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 ~ 0.3m for a midspectrum l of 0.55m

https://www.youtube.com/watch?v=n2asdncMYMo at~5:35-6:00
 Numerical Aperture (NA)
NA=1 - theoretical NA = n(sin )
maximum numerical
aperture of a lens n: refractive index of the
operating with air as imaging medium between
the imaging medium 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 focus (f mm) Depth of Field Ranges
(F (F
m)m)

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 NA f F
F is determined by NA.
M NA f F
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
Depth change
of focus in image sharpness.
(f mm)

NA f F
0.1 0.13 15.5
0.4 3.8 5.8
.95 80.0 0.19

25m

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: weaker diverging lens
The combination achromatic doublet
http://www.youtube.com/watch?v=yH7rbRu7Av8&list=PL02D1D436A44B521A chromatic aberration
http://www.youtube.com/watch?v=H8PQ9RMUoA8 at~3:30-4:30
 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 Olympus
splitter
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 to~3:30min
http://www.youtube.com/watch?v=E9qpbt0v5Hw
http://micro.magnet.fsu.edu/primer/java/polarizedlight/filters/index.html
 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://www.youtube.com/watch?v=WdrYRJfiUv0
 Anisotropic Optical Character a
(Birefringence) 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 a
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
character http://www.youtube.com/watch?v=ulNZ3u7_J5I to ~1:05

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 S
C= =
Sspecimen 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 at~2:17-3:46
Interaction of light with matter

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 C=
Imin 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 distance
http://www.youtube.com/watch?v=I4ZQm-CAgL8 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 at~0:50-5:20
http://www.youtube.com/watch?v=WvyCg1uzG5c
 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 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~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
Critical dimension measurements
in semiconductor metrology
w
Cross-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.
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
 1200C/30min
Grain Size Examination
Thermal Etching

a
1200C/2h

b 20m

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

5m 30m

Polycrystalline CaF2 Large grains in polycrystalline

illustrating normal grain spinel (MgAl2O4) growing by
growth. Better grain size secondary recrystallization
distribution. from a fine-grained matrix
Liquid Phase Sintering Reflective OM

Amorphous
phase

40m

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

(a) and (b) at 20oC, strongly

birefringent domains with extinction
n 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
T(oC) expense of vanishing strip domains.
 E-field Induced Phase Transition in
Pb(Zn1/3Nb2/3)O3-PbTiO3 Crystals

a b c
Single domain

Schematic diagram for Domain structures of PZN-PT

in situ domain observa- crystals as a function of E-field;
tions. (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.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 <15.5m
Rough surface
Low resolution ~0.2m
Shape of specimen
Thin section or polished surface
Cover glass
specimen
Glass slide
resin

20m

Lack of compositional and

crystallographic information
 Optical Microscopy vs Scanning
Electron Microscopy

25m
radiolarian
OM SEM
Small depth of field Large depth of field
Low resolution 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