Presentation on theme: "Lecture-3 Optical Microscopy"— Presentation transcript:
1 Lecture-3 Optical Microscopy IntroductionLens formula, Image formation and MagnificationResolution and lens defectsBasic components and their functionsCommon modes of analysisSpecialized Microscopy TechniquesTypical examples of applicationsat~0:46-1:33
2 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.
3 Resolution of Microscope – Numerical Aperture If the space between the specimen and the objective is filled with a medium of refractive index n, then wavelength in medium n = /nThe dmin = /2n sin = /2(N.A.)For circular aperturedmin= 1.22/2(N.A.)=0.61/(N.A.) where N.A. = n sin is called numerical apertureNA of an objective is a measure of its ability to gather light and resolve fine specimen detail at a fixed object distance.-Numerical Aperture Light ConesspecimenglassAir n=1.0Immersion oil n=1.515at~6:25-7:50at~5:12-6:00-oil immersion objective use in microscope at~0:33
4 Axial resolution – Depth of Field Depth of Field Ranges (F m)(F mm)Small F Large FDepth of focus (f mm)NA f FDepth of focus is important in photomicrography.The axial range through whichan object can be focused withoutany appreciable change in imagesharpnessM NA FF is determined by NA.
5 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 microscopereview of microscope parts and their functions
6 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)
7 Functions of the Major Parts of a Optical Microscope Lamp and Condenser: project a parallel beam of light onto the sample for illuminationSample 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 capabilityFocusing knobs: since the distance between objective and eyepiece is fixed, focusing is achieved by moving the sample relative to the objective lens
9 Condenser Light from the microscope light source -Condenser Alignment and Field Diaphragm Opening Size-Condenser Light Cones-Condenser Aperture Diaphragm - Control Of Specimen ContrastLight from the microscope light sourceCondenser gathers light and concentrates it into a cone of light that illuminates the specimen with uniform intensity over the entire viewfield~6:30 to 9:40
11 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)
12 Olympus BX51 Research Microscope Cutaway Diagram cameraBeamsplitterReflected lightOlympus BX51 Research Microscope Cutaway DiagramTransmitted light
13 Objective Lens dmin = 0.61l/NA Anatomy of an objective Objective specificationsricalture- Dry and oil immersion objective use in microscopeDIC-differential interference contrastObjectives are the most important components of alight microscope: image formation, magnification, thequality of images and the resolution of the microscopeObjectives to~5:26Grades of objectives to~2:30 & 3:25-4:50
14 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 microscopeobjectives to further magnify the intermediate image
15 Olympus BX51 Research Microscope Cutaway Diagram cameraBeamsplitterOlympus BX51 Research Microscope Cutaway Diagram
16 Common Modes of Analysis Depending on the nature of samples, different illumination methods must be usedTransmitted OM - transparent specimensthin section of rocks, minerals and single crystalsReflected OM - opaque specimensmost metals, ceramics, semiconductorsSpecialized Microscopy TechniquesPolarized LM - specimens with anisotropic opticalcharacterCharacteristics of materials can be determinedmorphology (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.
17 Anatomy of a modern OM Illumination System Reflected OM Transmitted OM Trans OM to~1:37Refle OM from 1:38-endIllumination SystemReflectedOMTransmitted 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.TransmittedOMIllumination Systemat~0:20-1:40 Field diaphragm
18 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 refractingPolarized 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.
19 http://www.youtube.com/watch?v=rbx3K1xBxVU polarized light Polarization of LightWhen 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
20 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.IsotropicanisotropicCaCO3Double Refraction (Birefringence)Anisotropic
21 Anisotropic Optical Character (Birefringence)CubicCrystals 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.tetragonalcAnisotropic 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
22 Polarized Optical Microscopy (POM) Reflected POM Transmitted POMSurface features of a microprocessor integrated circuitApollo 14 Moon rock
23 Specialized OM Techniques Enhancement of ContrastDarkfield MicroscopyPhase contrast microscopyDifferential interference contrast microscopyFluorescence microscopy-medical & organic materialsScanning confocal optical microscopy (relatively new)Three-Dimensional Optical Microscopyinspect and measure submicrometer features in semiconductors and other materialsHot- and cold-stage microscopymelting, freezing points and eutectics, polymorphs, twin and domain dynamics, phase transformationsIn situ microscopyE-field, stress, etc.Special environmental stages-vacuum or gasesat~3:50-4:30 Fluorescence
24 ContrastContrast 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 bySspecimen-Sbackgroud SC = =Sspecimen SASspecimen and Sbackgroud are intensities measured from specimen and backgroud, 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
25 Contrast in Optical Microscope at~2:17-3:46Interaction of light with matterContrast 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 microscopyDarkfield 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
26 http://www.youtube.com/watch?v=d6jsnLIsNwI at~3:40-5:20 Angle of IlluminationBright 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 featuresDark 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 stopContrast 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-IminImaxC=IminImaxC-contrastDark field microscopy
27 Condenser Light from the microscope light source at~9:00-10:10CondenserLight from the microscope light sourceCondenser gathers light and concentrates it into a cone of light that illuminates the specimen with uniform intensity over the entire viewfield
28 Transmitted Dark Field Illumination reflected DFTransmitted Dark Field IlluminationOblique raysspecimenreflected and transmitted dark field microscopeNo 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 beamIParallel beamIdistancedistanceat~5:24-8:14DF and BF images
29 Contrast EnhancementOM images of the green alga Micrasterias
30 Phase Contrast Microscopy Phase Contrast MicroscopyPhase 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
31 Crystals Growth by Differential Interference contrast microscopy (DIC) Growth spiral on cadmium iodide crystals growingFrom water solution (1025x).Fluorescence Microscopy Published on Jan 2, 2013Fluorescence 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~23:05-30:50Fluorescence microscopy - medical & organic materialsat~1:50-3:15
32 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.Why confocal? to~3:10at~0:40-1:36 & 2:40-2:56scanning
33 Scanning Confocal Optical Microscopy IntroductionScanning Confocal Optical MicroscopyThree-Dimensional Optical MicroscopyCritical dimension measurementsin semiconductor metrologywCross-sectional image with line scan at PR/Si interface of a sample containing 0.6m-wide lines and 1.0m-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 confocalinteractive tutorial
35 Grain Size Examination 1200C/30minThermal Etchinga1200C/2h20mbA 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.
36 Grain Size Examination Objective Lensx100Reflected OM
37 Grain Growth - Reflected OM 5mm30mmPolycrystalline CaF2 illustrating normal graingrowth. Better grain size distribution.Large grains in polycrystallinespinel (MgAl2O4) growing bysecondary recrystallizationfrom a fine-grained matrix
38 Liquid Phase Sintering – Reflective OM Amorphousphase40mmMicrostructure of MgO-2% kaolin body resultingfrom reactive-liquid phase sintering.
39 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).
40 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.
41 Specialized OM Techniques Enhancement of ContrastDarkfield MicroscopyPhase contrast microscopyDifferential interference contrast microscopyFluorescence microscopy-medical & organic materialsScanning confocal optical microscopy (relatively new)Three-Dimensional Optical Microscopyinspect and measure submicrometer features in semiconductors and other materialsHot- and cold-stage microscopymelting, freezing points and eutectics, polymorphs, twin and domain dynamics, phase transformationsIn situ microscopyE-field, stress, etc.Special environmental stages-vacuum or gasesCLEM Correlated light microscopy and electron microscopy
42 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.nT(oC)
43 E-field Induced Phase Transition in Pb(Zn1/3Nb2/3)O3-PbTiO3 Crystals Single domainSchematic diagram forin situ domain observa-tions.Domain structures of PZN-PTcrystals as a function of E-field;E=20kV/cm, (b) e=23.5kV/cm(c) E=27kV/cmRhombohedral at E=0 andTetragonal was induced at E>20kV/cm
44 Review - Optical Microscopy Use visible light as illumination sourceHas a resolution of ~o.2mRange of samples characterized - almost unlimited for solids and liquid crystalsUsually nondestructive; sample preparation may involve material removalMain 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
45 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.
46 Limits of Optical Microscopy Small depth of field <15.5mmRough surfaceLow resolution ~0.2mmShape of specimenThin section or polished surfaceCover glassspecimenGlass slideresin20mmLack of compositional and crystallographic information
47 Optical Microscopy vs Scanning Electron Microscopy 25mmradiolarianOMSEMSmall depth of fieldLow resolutionLarge depth of fieldHigh resolutionRadiolarian – marine protozoan
48 Scanning Electron Microscopy (SEM) What is SEM?Working principles of SEMMajor components and their functionsElectron beam - specimen interactionsInteraction volume and escape volumeMagnification, resolution, depth of field and image contrastEnergy Dispersive X-ray Spectroscopy (EDS)Wavelength Dispersive X-ray Spectroscopy (WDS)Orientation Imaging Microscopy (OIM)X-ray Fluorescence (XRF)