optical microscope and its details working

1. 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

2. 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

3. 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=1.515
http://www.youtube.com/watch?v=H8PQ9RMUoA8 at~6:25-7:50
http://www.youtube.com/watch?v=n2asdncMYMo at~5:12-6:00
Air n=1.0
specimen 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.

4. Depth of focus (f mm)
The axial range through which
an object can be focused without
any appreciable change in image
sharpness
(F mm)
M NA F
M NA F
Axial resolution – Depth of Field
Depth of Field Ranges (F mm)
F is determined by NA.
NA f F
0.1 0.13 15.5
0.4 3.8 5.8
.95 80.0 0.19
http://micro.magnet.fsu.edu/primer/java/nuaperture/index.html
Small F Large F

5. 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

6. 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)

7. 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

8. Light Sources

9. 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 9:40
http://micro.magnet.fsu.edu/primer/java/kohler/contrast/index.html

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

11.  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

12. Olympus
BX51
Research
Microscope
Cutaway
Diagram
Beam
splitter
camera
Reflected light
Transmitted light
http://micro.magnet.fsu.edu/primer/java/microassembly/index.html

13. Objective specifications
Objectives are the most important components of a
light microscope: image formation, magnification, the
quality of images and the resolution of the microscope
Objective Lens
Anatomy of an objective
rical
ture
dmin = 0.61/NA
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
DIC-differential interference contrast

14. (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

15. Olympus
BX51
Research
Microscope
Cutaway
Diagram
Beam
splitter
camera
http://micro.magnet.fsu.edu/primer/java/microassembly/index.html

16. 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
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.
Depending on the nature of samples, different illumination
methods must be used

17. Anatomy of a modern OM
Illumination System
Transmitted
OM
Reflected
OM
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

18. 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

19. 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~3:30min
http://www.youtube.com/watch?v=E9qpbt0v5Hw
http://www.youtube.com/watch?v=rbx3K1xBxVU polarized light

20. 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

21. (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

22. 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

23. 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

24. 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 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

25. 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

26. 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
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

27. 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

28. Transmitted Dark Field Illumination
specimen
I I
distance distance
Oblique rays
http://micro.magnet.fsu.edu/primer/java/darkfield/cardioid/index.html
http://micro.magnet.fsu.edu/primer/techniques/darkfieldreflect.html reflected DF
http://www.youtube.com/watch?v=I4ZQm-CAgL8 at~5:24-8:14
Reflected beam
Parallel beam

29. Contrast Enhancement
OM images of the green alga Micrasterias

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

31. 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

32. 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~3:10
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

33. Scanning Confocal Optical Microscopy
Critical dimension measurements
in semiconductor metrology
Cross-sectional image with line scan
at PR/Si interface of a sample
containing 0.6mm-wide lines and
1.0mm-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 confocal
http://micro.magnet.fsu.edu/primer/virtual/confocal/index.html interactive tutorial
http://www.olympusconfocal.com/theory/confocalintro.html Introduction

34. Typical Examples of
OM Applications

35. 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
20mm
1200C/30min
1200C/2h

36. Grain Size Examination
Objective Lens
x100
Reflected OM

37. Grain Growth - Reflected OM
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
30mm
5mm

38. Liquid Phase Sintering – Reflective OM
Microstructure of MgO-2% kaolin body resulting
from reactive-liquid phase sintering.
Amorphous
phase
40mm

39. 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).

40. Phase Identification by Reflected
Polarized Optical Microscopy
YBa2Cu307-x superconductor material: (a) tetragonal phase and
(b) orthorhombic phase with multiple twinning (arrowed) (100 x).

41. 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

42. 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)

43. E-field Induced Phase Transition in
Pb(Zn1/3Nb2/3)O3-PbTiO3 Crystals
Schematic diagram for
in situ domain observa-
tions.
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
a b c
Single domain

44. Review - Optical Microscopy
• Use visible light as illumination source
• Has a resolution of ~o.2mm
• 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

45. 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.

46. Limits of Optical Microscopy
• Small depth of field <15.5mm
Rough surface
• Low resolution ~0.2mm
• Shape of specimen
Thin section or polished surface
Glass slide
specimen
Cover glass
resin
20mm
• Lack of compositional and
crystallographic information

47. Optical Microscopy vs Scanning
Electron Microscopy
25mm
OM SEM
Small depth of field
Low resolution
Large depth of field
High resolution
radiolarian
http://www.mse.iastate.edu/microscopy/
Radiolarian – marine protozoan

48. 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/