2O4) growing by secondary recrystallization from a fine-grained matrix

Liquid Phase Sintering – Reflective OM
Amorphous phase 40mm Microstructure of MgO-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
YBa2Cu307-x superconductor material: (a) tetragonal phase and (b) orthorhombic phase with multiple twinning (arrowed) (100 x). Under plane-polarized light, i.e., only analyzer was used and incident beam is unpolarized. The color is due to anisotropic absorption.

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 CLEM Correlated light microscopy and electron microscopy

Hot-stage POM of Phase Transformations in Pb(Mg1/3Nb2/3)O3-PbTiO3 Crystals
(a) and (b) at 20oC, strongly birefringent domains with extinction directions along <100>cubic, indicating a tetragonal symmetry; (c) at 240oC, phase transition from the tetragonal into cubic phase with increasing isotropic areas at the expense of vanishing strip domains. n T(oC)

E-field Induced Phase Transition in Pb(Zn1/3Nb2/3)O3-PbTiO3 Crystals
Single domain Schematic diagram for in situ domain observa- tions. Domain structures of PZN-PT crystals as a function of E-field; E=20kV/cm, (b) e=23.5kV/cm (c) E=27kV/cm Rhombohedral at E=0 and Tetragonal was induced at E>20kV/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 charac-terization 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 <15.5mm Rough surface Low resolution ~0.2mm Shape of specimen Thin section or polished surface Cover glass specimen Glass slide resin 20mm Lack of compositional and crystallographic information

Optical Microscopy vs Scanning Electron Microscopy
25mm radiolarian OM SEM Small depth of field Low resolution Large depth of field High resolution 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)

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