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
http://micro.magnet.fsu.edu/primer/java/microscopy/immersion/index.html
Resolution of a Microscope (lateral)
The smallest distance between two specimen points
that can still be distinguished as two separate entities
dmin = 0.61l/NA
NA=nsin()
l – illumination wavelength (light)
NA – numerical aperture
-one half of the objective angular aperture
n-imaging medium refractive index
dmin ~ 0.3m for a midspectrum l of 0.55m
https://www.youtube.com/watch?v=n2asdncMYMo at~5:35-6:00
Numerical Aperture (NA)
NA=1 -
NA = n(sin )
theoretical
maximum numerical
aperture of a lens
operating with air as
the imaging medium
n: refractive index of the
imaging medium between
the front lens of objective
and specimen cover glass
Objective lens

Angular aperture
(72 degrees)
One half of A-A
Specimen
cover glass
NA of an objective is a measure of its ability to gather
light and resolve fine specimen detail at a fixed object
distance.
https://en.wikipedia.org/wiki/Angular_aperture
http://micro.magnet.fsu.edu/primer/java/microscopy/immersion/index.html
Numerical Aperture
NA = n(sin )
Imaging Medium
Air
n=1.0
Immersion oil
n=1.515
http://www.youtube.com/watch?v=RSKB0J1sRnU
oil immersion objective use in microscope at~0:33
Axial resolution – Depth of Field
Depth of Field Ranges
m)
(F (F
m)
Depth of focus (f mm)
NA
f
F
0.1 0.13 15.5
0.4 3.8 5.8
.95 80.0 0.19
The distance above and below The axial range through which
geometric image plane within an object can be focused without
which the image is in focus
any appreciable change in image
sharpness
M
M
NA
NA
f
f
F
F
F is determined by NA.
http://www.matter.org.uk/tem/depth_of_field.htm
http://www.youtube.com/watch?v=FvC2WLUqEug
at~3:40
Depth of Focus
The distance above and below geometric image plane within
which the image is in focus.
Depth of focus (f mm)
CCD camera
Axial resolution – Depth of Field
The axial range through which an object can be focused without
any
appreciable
change
in image sharpness.
Depth
of focus
(f mm)
NA
f
F
0.1 0.13 15.5
0.4 3.8 5.8
.95 80.0 0.19
25m
Small F
Large F
Basic components and their functions
http://www.youtube.com/watch?v=RKA8_mif6-E
Microscope 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&featur
e=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=related
How to use a microscope (specimen preparation at~1:55-2:30)
http://www.youtube.com/watch?v=bGBgABLEV4g
How 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 ~8:08 to 9:40
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
Reflected light
Olympus
BX51
Research
Microscope
Cutaway
Diagram
Transmitted light
http://micro.magnet.fsu.edu/primer/java/microassembly/index.html
Objective Lens
dmin = 0.61l/NA
Objective specifications
Anatomy of an objective
rical
ture
DIC-differential interference contrast
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?v=P0Z4H2O_Stg Objectives to~5:26
http://www.youtube.com/watch?v=H8PQ9RMUoA8 Grades of objectives to~2:30 & 3:25-4:50
https://www.youtube.com/watch?v=FwBjpi8ck1Y
Alignment of OM
Defects in Lens


Curvature of Field
- When visible light
is focused through a
curved lens, the
image plane
produced by the lens
will be curved
The image appears
sharp and crisp
either in the center
or on the edges of
the viewfield but not
both
http://micro.magnet.fsu.edu/primer/java/aberrations/curvatureoffield/index.html
Defects in Lens
 Chromatic Aberration


Axial - Blue light is refracted to
the greatest extent followed by
green and red light, a
phenomenon commonly referred
to as dispersion
Lateral - chromatic difference of
magnification: the blue image of a
detail was slightly larger than the
green image or the red image in
white light, thus causing color
ringing of specimen details at the
outer regions of the field of view
A converging lens can be combined
with a weaker diverging lens, so that
the chromatic aberrations cancel for
certain wavelengths:
The combination – achromatic doublet
weaker diverging lens
http://www.youtube.com/watch?v=yH7rbRu7Av8&list=PL02D1D436A44B521A
http://www.youtube.com/watch?v=H8PQ9RMUoA8
at~3:30-4:30
chromatic aberration
Eyepiece Lens
(Diaphragm)
M=(L/fo)(25/fe)
Eyepieces (Oculars) work in combination with microscope
objectives to further magnify the intermediate image
http://micro.magnet.fsu.edu/primer/anatomy/oculars.html
http://www.birdwatching.com/optics/diopter_set.html
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
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://www.youtube.com/watch?v=7-Tlyd7piSM
Trans OM to~1:37
Refle OM from 1:38-end
Illumination System
Reflected
OM
Transmitted
OM
Illumination System
http://www.youtube.com/watch?v=zq13e36cs3s
at~0:20-1:40 Field diaphragm
camera
Beam
splitter
Olympus
BX51
Research
Microscope
Cutaway
Diagram
http://micro.magnet.fsu.edu/primer/java/microassembly/index.html
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.
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
http://www.youtube.com/watch?v=rbx3K1xBxVU
polarized 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
http://www.youtube.com/watch?v=E9qpbt0v5Hw
http://micro.magnet.fsu.edu/primer/java/polarizedlight/filters/index.html
to~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://www.youtube.com/watch?v=WdrYRJfiUv0
Anisotropic Optical Character
Cubic
(Birefringence)
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
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 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
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
http://www.youtube.com/watch?v=ulNZ3u7_J5I to ~1:05
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.
http://www.youtube.com/watch?v=Iw734z1e6wQ to~1:30
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
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
http://micro.magnet.fsu.edu/primer/techniques/contrast.html
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 (Smax) and Sbackgroud (Smin)
are intensities measured from
specimen and background, 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
Contrast in Optical Microscope
https://www.youtube.com/watch?v=L3SsxIUm0As
Interaction of light with matter
at~2:17-3:46
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
http://www.youtube.com/watch?v=P2teE17zT4I&list=PLKstG-8VPWKzOe4TkvA7F6qMlG2HH8meX
at~1:33-2:21
http://www.youtube.com/watch?v=d6jsnLIsNwI



at~3:40-5:20
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://www.youtube.com/watch?v=7V3nyRGeha4
Dark field microscopy
http://micro.magnet.fsu.edu/primer/techniques/darkfieldreflect.html
Condenser
Oblique hollow cone of light
Cone of light
Reflected light
Light stop
Bright field illumination
Dark field illumination
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/darkfield/cardioid/index.html
http://micro.magnet.fsu.edu/primer/techniques/darkfieldreflect.html reflected DF
Transmitted Dark Field Illumination
Oblique rays
specimen
Reflected beam
I
Parallel beam
I
distance
http://www.youtube.com/watch?v=I4ZQm-CAgL8
distance
at~5:24-8:14
Contrast Enhancement
OM images of the green alga Micrasterias
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
http://www.microscopyu.com/articles/phasecontrast/phasemicroscopy.html
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.youtube.com/watch?v=I4ZQm-CAgL8
http://www.youtube.com/watch?v=WvyCg1uzG5c
at~0:50-5:20
Crystals Growth by Differential
Interference contrast microscopy (DIC)
Growth spiral on
cadmium iodide
crystals growing
From water
solution (1025x).
http://www.youtube.com/watch?v=P2teE17zT4I at~23:05-30:50
http://micro.magnet.fsu.edu/primer/techniques/dic/dichome.html
Fluorescence microscopy - medical & organic materials
http://www.youtube.com/watch?v=iPrZ84kHH2U at~1:50-3:15
http://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorhome.html
http://micro.magnet.fsu.edu/primer/techniques/confocal/index.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 infocus 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~1:05
http://www.youtube.com/watch?v=puT1ccMWKyQ
at~0:40-1:36 & 2:40-2:56
http://www.youtube.com/watch?v=Axrst4T__YY
scanning
http://www.olympusconfocal.com/theory/confocalintro.html
Introduction
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.youtube.com/watch?v=oluJW7uK7rw&index=12&list=PL200E1A86911B0422 to~2.44 coral under confocal
http://micro.magnet.fsu.edu/primer/virtual/confocal/index.html
interactive tutorial
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
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
Reflected OM
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).
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 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
http://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
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/
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
http://science.howstuffworks.com/scanning-electron-microscope.htm/printable