CHARACTERISATIONS OF

MATERIALS

OPTICAL
MICROSCOPY

ELECTRON MICROSCOPY

CONTENTS
1.0 Introduction and History
1.1 Brief Review of Light Physics
1.2 Characteristic Information
2.0 Basic Principles
2.1 Ray Optics of the Optical Microscope
2.2 Summary

TOOLS TO SEE SMALL MATTERS

A GLANCE OF HISTORY IN OPTICS

Medieval Islamic Contribution in optics
The Islamic contribution to the science of optics within the medieval
Islamic World should be measured not by the number of practitioners,
which was small, but the quality of the contributions, which was great

Linberg

Contribution of Muslim Scholars in the

theory of optics
Yaqub Ibn Ishaq Ibn Sabah Al-Kindi (c 801 873)
One of the earliest optic scientist
His theory of the active power of rays, stating that
luminous object emits ray in every direction, influence
several European Scientist

Hunyn Ibn Ishaq (Isaac)

Contemporary and Neighbour to Al Kindi
He wrote ten Treatise on the Eye and claimed that
sensitive organ of the eye is the crystalline lens located
in the center of the eye.

Contribution of Muslim Scholars in the theory of

optics
Abu Sad Al Alla Ibn Sahl
Excelled in optics, Author of a treatise on Burning
Mirrors and Lenses
Wrote his textbook in 984 where he set out his
understanding of how curves mirrors and lenses bend
and focus light,
R. Rasheed credited Ibn Sahl with discovering the law
of reflection usually called Snells Law

Abu Ali Hasan Ibn Haitham ( c. 965 1039)

Known as Alhazen, Born in Basra
The field of optics reached its peak with ibn Hytham
He rejected Aristotles theory (384 322 B.C) claiming
that there is difference between the laws governing
events on earth and those pertaining to celestial bodies.

Ibn Hytham Attempts and Achievements

Human Eye
Cornea, retina, vitreous humor are among names given by
him
He was able to identify the eye layers with great precision
and to define his lens system as comprising of the aques
and the vitreous humours and the lens.

Current Finding

Normal viewing distance -250 mm

Angular resolution min 1
Spatial resolution hmin 80 m
Nodal distance -17 mm
Average retinal cell distance 1.5 m
Spectral range 400 nm -800 nm
Can resolve contrast about 5%
High dynamic range 10 decades
Max sensitivity at 505 nm (night, rods)
Max sensitivity at 555 nm (day, cones)
More sensitive to color than to intensity

Most perfect
sensor for
light detection
up to now;
It is the
greatest
creation by
Allah SWT

Ibn Hytham Attempts and Achievements

Light Dispersion
He carried out the first experiments of light into its
constituents colours.
Made the first experiment to disperse light, to break white
light into its constituent colours
He realised that each band in the resulting multicolours
beam had been refracted at measurable angles and each
colours always occur at the same angle.

The angle of deviation

Ibn Hytham Attempts and Achievements

Camera Obscura
The first camera in history, shuttered room with a narrow
aperture that admits light, Which the idea propogate to
microscope after certain time.

Refraction theory
He studied a phenomenon in which light rays bend when
travelling from one medium to another
The effect causes an object to appear to be in a location
other than where it actually is.
He contended that magnification was due to refraction.
He made the link between glass curvature and
magnification.
He is then credited with discovering that the magnifying
effect take places at the surface of the optical elements
rather than within it.

Microscope and Its Working-Science

MAIN ISSUES OF MICROSCOPY

In order to observe small objects, three preconditions have to be
fulfilled

Magnification
Resolution
Microscopy Resolution and Magnification
Microscopy Field of View (FOV)

Contrast
Only fulfillment of these three conditions allows translation of
information as accurately as possible from object into an image which
represents that object.

Microscopy Resolution and Magnification

Microscopy Field of View

MAGNIFICATION
Magnification is the process

of enlarging something only

in
appearance,
not
in
physical
size.
This
enlargement is quantified by
a calculated number also
called "magnification". When
this number is less than one,
it refers to a reduction in size,
sometimes
called
"minification"
or
"demagnification"

Calculating The Magnification Of Optical Systems

Single lens: The linear magnification of a thin lens is

where f is the focal length and do is the distance from the lens to the

object. Note that for real images, M is negative and the image M is
inverted. For virtual images, is positive and the image is
upright.With di being the distance from the lens to the image, the hi
and ho height of the image and the height of the object, the
magnification can also be written as:

Magnification of microscope
Microscope: The angular magnification is given by

where Mo is the magnification of the objective and Me the

magnification of the eyepiece. The magnification of the objective

depends on its focal length fo and on the distance d between objective
back focal plane and the focal plane of the eyepiece (called the tube
length)

RESOLUTION
The resolution of a microscope is
the ability to clearly determine two
separate points, or objects, as
singular, distinguished entities. If the
object are closer together than
appropriate for your resolution, they
blur together, making it impossible
to differentiate. Use the resolving
power of the lens on the microscope
to adjust the resolution. Resolution
is not magnification. Magnification is
a microscope's ability to increase
size -- it does not improve clarity.
Magnification also utilizes lenses,
but if the resolving power is poor,
increasing
magnification
only
magnifies a blurry specimen.

Calculating Resolutions
Maximum resolution:

R = (0.61 X )/ N.A
where:
0.61 is a geometrical term, based on the average 20-20
eye,
= wavelength of illumination,
N.A. = Numerical Aperture,
The N.A. is a measure of the light gathering capabilities of an objective
lens.

N.A. = n sin ,
where:
n = index of refraction of medium, = < subtended by the lens

Factors affecting resolution

Resolution (dmin) improves (smaller dmin) if or n or
Assuming that sin = 0.95 ( = 71.8)

(The eye is more sensitive to blue than violet)

CONTRAST
Contrast is defined as the difference in light intensity between the

image and the adjacent background relative to the overall background

intensity. In general, a minimum contrast value of 0.02 (2 percent) is
needed by the human eye to distinguish differences between the image
and its background

Calculating Contrast
Contrast produced in the specimen by the absorption of light, brightness,

reflectance, birefringence, light scattering, diffraction, fluorescence, or color

variations has been the classical means of imaging specimens in brightfield
microscopy. The ability of a detail to stand out against the background or other
adjacent details is a measure of specimen contrast. In terms of a simple
formula, contrast can be described as :
Percent Contrast (C) = ((I(s) - I(b)) x 100)/I(b)
Where I(b) is the intensity of the background and

I(s) is the specimen intensity.

From this equation, it is evident that specimen contrast refers to the
relationship between the highest and lowest intensity in the image.

Factor affecting contrast

The graph shown illustrates the

effect of background intensity on

contrast. When the background is a
very dark gray color (I(b)equals
0.01), a small change in image
intensity produces a large change
in contrast. By lightening the
background to a somewhat lighter
gray color (I(b) equals 0.10), small
changes in image intensity provide
a useful range of contrast. At still
lighter background colors (I(b) >
0.20), image contrast is relatively
insensitive to background intensity
and large changes in I(b) produce
only small increases or decreases
in image contrast.

DEFECTS IN LENSES

Spherical Aberration
Peripheral rays and axial rays have different focal points.
This causes the image to appear hazy or blurred and slightly out of focus.
This is very important in terms of the resolution of the lens because it
affects the coincident imaging of points along the optical axis and degrades
the performance of the lens

DEFECT IN LENSES
Chromatic Aberration
Axial - Blue light is refracted to the greatest extent followed by green and
red light, a phenomenon commonly referred to as dispersion
Lateral - chromatic difference of magnification: the blue image of a detail
was slightly larger than the green image or the red image in white light,
thus causing color ringing of specimen details at the outer regions of the
field of view
A converging lens can be combined with a weaker diverging lens, so that
the chromatic aberrations cancel for certain wavelengths:
The combination achromatic doublet

DEFECT IN LENSES
Astigmatism - The off-axis image of a specimen point appears as a disc or
blurred lines instead of a point.
Depending on the angle of the off-axis rays entering the lens, the line
image may be oriented either tangentially or radially

DEPTH OF FOCUS
We also need to consider the depth of focus (vertical resolution). This is
the ability to produce a sharp image from a non-flat surface.

Depth of Focus is increased by inserting the objective aperture (just an

iris that cuts down on light entering the objective lens). However, this
decreases resolution.

SUMMARY
1.

All microscopes are similar in the way lenses work and they all suffer
from the same limitations and problems.

2.

Magnification is a function of the number of lenses. Resolution is a

function of the ability of a lens to gather light.

3.

Apertures can be used to affect resolution and depth of field if you

know how they affect the light that enters the lens.