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=P2teE17zT4I&list=PLKstG-8VPWKzOe4TkvA7F6qMlG2HH8meX 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

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 = /n

The dmin = /2n sin = /2(N.A.)

For circular aperture

dmin= 1.22/2(N.A.)=0.61/(N.A.)

where N.A. = n sin is called numerical aperture

Immersion oil n=1.515http://www.youtube.com/watch?v=H8PQ9RMUoA8 at~6:25-7:50http://www.youtube.com/watch?v=n2asdncMYMo at~5:12-6:00

Air n=1.0specimen glass

http://www.youtube.com/watch?v=RSKB0J1sRnU -oil immersion objective use in microscope at~0:33

NA of an objective is a measure of its ability to gather light and resolve fine specimen detail at a fixed object distance.

Depth of focus (f mm)

The axial range through whichan object can be focused withoutany appreciable change in imagesharpness

(F m)

M NA FM NA F

Axial resolution – Depth of FieldDepth of Field Ranges (F m)

F is determined by NA.

NA f F0.1 0.13 15.50.4 3.8 5.8.95 80.0 0.19

http://micro.magnet.fsu.edu/primer/java/nuaperture/index.html

Small F Large F

Basic components and their functions

http://www.youtube.com/watch?v=RKA8_mif6-EMicroscope Review (simple, clear)

http://www.youtube.com/watch?v=b2PCJ5s-iyk Microscope working in animation (How to use a microscope)

http://www.youtube.com/watch?annotation_id=annotation_100990&feature=iv&src_vid=L6d3zD2LtSI&v=ntPjuUMdXbg (I) http://www.youtube.com/watch?v=VQtMHj3vaLg (II)Parts and Function of a Microscope (details)

http://www.youtube.com/watch?v=X-w98KA8UqU&feature=relatedHow to use a microscope (specimen preparation at~1:55-2:30)

http://www.youtube.com/watch?v=bGBgABLEV4gHow 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=L6d3zD2LtSI&v=ntPjuUMdXbg ~6:30 to 9:40http://micro.magnet.fsu.edu/primer/java/kohler/contrast/index.html

Specimen Stage

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

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)

Functions of the Major Parts of a Optical Microscope

Olympus BX51

Research Microscope

Cutaway Diagram

Beam splitter

camera

Reflected light

Transmitted lighthttp://micro.magnet.fsu.edu/primer/java/microassembly/index.html

Objective specifications

Objectives are the most important components of alight microscope: image formation, magnification, thequality of images and the resolution of the microscope

Objective Lens

Anatomy of an objective

ricalture

dmin = 0.61/NA

http://www.youtube.com/watch?v=P0Z4H2O_Stg Objectives to~5:26http://www.youtube.com/watch?v=H8PQ9RMUoA8 Grades of objectives to~2:30 & 3:25-4:50

DIC-differential interference contrast

(Diaphragm)

Eyepieces (Oculars) work in combination with microscope objectives to further magnify the intermediate image

Eyepiece Lens

M=(L/fo)(25/fe)

http://micro.magnet.fsu.edu/primer/anatomy/oculars.html

Olympus BX51

Research Microscope

Cutaway Diagram

Beam splitter

camera

http://micro.magnet.fsu.edu/primer/java/microassembly/index.html

Common Modes of Analysis

• 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 characterCharacteristics 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.

Depending on the nature of samples, different illumination methods must be used

Anatomy of a modern OM

Illumination System

TransmittedOM

ReflectedOM

Illumination System

http://www.youtube.com/watch?v=zq13e36cs3s at~0:20-1:40 Field diaphragm

http://www.youtube.com/watch?v=7-Tlyd7piSM Trans OM to~1:37 Refle OM from 1:38-end

Polarized Light MicroscopyPolarized 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

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.

Polarization of Light

http://micro.magnet.fsu.edu/primer/java/polarizedlight/filters/index.html

http://www.youtube.com/watch?v=lZ-_i82s16E&feature=endscreen&NR=1 to~3:30minhttp://www.youtube.com/watch?v=E9qpbt0v5Hw

http://www.youtube.com/watch?v=rbx3K1xBxVU polarized light

Birefringence

Isotropic

anisotropic

CaCO3 Double Refraction (Birefringence)

Birefringence is optical property of a material having a refractive index that depends on the polarization and propagation direction of light.

Anisotropic

http://www.youtube.com/watch?v=WdrYRJfiUv0

(Birefringence)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.

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.

Cubic a

tetragonal c

a

Anisotropic Optical Character

http://micro.magnet.fsu.edu/primer/java/polarizedlight/crystal/index.html

Polarized Optical Microscopy (POM)

(a) Surface features of a microprocessor integrated circuit(b)Apollo 14 Moon rock

Reflected POM Transmitted POM

http://micro.magnet.fsu.edu/primer/virtual/polarizing/index.html

Specialized OM Techniques• Enhancement of Contrast

Darkfield Microscopy Phase contrast microscopyDifferential 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 microscopymelting, freezing points and eutectics, polymorphs, twin and domain dynamics, phase transformations

• In situ microscopyE-field, stress, etc.

• Special environmental stages-vacuum or gases

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 by

Sspecimen-Sbackgroud SC = = 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. http://www.youtube.com/watch?v=SVK4OkUK0Yw at~1:47-3:04

http://micro.magnet.fsu.edu/primer/techniques/contrast.html

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.

Contrast in Optical Microscope

http://micro.magnet.fsu.edu/primer/virtual/virtualzoo/index.html

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://www.youtube.com/watch?v=P2teE17zT4I&list=PLKstG-8VPWKzOe4TkvA7F6qMlG2HH8meX at~1:33-2:21

https://www.youtube.com/watch?v=L3SsxIUm0As at~2:17-3:46 Interaction of light with matter

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

ImaxImin

C=Imax-Imin

Imax

C-contrast

http://www.youtube.com/watch?v=d6jsnLIsNwI at~3:40-5:20

http://www.youtube.com/watch?v=7V3nyRGeha4 Dark field microscopy

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=L6d3zD2LtSI&v=ntPjuUMdXbg at~9:00-10:10

Transmitted Dark Field Illumination

specimen

I I

distance distance

Oblique rays

http://micro.magnet.fsu.edu/primer/java/darkfield/cardioid/index.htmlhttp://micro.magnet.fsu.edu/primer/techniques/darkfieldreflect.html reflected DF

http://www.youtube.com/watch?v=I4ZQm-CAgL8 at~5:24-8:14http://www.youtube.com/watch?v=J2e0x7iTqTU DF and BF images

Reflected beam

Parallel beam

Contrast Enhancement

OM images of the green alga Micrasterias

Phase Contrast Microscopy

http://www.youtube.com/watch?v=I4ZQm-CAgL8 at~0:50-5:20http://www.youtube.com/watch?v=WvyCg1uzG5c

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.microscopyu.com/articles/phasecontrast/phasemicroscopy.html

Crystals Growth by Differential Interference contrast microscopy (DIC)

Growth spiral on cadmium iodide crystals growingFrom water solution (1025x).

http://www.youtube.com/watch?v=P2teE17zT4I at~23:05-30:50http://micro.magnet.fsu.edu/primer/techniques/dic/dichome.html

Fluorescence microscopy - medical & organic materialshttp://www.youtube.com/watch?v=iPrZ84kHH2U at~1:50-3:15 http://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorhome.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 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.

http://www.youtube.com/watch?v=mrjgNyKX8-w Why confocal? to~3:10http://www.youtube.com/watch?v=puT1ccMWKyQ at~0:40-1:36 & 2:40-2:56

http://www.youtube.com/watch?v=Axrst4T__YY scanning

Scanning Confocal Optical Microscopy

Critical dimension measurementsin semiconductor metrologyCross-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.

w

Three-Dimensional Optical Microscopy

http://www.youtube.com/watch?v=oluJW7uK7rw&index=12&list=PL200E1A86911B0422 to~2.44 coral under confocalhttp://micro.magnet.fsu.edu/primer/virtual/confocal/index.html interactive tutorial

http://www.olympusconfocal.com/theory/confocalintro.html Introduction

Typical Examples of OM Applications

Grain Size Examination

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.

a

b

Thermal Etching

20m

1200C/30min

1200C/2h

Grain Size Examination

Objective Lens

x100

Reflected OM

Grain Growth - Reflected OM

Polycrystalline CaF2 illustrating normal graingrowth. Better grain size distribution.

Large grains in polycrystallinespinel (MgAl2O4) growing bysecondary recrystallization from a fine-grained matrix

30m5m

Liquid Phase Sintering – Reflective OM

Microstructure of MgO-2% kaolin body resultingfrom reactive-liquid phase sintering.

Amorphousphase

40m

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).

Specialized OM Techniques• Enhancement of Contrast

Darkfield Microscopy Phase contrast microscopyDifferential 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 microscopymelting, freezing points and eutectics, polymorphs, twin and domain dynamics, phase transformations

• In situ microscopyE-field, stress, etc.

• Special environmental stages-vacuum or gaseshttp://www.nature.com/nmeth/journal/v12/n6/full/nmeth.3400.html

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

Schematic diagram forin situ domain observa-tions.

Domain structures of PZN-PTcrystals as a function of E-field; (a) E=20kV/cm, (b) e=23.5kV/cm(c) E=27kV/cmRhombohedral at E=0 andTetragonal was induced at E>20kV/cm

a b cSingle domain

Review - Optical Microscopy• Use visible light as illumination source• Has a resolution of ~o.2m• 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 <15.5mRough surface

• Low resolution ~0.2m• Shape of specimen

Thin section or polished surface

Glass slide

specimenCover glass

resin

20m

• Lack of compositional and crystallographic information

Optical Microscopy vs Scanning Electron Microscopy

m

OM SEM

Small depth of field Low resolution

Large depth of field High resolution

radiolarian

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/