AP 5301/8301Instrumental Methods of Analysis

and Laboratory

Lecture 2

Microscopy (I) Optical

Prof YU Kin Man

E-mail: kinmanyu@cityu.edu.hk

Tel: 3442-7813

Office: P6422

1

http://www6.cityu.edu.hk/appkchu/AP5301/Notes.htm

Lecture 2: Outline Introduction:

─ Materials characterization techniques

─ Microscopy

Optical microscopy basics

─ Basic concepts

─ Terminologies

─ Resolution

─ Aberrations

Optical microscope

─ Major parts and functions

─ Common modes of analysis Bright and dark field imaging

Polarized microscopy

Phase-contrast microscopy

Differential interference contrast microscopy

Fluorescence microscopy

Scanning confocal optical microscopy

Some examples of applications

2

Introduction: Materials CharacterizationWikipedia:

Characterization, when used in materials science, refers to the broad and

general process by which a material's structure and properties are probed and

measured. It is a fundamental process in the field of materials science, without

which no scientific understanding of engineering materials could be ascertained.

The structure of a material is

determined by its chemical

composition and how it was

synthesized (processed)

A material’s properties will

determine what it can be used

for (applications) and the

performance of the final device.

At the CORE of this tetrahedron

is material characterization

Performance is the ultimate end use function

of the material and is resulted from properly

tuning properties of materials by optimizing

the structure down to the atomic level

through material processing (synthesis).

3

Introduction: this course

Introduces a broad range of advanced materials characterization

techniques─ Basic theory

─ Equipment and operation

─ Applications

These techniques study a wide range of material properties (thin film

and bulk)─ Chemical composition

─ Crystal structure

─ Surface and interfaces

─ Defects: point and extended defects

─ Morphology

─ Electrical, thermal and optical properties

At the end of the course, you should be able to─ Determine the appropriate method(s) to use

─ Understand experimental papers and determine if the data presented by

others are valid

4

Materials characterization techniques

We can broadly classify materials characterization techniques as

imaging and spectroscopic techniques

─ Imaging (microscopy): Optical microscopy, scanning electron

microscopy (SEM), Transmission Electron Microscope (TEM),

Scanning Tunneling Microscope (STM), Scanning probe microscopy

(SPM)

─ Spectroscopy/spectrometry: involves interaction of radiation

(radiative energy) with matter resulting in a plot of the response of

interest as a function of wavelength or frequency (energy)─spectrum:

Energy/Wavelength-Dispersive X-ray spectroscopy (EDX/WDX), X-

Ray Diffraction (XRD), Secondary Ion Mass Spectrometry (SIMS),

Electron Energy Loss Spectroscopy (EELS), Auger electron

spectroscopy (AES), X-ray photoelectron spectroscopy (XPS),

Ultraviolet-visible-near infrared spectroscopy (UV-vis-NIR),

spectroscopic ellipsometry, Photo/Cathodo-luminescence (PL/CL)

─ Electrical/thermal probes: Four point probe, Hall effect, Capacitance-

Voltage (CV), Thermal Power (Seeback)

5

Course Outline

Week Date Topic

1 8-30 Introduction (Chu)

2 9-06 Microscopy (I): Optical microscopy

3 9-13 Microscopy (II): SEM and Scanning probe

4 9-20 TEM & Electron probe microanalysis

5 9-27 X ray diffraction

6 10-04 Electrical measurements: four point probe, Hall, C-V, thermal

probe, minority carrier lifetime

7 10-11 Optical spectroscopies: spectrophotometry,

photoluminescence, spectroscopic ellipsometry, modulation

spectroscopy

8 10-18 Secondary Ion Mass Spectrometry (SIMS)

9 10-25 Auger Electron Spectroscopy (AES) (Chu)

10 11-01 X-ray Photoelectron Spectroscopy (XPS) (Chu)

11 11-08 Ion Beam techniques: RBS, PIXE, channeling

12 11-15 review

13 11-22 open

FINAL EXAM

6

Microscopy7

To provide a magnified image

To observe features that are beyond the resolution of the human

eyes (~100mm)

─ directly by light (optical) microscope using visible light

─ other microscopy imaging techniques use some other interaction probe

and response signal (usually electrons) to provide the contrast that

produces an image.

Able to “sense” depth

─ in the light microscope, topological contrast is provided largely by

shadowing in reflection,

─ in Scanning Electron Microscopy (SEM) secondary electrons are

generated from different depths, giving rise to topological contrast,

─ in Transmission Electron Microscopy (TEM), no depth information is

obtained.

For materials characterization, light Microscope is likely to be the

first imaging instrument to use

─ the cheapest “modern” instrument and take up the least physical space

Biomotor using ATP

DNA

~2 nm wide

The scale of things

The Microworld

0.1 nm

1 nan

om

eter (nm

)

0.01 mm

10 nm

0.1 mm

100 nm

1 micro

meter (m

m)

0.01 mm

10 mm

0.1 mm

100 mm

1 millim

eter (mm

)

0.01 m

1 cm

10 mm

0.1 m

100 mm

1 meter (m

)10

0m

10-1

m

10-2

m

10-3

m

10-4

m

10-5

m

10-6

m

10-7

m

10-8

m

10-9

m

10-10

m

Visiblespectrum

The Nanoworld

Cat~ 0.3 mMonarch butterfly

~ 0.1 mDust mite300 mm

Bee~ 15 mm

Human hair~ 50 mm wide

Magnetic domains garnet film

11 mm wide stripes

Red blood cellswith white cell

~ 2-5 mm

10

nm

Cell membrane

ATP synthaseSchematic, central core

Atoms of siliconspacing ~tenths of nm

Quantum corral of 48 iron atoms on copper surfacepositioned one at a time with an STM tip

Corral diameter 14 nm

Self-assembled “mushroom”

MEMS (MicroElectroMechanical Systems) Devices10 -100 mm wide

Red blood cellsPollen grain

Head of a pin1-2 mm

Microelectronics

Objects fashioned frommetals, ceramics, glasses, polymers ...

8

Optical vs. electron microscopy9

Microstructure of steel D2

(Metal Ravne Steel Selector)

Glass ceramic transmission microscope image

made with polarized light and full wave plate

Exfoliated molybdenum

disulfide on a perforated gridOptical Microscope

Electron Microscope

Atomic resolution TEM image of

nanocrystalline palladium. H. Rösner and C.

Kübel et al., Acta Mat., 2011, 59, 7380-7387.

Cross-section TEM image of MOCVD

grown InGaAs/GaAs quantum dot

superlattice solar cell (NREL)SEM micrographs of SMNb0.05%

Mat. Res. vol.6 no.2 São Carlos

Apr./June 2003.

Scanning electron microscopy (SEM) image

of as-grown p-type gallium nitride (p-GaN)

nanowire arrays on a silicon (111) substrate

Optical (light) microscopy

Introduction

Basic principles

─ Lens formula, Image formation and Magnification

─ Resolution and lens defects

Basic components and their functions

Common modes of analysis: bright field and dark field

Specialized Microscopy Techniques

─ Polarized light microscopy

─ Phase contrast microscopy

─ Differential interference contrast (DIC) microscopy

─ Fluorescence microscopy

─ Scanning confocal optical microscopy (SCOM)

Typical examples of applications

http://micro.magnet.fsu.edu/primer

http://www.doitpoms.ac.uk/tlplib/optical-microscopy/index.php

10

Introduction

An optical microscope:

uses visible light as the illumination source,

has lateral resolution down to 0.1mm (typically a few mm),

can be used for almost all solids and liquid crystals,

is typically nondestructive; sample preparation may involve

material removal,

is mainly used for direct visual observation; preliminary

observation for final characterization with applications in

geology, medicine, materials research and engineering,

industries, etc.

Typical microstructural features observed in materials science

are grains, precipitates, inclusions, pores, whiskers, defects,

twin boundaries, etc.

http://www.youtube.com/watch?v=bGBgABLEV4g&feature=endscreen&NR=1 using a microscope

11

Light microscope: parts12

Base – Base supports the microscope

which is horseshoe shaped

Illuminator - This is the light source located

below the specimen.

Iris diaphragm - Regulates the amount of

light into the condenser.

Condenser - Focuses the ray of light

through the specimen.

Stage - The fixed stage is a horizontal

platform that holds the specimen.

Nosepiece - The portion of the body that

holds the objectives over the stage.

Objective - The lens that is directly above the stage.

Coarse focusing knob - Used to make relatively wide focusing adjustments to

the microscope.

Fine focusing knob - Used to make relatively small adjustments to the

microscope.

Body - The microscope body.

Ocular eyepiece - Lens on the top of the body tube. It has a magnification of 10×

normal vision.

Light microscopes: then and now

http://www.youtube.com/watch?annotation_id=annotation_100990&feature=iv&src_vid=L6d3zD2LtSI&v=ntPjuUMdXbghttp://www.youtube.com/watch?v=X-w98KA8UqU&feature=related

http://www.youtube.com/watch?v=sCYX_XQgnSA&feature=related <2min

http://www.youtube.com/watch?v=1k659rtLrhk <2min

13

Basic concepts14

Magnification: the ratio of the size of an object seen under the microscope

to the actual size observed with unaided eye.

─ The total magnification of a microscope is calculated by multiplying the

magnifying power of the objective lens by that of eye piece

Resolving power: the ability to differentiate two close points as separate

─ The resolving power of human eye is 0.25 mm

─ The light microscope can separate dots that are 0.25µm apart.

─ The electron microscope can separate dots that are 0.5nm apart

Limit of resolution (resolving power) : is the minimum distance

between two points to identify them separately.

Abbe diffraction limit:

resolving power, RP=𝑤𝑎𝑣𝑒𝑙𝑒𝑛𝑔𝑡ℎ 𝑜𝑓 𝑙𝑖𝑔ℎ𝑡

2 × 𝑛𝑢𝑚𝑒𝑟𝑖𝑐𝑎𝑙 𝑎𝑝𝑒𝑟𝑡𝑢𝑟𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑜𝑏𝑗𝑒𝑐𝑡𝑖𝑣𝑒 𝑙𝑒𝑛𝑠

Numerical aperture(NA): 𝑁𝐴 = 𝑛 sin 𝜃, where

n is the index of refraction of the medium, 𝜃 is the

maximal half-angle of the cone of light that can

enter or exit the lens

Focal length

Numerical Aperture (NA)

http://www.youtube.com/watch?v=RSKB0J1sRnU

oil immersion objective use in microscope at~0:33

𝑁𝐴 = 𝑛(sin )

Immersion oil

n=1.515

Air

n=1.0

Imaging Medium

15

Basic concept: Absorption16

When light passes through an object the intensity is reduced

depending upon the color absorbed. Thus the selective absorption of

white light produces colored light.

The Beer–Lambert law relates the attenuation of light to the

properties of the material through which the light is traveling.

𝐼𝑡(𝜆) = 𝐼𝑜(𝜆)𝑒− 𝛼(𝜆)𝑥

where 𝐼𝑡(𝜆)𝑎𝑛𝑑 𝐼𝑜(𝜆) are the transmitted and incident light intensity and

wavelength 𝜆, respectively, 𝛼(𝜆) is the absorption coefficient of the material

and 𝑥 is the thickness of the material.

The absorbance of an object quantifies

how much of the incident light is

absorbed by it. It is a measure of

attenuation. Absorbance of a

material, denoted A, is given by

𝐴 = 𝑙𝑜𝑔10𝐼𝑜𝐼𝑡

Basic concept: Refraction17

The bending of a wave when it enters a medium where its speed is

different due to difference in the index of refraction (densities).

A ray from less to more dense medium is bent toward the surface

normal, with greater deviation for shorter wavelengths.

The index of refraction is defined as the speed of light in vacuum

divided by the speed of light in the medium: 𝑛 =𝑐

𝑣≥ 1, where c and v

are the speed of light in vacuum and in the medium, respectively.

Snell's Law relates the indices of

refraction n of the two media to the

directions of propagation in terms of

the angles to the normal.

𝑛1 sin 𝜃1 = 𝑛2 sin 𝜃2

𝑛1

𝑛2=

sin 𝜃2

sin 𝜃1=

𝑣2

𝑣1

Basic concept: Refraction

http://micro.magnet.fsu.edu/primer/java/refraction/refractionangles/index.html

http://www.youtube.com/watch?v=jQDRNb-E-cY ~1.00–2:20

18

Material n

Vacuum 1.0000

Air 1.000277

Ice 1.31

Water 1.333

Ethyl alcohol 1.362

Lucite 1.47

Glass 1.52

Polystyrene 1.59

Diamond 2.417

Basic concept: Diffraction19

Diffraction manifests itself in the apparent bending of waves

around small obstacles and the spreading out of waves past

small openings.

Diffraction provides a powerful tool for studying the geometry of

objects that are too small to be viewed directly.

Will be covered in more detail when we talk about x-ray diffraction

Basic concept: Dispersion20

Chromatic dispersion is the change of index of refraction with

wavelength.

─ Generally the index decreases as wavelength increases (𝒏 ↓ 𝝀 ↑)

─ Blue light (~400 nm) travels slower in the material than red light (~700nm).

Dispersion is the phenomenon which gives you the separation of colors

in a prism. It also gives the generally undesirable chromatic

aberration in lenses.

http://hyperphysics.phy-astr.gsu.edu/hbase/hph.html

Light microscope: a little history21

In 1590 F.H Janssen & Z. Janssen

constructed the first simple compound light

microscope.

In 1665 Robert Hooke developed a first

laboratory compound microscope.

Later, Kepler and Galileo developed a modern

class room microscope.

In 1672 Leeuwenhoek developed a simple

microscope with a magnification of 275x. He is

mistakenly called "the inventor of the microscope“

In 1880 Abbe and Zeiss developed oil immersion

systems and were able to make the a Numeric

Aperture (N.A.) to the maximum of 1.4 allowing

light microscopes to resolve two points distanced

only 0.2 microns apart.

Remember that: RP=𝜆

2×𝑁𝐴

A typical light microscope22

Basics: focusing by a curved surface

In entering an optically more dense medium (𝑛𝟐 > 𝑛𝟏), rays are bent

toward the normal to the interface at the point of incidence.

Curved (converging) glass surface

F - focal point f – focal length

normal

F

f

𝑛𝟐𝑛𝟏

Focal plane

Air

23

Basics: principal focal length24

For a thin double convex lens, refraction acts to focus all parallel

rays to a point referred to as the principal focal point.

The distance from the lens to that point is the principal focal length

f of the lens

For a double concave lens where the rays are diverged, the

principal focal length is the distance at which the back-projected rays

would come together and it is given a negative sign.

The lens strength in diopters is defined as the inverse of the focal

length in meters.

𝑃(𝑑𝑖𝑜𝑝𝑡𝑒𝑟) =1

𝑓(𝑚)

http://hyperphysics.phy-astr.gsu.edu/hbase/geoopt/foclen.html

Basics: converging (Bi-Convex) lens25

Most lenses are spherical lenses: their two surfaces are parts of the surfaces

of spheres.

A lens is biconvex (or double convex, or just convex) if both surfaces are

convex.

The line joining the centers of the spheres making up the lens surfaces is

called the axis of the lens.

If the lens is biconvex, a collimated beam of light passing through the lens

converges to a spot (a focus) behind the lens─a positive or converging lens.

𝒇 is the focal length of the lens,

𝒏 is the refractive index of the lens

material,

𝑹𝟏 is the radius of curvature (with sign)

of the lens surface closest to the light

source,

𝑹𝟐 is the radius of curvature of the lens

surface farthest from the light source,

𝒅 is the thickness of the lens (the

distance along the lens axis between

the two surface vertices)

+ve-ve

Basics: Lensmaker’s equation26

1

𝑓= (𝑛 − 1)

1

𝑅1−

1

𝑅2+

𝑛 − 1 𝑑

𝑛𝑅1𝑅2

The reciprocal of the focal length,

1/𝑓, is the optical power of the

lens. If the focal length is in meters,

this gives the optical power in

diopters (inverse meters)

For a thin lens where 𝑑 ≪ 𝑅1and 𝑅2:1

𝑓≈ (𝑛 − 1)

1

𝑅1−

1

𝑅2

For a thin bi-convex lens with equal curvatures:1

𝑓≈

2(𝑛−1)

𝑅

http://www.youtube.com/watch?v=R-uMcngNsSk converging (convex) lens<6:10

http://www.youtube.com/watch?v=KYrsmzM9I_8 diverging (concave) lens

http://www.youtube.com/watch?v=Am5wJUEiNAI how it’s made: optical lenses

Image formation27

For a lens of negligible thickness, in air, the distances are

related by the thin lens formula

1

𝑆1+

1

𝑆2=

1

𝑓or

1

𝑖+

1

𝑜=

1

𝑓

http://www.youtube.com/watch?v=-k1NNIOzjFo&feature=related to~3:42

Principal ray

F

F

𝑜 𝑖

Magnification: angular28

The standard close focus distance is

taken as 25 cm

A simple magnifier achieves angular

magnification by permitting the

placement of the object closer to the eye

than the eye could normally focus.

The angular magnification is given

by: 𝑀𝛼=𝛼′

𝛼

http://hyperphysics.phy-astr.gsu.edu/hbase/geoopt/simmag.html

For small angles: 𝛼′

𝛼≈

ℎ′

25ℎ

25

=ℎ′

ℎ=

25

𝑜; using the lens formula:

25

𝑜=

25

𝑓+

25

25

𝛼′

𝛼≈

25

𝑓+ 1; angular magnification 𝑀𝛼=

25

𝑓+ 1

(when the image is at the close focus point of 25 cm)

𝑆1

−𝑆2i

Our eyes are most relaxed while focus at infinity, i.e. 𝑖 = ∞

𝑀𝛼=25

𝑓

A large magnification requires a lens with a small focal length

Magnification: Linear29

The linear magnification or transverse magnification is the ratio of the

image size to the object size. If the image and object are in the same

medium it is just the image distance divided by the object distance.

ℎ𝑜

ℎ𝑖

𝑜 𝑖

𝑀 =ℎ𝑖ℎ𝑜

=−𝑖

𝑜(𝑜𝑟

−𝑆2𝑆1

)

A negative sign is used on the linear magnification equation as a reminder that all

real images are inverted.

𝑠1 𝑠1′

𝑓𝑜

𝑓𝑒

Compound microscope30

A compound microscope uses a very short focal length objective lens to

form a greatly enlarged image. This image is then viewed with a short focal

length eyepiece (Ocular) used as a simple magnifier.

Magnification by the objective 𝑚0 = 𝑠1′/𝑠1

Since 𝑠1′ 𝐿 and 𝑠1 𝑓𝑜, therefore magnification of objective 𝑚𝑜 = −

𝐿

𝑓𝑜

Magnification of the eyepiece 𝑚𝑒 =25

𝑓𝑒(assuming the final image forms at )

Overall magnification 𝑀 = 𝑚𝑜𝑚𝑒 = −𝐿

𝑓𝑜

25

𝑓𝑒

http://www.youtube.com/watch?v=kcyF4kLKQTQ at~1:57

http://www.youtube.com/watch?v=RKA8_mif6-E

Microscope resolution31

The resolution of an optical microscope is

defined as the shortest distance between two

points on a specimen that can still be

distinguished by the observer as separate

entities.

The diffraction pattern resulting from a uniformly-

illuminated circular aperture has a bright region

in the center, known as the Airy disk, which

together with the series of concentric bright rings

around is called the Airy pattern.

The lens' circular aperture is analogous to a two-

dimensional version of the single-slit

experiment. Light passing through the lens

interferes with itself creating a ring-shape Airy

pattern

The limit of resolution of a microscope objective

refers to its ability to distinguish between two

closely spaced Airy disks in the diffraction

pattern.

𝐬𝐢𝐧 = /𝒅

Resolution– Rayleigh Criteria32

The angular resolution of an optical system can be

estimated (from the diameter of the aperture and the

wavelength of the light) by the Rayleigh criterion: Two

point sources are regarded as just resolved when the

principal diffraction maximum of one image coincides

with the first minimum of the other.

𝜃 = 1.22𝜆

𝑑

θ is the angular resolution (radians),

λ is the wavelength of light, and

d is the diameter of the lens'

aperture.

The factor 1.22 is derived from a

calculation of the position of the

first dark circular ring surrounding

the central Airy disc of the

diffraction pattern

Resolution –Linear separation

To express the resolution in terms of a linear

separation r, we have to consider the

Abbe’s theory

Path difference between the two beams

passing the two slits is

𝑑 sin 𝑖 + 𝑑 sin 𝛼 = 𝜆

Assuming that the two beams are just

collected by the objective, then i = and

𝑑𝑚𝑖𝑛 = /2sin

If the space between the specimen and the

objective is filled with a medium of refractive

index n, then wavelength in medium 𝜆𝑛 =𝜆

𝑛

𝑑𝑚𝑖𝑛 =

2𝑛 sin=

𝜆

2(𝑁𝐴)

𝑁𝐴 = 𝑛 sin 𝛼 𝑖𝑠 called numerical aperture

For circular aperture: 𝑑𝑚𝑖𝑛 =1.22𝜆

2(𝑁𝐴)=

0.61𝜆

(𝑁𝐴)

33

𝑑𝑚𝑖𝑛 ~0.3 𝜇𝑚 for a mid-

spectrum of 0.55mm

Axial resolution – Depth of Field34

Another important aspect to resolution is the axial (or longitudinal)

resolving power of an objective, which is measured parallel to the optical

axis and is most often referred to as depth of field.

Depth of field (F in mm) is the range of

distance at the specimen parallel to the

illuminating beam in which the object

appears to be in focus

Depth of focus (f in mm) is the range of

distance at the image plane in which an

object appears to be in focus

Depth of focus varies with numerical

aperture (NA) and magnification (M) of

the objective

─ high NA systems have deeper focus

depths but lower depth of field

M NA f F

M NA f F

http://www.youtube.com/watch?v=FvC2WLUqEug at~3:40

http://micro.magnet.fsu.edu/primer/java/nuaperture/index.html

Optical aberrations35

Aberration Character Correction

Spherical Monochromatic, on-and -off axis, image blur

Bending, high index, aspherics, gradient index, doublet

Coma Monochromatic, off-axis only, blur Bending, spaced doublet with central stop

Oblique astigmatism

Monochromatic, off-axis, blur Spaced doublet with stop

Curvature of field

Monochromatic, off-axis Spaced doublet

distortion Monochromatic, off-axis Spaced doublet with stop

chromatic Heterochromatic, on- and off-axis, blur Contact doublet, spaced doublet

The influences which cause

different rays to converge to

different points are called

aberrations

Aberrations reduce resolution

of a microscope

http://hyperphysics.phy-astr.gsu.edu/hbase/geoopt/aberrcon.html

Spherical aberration For lenses made with spherical surfaces, rays which are parallel to the optic

axis but at different distances from the optic axis fail to converge to the same point

The image appears hazy or blur and slightly out of focus.

For a single lens, it can be minimized by bending the lens into its best form.

For multiple lenses, spherical aberrations can be canceled by overcorrecting some elements.

36

The focal length depends on refraction and the index of refraction 𝑛 for blue light (short wavelengths) is larger than that of red light (long wavelengths)

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.

Chromatic Aberration37

Achromatic doublet─a strong positive lens made from a low dispersion

glass like crown glass coupled with a weaker negative high dispersion glass

like flint glass calculated to match the focal lengths

An achromatic doublet does not completely eliminate chromatic aberration,

but can eliminate it for two colors

Astigmatism occurs when rays travelling along two perpendicular planes have different image distances for a sharp focus

─ The off-axis image of a specimen point appears as a disc or blurred lines instead of a point.

Comatic aberration occurs due to imperfection in the lens or other components resulting in off-axis point sources such as stars appearing distorted, appearing to have a tail (coma) like a comet.

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

Aberrations due to lens imperfection38

Light microscope: basic components and functions

1. Eyepiece (ocular lens)

2. Revolving nose piece (to hold

multiple objective lenses)

3. Objective lenses

4. Focus knobs coarse

5. Focus knobs Fine

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)

39

Functions of the Major Parts

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

40

Major Parts: objective lensObjective: does the main part of magnification and resolves the

fine details on the samples (𝑚𝑜~10 – 100)

Objectives are the most important components of a light microscope:

they are responsible for image formation, magnification, the quality

of images and the resolution of the microscope

𝑑𝑚𝑖𝑛 = 0.61𝜆/𝑁𝐴

41

Eyepieces (Oculars) work in combination with microscope

objectives to further magnify the intermediate image

Major parts: eyepiece Lens

𝑴 = (𝑳/𝒇𝒐)(𝟐𝟓/𝒇𝒆)

Eyepiece: is a cylinder containing two or more lenses; its function is to

bring the image into focus for the eye with typical magnification up to 20x

42

Common light microscopes

Transmitted OM -transparent specimens

─ thin section of rocks,

minerals and single

crystals

Reflected OM - opaque

specimens

─ most metals, ceramics,

semiconductors

Depending on the nature

of samples, either a

transmitted or reflected

optical microscope can be

used

43

Common modes of analysisInstead of using the full illumination of the light source (bright field), it is

sometimes useful to illuminate the sample with peripheral light by blocking the

axial rays─dark field microscopy. This produces the classic appearance of a

dark, almost black, background with bright objects on it

Advantages:

─ A simple procedure which can be used

on live transparent specimens

─ The images appear spectacular and

are visually impressive.

─ Even allows for the visualization of

objects that are below (!) the

resolution of the microscope.

Disadvantages:

─ very sensitive to dirt and dust located

in the light path

─ not suitable for all specimens

─ The intensity of the illumination

system must be high

44

Polarized Light Microscopy45

Polarized light microscopy involves

illumination of the sample with polarized

light.

Designed for specimens that are visible

primarily due to their optically anisotropic

character.

Image contrast arises from the interaction

of plane-polarized light with a

birefringent (or doubly-refracting)

specimen to produce two individual wave

components that are each polarized in

mutually perpendicular planes.

The light components are recombined

with constructive and destructive

interference when they pass through the

analyzer.

Reveals detailed information concerning

the structure and composition of

materials

Phase contrast microscopy46

Phase contrast microscopy uses a special condenser and objective lenses to convert phase differences (not visible) into amplitude differences (visible)

The image contrast is improved in two steps:

─ The background light is phase-shifted by −90° by passing it through a phase-shift ring, leading to an increased intensity between foreground and background

─ To further increase contrast, the background is dimmed by a gray filter ring

Differential interference contrast microscopy Differential interference contrast (DIC) microscopy enhances contrast by creating

artificial shadows (pseudo three-dimensional) using polarized light as if the

object is illuminated from the side

Differential interference contrast converts gradients in specimen optical path

length into amplitude differences that can be visualized as improved contrast

in the resulting image.

47

Blue-green Algae

Phase contrast DIC

The specimen optical path difference is determined by the product of the

refractive index difference (between the specimen and its surrounding medium)

and the geometrical distance (thickness) traversed by a light beam between two

points on the optical path.

Fluorescence microscopy48

A fluorescence microscope uses fluorescence to generate an image

The specimen is illuminated with light of a specific wavelength (or wavelengths) which is absorbed by the fluorophores, causing them to emit light of longer wavelengths (i.e., of a different color than the absorbed light).

The illumination light is separated from the much weaker emitted fluorescence through the use of a spectral emission filter.

Only allows observation of the specific structures which have been labeled for fluorescence

Fluorescent micrograph of an amphibian cell during anaphase

when the chromatids comprising each chromosome disjoin

and move towards their respective poles

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 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.

49

Some examples of

applications in materials

science

50

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 Etching1200C/30min

20mm

1200C/2h

51

Grain size examination

Objective Lens

x100

Reflected OM

52

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

30mm5mm

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Liquid phase sintering – reflective OM

Microstructure of MgO-2% kaolin body resulting

from reactive-liquid phase sintering.

Amorphousphase

40mm

54

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).

55

Phase identification by reflective polarized OM

YBa2Cu307-x superconductor material: (a) tetragonal phase and (b) orthorhombic phase with multiple twinning (arrowed) (100 x).

56

Internet resources57

http://www.youtube.com/watch?v=4RijnutOU4o

http://micro.magnet.fsu.edu/primer/java/aberrations/astigmatism/index.html

http://www.youtube.com/watch?v=yQ4rDNOX7So at~3:27-4:15

Astigmatism

http://www.youtube.com/watch?v=EXmaY2txEBo&list=PL02D1D436A44B521A&index=4

http://micro.magnet.fsu.edu/primer/java/aberrations/coma/index.html

Comatic aberration

http://www.youtube.com/watch?v=E85FZ7WLvao

http://micro.magnet.fsu.edu/primer/java/aberrations/spherical/index.html

http://www.youtube.com/watch?v=MKNFW0YwDYw -Canon lens production

Spherical aberration

Chromatic aberration

http://www.youtube.com/watch?v=yH7rbRu7Av8&list=PL02D1D436A44B521A chromatic aberration

http://www.youtube.com/watch?v=H8PQ9RMUoA8 at~3:30-4:30

http://micro.magnet.fsu.edu/primer/java/aberrations/curvatureoffield/index.html

Curvature of field

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&feature=iv&src_vid=L6d3zD2

LtSI&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

58

Do review problems (1-9) on OM

http://www.doitpoms.ac.uk/tlplib/optical-microscopy/questions.php

59