Lecture 2 optical microscopy
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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. Basic components and their functions.

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Lecture-2 Optical Microscopy

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Lecture 2 optical microscopy

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


Basic components and their functions

Basic components and their functions

http://www.youtube.com/watch?v=PMIU1fkIPQsMicroscope Review (simple, clear)

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

http://www.youtube.com/watch?v=VQtMHj3vaLg (II)

http://www.youtube.com/watch?v=X-w98KA8UqU&feature=relatedHow to use a microscope

http://www.youtube.com/watch?v=bGBgABLEV4g


Basic components and their functions1

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

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

Light Sources


Condenser

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://micro.magnet.fsu.edu/primer/java/kohler/condensercones/index.html

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


Specimen stage

Specimen Stage

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


Functions of the major parts of a optical microscope1

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

Olympus BX51Research Microscope Cutaway Diagram

Beam

splitter


Objective lens

Objective Lens

dmin = 0.61l/NA

Anatomy of an objective

Objective specifications

rical

ture

Objectives are the most important components of a

light microscope: image formation, magnification, the

quality of imagesand the resolutionof the microscope

http://www.youtube.com/watch?annotation_id=annotation_100990&feature=iv&src_vid=L6d3zD2LtSI&v=ntPjuUMdXbg

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

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


Eyepiece lens

Eyepiece Lens

(Diaphragm)

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

Eyepieces (Oculars) work in combination with microscope

objectives to further magnify the intermediate image


Common modes of analysis

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

Anatomy of a modern OM

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

Illumination System

Reflected

OM

Transmitted

OM

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

Illumination System

http://micro.magnet.fsu.edu/primer/java/microscopy/transmitted/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.

Polarized Light Microscopy

birefringent - doubly refracting


Polarization of 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.

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

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


Birefringence

Birefringence

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

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


Birefringence1

a

Cubic

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.

tetragonal

c

a

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.

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


Olympus bx51 research microscope cutaway diagram1

camera

Olympus BX51Research Microscope Cutaway Diagram

Beam

splitter

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


Specialized om techniques

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


Contrast

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

Sspecimenand 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.


Lecture 2 optical microscopy

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.

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


Angle of illumination

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

C=

Imax

Imin

C-contrast

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


Transmitted dark field illumination

Transmitted Dark Field Illumination

Oblique rays

specimen

I

I

distance

distance

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


Contrast enhancement

Contrast Enhancement

OM images of the green alga Micrasterias


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).

http://www.microscopyu.com/articles/phasecontrast/phasemicroscopy.html


Crystals growth by differential interference contrast microscopy

Crystals Growthby Differential Interference contrast microscopy

Growth spiral on cadmium iodide crystals growing

From water solution (1025x).

http://micro.magnet.fsu.edu/primer/techniques/dic/dichome.html

Fluorescence microscopy - medical & organic materials

http://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorhome.html


Scanning confocal optical microscopy

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.

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

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


Typical examples of om applications

Typical Examples of OM Applications


Grain size examination

1200C/30min

Grain Size Examination

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 examination1

Grain Size Examination

Objective Lens

x100


Grain growth 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

Liquid Phase Sintering – Reflective OM

Amorphous

phase

40mm

Microstructure of MgO-2% kaolin body resulting

from reactive-liquid phase sintering.


Image of magnetic domains

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).


Polarized optical microscopy pom

Polarized Optical Microscopy (POM)

Reflected POM Transmitted POM

  • Surface features of a microprocessor integrated circuit

  • Apollo 14 Moon rock

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


Phase identification by reflected polarized optical microscopy

Phase Identification by Reflected Polarized Optical Microscopy

YBa2Cu307-x superconductor material: (a) tetragonal phase and (b) orthorhombic phase with multiple twinning (arrowed) (100 x).


Hot stage pom of phase transformations in pb mg 1 3 nb 2 3 o 3 pbtio 3 crystals

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 zn 1 3 nb 2 3 o 3 pbtio 3 crystals

E-field Induced Phase Transition in Pb(Zn1/3Nb2/3)O3-PbTiO3 Crystals

c

a

b

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

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

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

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

Optical Microscopy vs Scanning Electron Microscopy

25mm

radiolarian

OM

SEM

Small depth of field

Low resolution

Large depth of field

High resolution

http://www.mse.iastate.edu/microscopy/


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