1. 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
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.

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
NA of an objective is a measure of its ability to gather light and resolve fine specimen detail at a fixed object distance.
-Numerical Aperture Light Cones
specimen
glass
Air n=1.0
Immersion oil n=1.515
at~6:25-7:50
at~5:12-6:00
-oil immersion objective use in microscope at~0:33

4. Axial resolution – Depth of Field
Depth of Field Ranges (F m)
(F mm)
Small F Large F
Depth of focus (f mm)
NA f F
Depth of focus is important in photomicrography.
The axial range through which
an object can be focused without
any appreciable change in image
sharpness
M NA F
F is determined by NA.

5. Basic components and their functions
Microscope Review (simple, clear)
Microscope working in animation (How to use a microscope)
(I) (II)
Parts and Function of a Microscope (details)
How to use a microscope (specimen preparation at~1:55-2:30)
How to care for and operate a microscope
review of microscope parts and their functions

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 Alignment and Field Diaphragm Opening Size
-Condenser Light Cones
-Condenser Aperture Diaphragm - Control Of Specimen Contrast
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
~6:30 to 9:40

10. Specimen Stage

11. 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)

12. Olympus BX51 Research Microscope Cutaway Diagram
camera
Beam
splitter
Reflected light
Olympus BX51 Research Microscope Cutaway Diagram
Transmitted light

13. Objective Lens dmin = 0.61l/NA Anatomy of an objective
Objective specifications
rical
ture
- Dry and oil immersion objective use in microscope
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
Objectives to~5:26
Grades of objectives to~2:30 & 3:25-4:50

14. Eyepiece Lens M=(L/fo)(25/fe)
(Diaphragm)
Diopter – optics. A unit of measure of the refractive power of a lens, having the dimension of the reciprocal of length and a unit equal to the reciprocal of one meter.
M=(L/fo)(25/fe)
Eyepieces (Oculars) work in combination with microscope
objectives to further magnify the intermediate image

15. Olympus BX51 Research Microscope Cutaway Diagram
camera
Beam
splitter
Olympus BX51 Research Microscope Cutaway Diagram

16. 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.

17. Anatomy of a modern OM Illumination System Reflected OM Transmitted OM
Trans OM to~1:37
Refle OM from 1:38-end
Illumination System
Reflected OM
Transmitted light microscopy is the general term used for any type of microscopy where the light is transmitted from a source on the opposite side of the specimen from the objective. Usually the light is passed through a condenser to focus it on the specimen to get very high illumination. After the light passes through the specimen, the image of the specimen goes through the objective lens and to the oculars where the enlarged image is viewed.
Reflected light microscopy is often referred to as incident light or metallurgical microscopy, and is the method of choice for imaging specimens that remain opaque even when ground to a thickness of 30 microns.
Transmitted OM
Illumination System
at~0:20-1:40 Field diaphragm

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
Polarized light microscopy is a useful method to generate contrast in birefringent specimens and to determine qualitative and quantitative aspects of crystallographic axes present in various materials.

19. 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.
to~3:30min

20. Birefringence anisotropic Isotropic CaCO3
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

21. Anisotropic Optical Character
(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 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. a

22. Polarized Optical Microscopy (POM)
Reflected POM Transmitted POM
Surface features of a microprocessor integrated circuit
Apollo 14 Moon rock

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
at~3:50-4:30 Fluorescence

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.
at~1:47-3:04

25. Contrast in Optical Microscope
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.
When imaging specimens in the optical microscope, differences in intensity and/or color create image contrast, which allows individual features and details of the specimen to become visible.
at~1:33-2:21

26. 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
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 transmitted optical microscope. Different phases or variations in thickness absorb light to differing extends which causes them to differ in brightness in specimens of uniform thickness in the former and in specimens with different thickness in the latter. Furthermore, in some cases selective absorption of a particular wavelength or wavelengths of light occurs causing the phases to appear coloured.), brightness, reflectance (light from the surfaces that are perpendicular to the incident beam is reflected back into the objective, while light from tilted areas of the surfaces or grain boundaries is reflected away from the objective, so that they appear dark. Conversely with DF illumination, light reflected from grain surfaces which are almost perpendicular to the optical axis of the microscope is reflected away from the objective, while light from grain boundaries and tilted surfaces is reflected into it. Mainly for opaque specimens.), birefringence, light scattering, diffraction, fluorescence, or color variations has been the classical means of imaging specimens in brightfield microscopy.
Imax-Imin
Imax C=
Imin
Imax
C-contrast
Dark field microscopy

27. Condenser Light from the microscope light source
at~9:00-10:10
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

28. Transmitted Dark Field Illumination
reflected DF
Transmitted Dark Field Illumination
Oblique rays
specimen
reflected and transmitted dark field microscope
No zeroth order light reaches the specimen, only light that has been diffracted, refracted and reflected from the surface of the specimen is able to enter the front lens of the objective to form an image. In the absence of a specimen, the viewfield appears totally back, because no light is reflected or diffracted into the objective.
Reflected beam I
Parallel beam I
distance
distance
at~5:24-8:14
DF and BF images

29. Contrast Enhancement
OM images of the green alga Micrasterias

30. 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).
at~0:50-5:20

31. Crystals Growth by Differential Interference contrast microscopy (DIC)
Growth spiral on cadmium iodide crystals growing
From water solution (1025x).
Fluorescence Microscopy Published on Jan 2, 2013
Fluorescence is a physical phenomenon in which a compound absorbs light and re-emits this as light of a usually higher wavelength. Since the wavelengths of the excitation light source and the emitted fluorescence can be separated very well, we can detect fluorescence with very high sensitivity, making it possible to visualize even single molecules. Many different fluorescent probes for cellular components have been developed, including genetically encoded probes like the Green Fluorescent Protein (GFP). For these reasons, fluorescence microscopy is a very powerful tool in Cell Biology research.
at~23:05-30:50
Fluorescence microscopy - medical & organic materials
at~1:50-3:15

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.
Why confocal? to~3:10
at~0:40-1:36 & 2:40-2:56
scanning

33. Scanning Confocal Optical Microscopy
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 0.6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.
Confocal microscopy offers several advantages over conventional optical microscopy, including controllable depth of field, the elimination of image degrading out-of-focus information, and the ability to collect serial optical sections from thick specimens. The key to the confocal approach is the use of spatial filtering to eliminate out-of-focus light or flare in specimens that are thicker than the plane of focus.
to~2.44 coral under confocal
interactive tutorial

34. Typical Examples of OM Applications

35. Grain Size Examination
1200C/30min
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.

36. Grain Size Examination
Objective Lens
x100
Reflected OM

37. 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

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

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).
Under plane-polarized light, i.e., only analyzer was used and incident beam is unpolarized. The color is due to anisotropic absorption.

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
CLEM Correlated light microscopy and electron microscopy

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
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

44. 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

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
Cover glass
specimen
Glass slide
resin
20mm
Lack of compositional and crystallographic information

47. Optical Microscopy vs Scanning Electron Microscopy
25mm
radiolarian OM
SEM
Small depth of field
Low resolution
Large depth of field
High resolution
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)