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Lecture-2 Optical Microscopy

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Presentation on theme: "Lecture-2 Optical Microscopy"— Presentation transcript:

1 Lecture-2 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

2 Basic components and their functions
Microscope Review (simple, clear) (I) Parts and Function of a Microscope (details) (II) How to use a microscope

3 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)

4 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

5 Light Sources

6 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

7 Specimen Stage

8 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)

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

10 Objective Lens dmin = 0.61l/NA Anatomy of an objective
Objective specifications rical ture - Dry and oil immersion objective use in microscope -Specifications and Identification -Numerical Aperture Light Cones Objectives are the most important components of a light microscope: image formation, magnification, the quality of images and the resolution of the microscope

11 Eyepiece Lens M=(L/fo)(25/fe)
(Diaphragm) M=(L/fo)(25/fe) Eyepieces (Oculars) work in combination with microscope objectives to further magnify the intermediate image

12 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.

13 Anatomy of a modern OM Illumination System Reflected OM Transmitted OM
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

14 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.

15 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. ~3:30min

16 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

17 Birefringence a 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

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

19 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

20 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 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.

21 Formation of Contrast 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.

22 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

23 Transmitted Dark Field Illumination
Oblique rays specimen 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. I I distance distance

24 Contrast Enhancement OM images of the green alga Micrasterias

25 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).

26 Crystals Growth by Differential Interference contrast microscopy
Growth spiral on cadmium iodide crystals growing From water solution (1025x). Fluorescence microscopy - medical & organic materials

27 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.

28 Typical Examples of OM Applications

29 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.

30 Grain Size Examination
Objective Lens x100

31 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

32 Liquid Phase Sintering – Reflective OM
Amorphous phase 40mm Microstructure of MgO-2% kaolin body resulting from reactive-liquid phase sintering.

33 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).

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

35 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.

36 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)

37 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

38 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

39 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.

40 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

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

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