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
http://www.youtube.com/watch?v=PMIU1fkIPQs
Microscope Review (simple, clear)
http://www.youtube.com/watch?annotation_id=annotation_100990&feature=iv&src_vid=L6d3z
D2LtSI&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=related
http://www.youtube.com/watch?v=bGBgABLEV4g
How to use 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://micro.magnet.fsu.edu/primer/java/kohler/condensercones/index.html
http://micro.magnet.fsu.edu/primer/java/kohler/contrast/index.html
Specimen Stage
http://micro.magnet.fsu.edu/primer/flash/stage/index.html
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)
camera
Beam
splitter
Olympus
BX51
Research
Microscope
Cutaway
Diagram
Objective Lens
dmin = 0.61l/NA
Objective specifications
Anatomy of an objective
rical
ture
Objectives are the most important components of a
light microscope: image formation, magnification, the
quality of images and the resolution of 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
(Diaphragm)
M=(L/fo)(25/fe)
Eyepieces (Oculars) work in combination with microscope
objectives to further magnify the intermediate image
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
http://micro.magnet.fsu.edu/primer/java/microscopy/reflected/index.html
Illumination System
Reflected
OM
Transmitted
OM
http://micro.magnet.fsu.e
du/primer/java/microscop
y/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.
birefringent - doubly refracting
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
http://micro.magnet.fsu.edu/primer/java/polarizedlight/filters/index.html
~3:30min
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
Anisotropic
Double Refraction (Birefringence)
http://micro.magnet.fsu.edu/primer/java/polarizedlight/icelandspar/index.html
Birefringence
Cubic
a
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
camera
Beam
splitter
Olympus
BX51
Research
Microscope
Cutaway
Diagram
http://micro.magnet.fsu.edu/primer/java/microassembly/index.html
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 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
C=
Sspecimen
S
=
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.
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
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-Imin
C=
Imax
C-contrast
http://micro.magnet.fsu.edu/primer/java/darkfield/reflected/index.html
Transmitted Dark Field Illumination
Oblique rays
specimen
I
I
distance
distance
http://micro.magnet.fsu.edu/primer/java/darkfield/cardioid/index.html
Contrast Enhancement
OM images of the green alga Micrasterias
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
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
Three-Dimensional Optical Microscopy
w
Critical dimension measurements
in semiconductor metrology
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
Grain Size Examination
1200C/30min
Thermal Etching
a
1200C/2h
b
20m
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
Grain Growth - Reflected OM
5m
Polycrystalline CaF2
illustrating normal grain
growth. Better grain size
distribution.
30m
Large grains in polycrystalline
spinel (MgAl2O4) growing by
secondary recrystallization
from a fine-grained matrix
Liquid Phase Sintering – Reflective OM
Amorphous
phase
40m
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).
Polarized Optical Microscopy (POM)
Reflected POM
Transmitted POM
(a)Surface features of a microprocessor integrated circuit
(b)Apollo 14 Moon rock
http://micro.magnet.fsu.edu/primer/virtual/polarizing/index.html
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(Mg1/3Nb2/3)O3-PbTiO3 Crystals
n
T(oC)
(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.
E-field Induced Phase Transition in
Pb(Zn1/3Nb2/3)O3-PbTiO3 Crystals
a
Schematic diagram for
in situ domain observations.
b
c
Single domain
Domain structures of PZN-PT
crystals as a function of E-field;
(a)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 characterization 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 <15.5m
Rough surface
• Low resolution ~0.2m
• Shape of specimen
Thin section or polished surface
Cover glass
specimen
Glass slide
resin
20m
• Lack of compositional and
crystallographic information
Optical Microscopy vs Scanning
Electron Microscopy
radiolarian
25m
OM
Small depth of field
Low resolution
SEM
Large depth of field
High resolution
http://www.mse.iastate.edu/microscopy/