OPTICAL MICROSCOPY

ELECTRON MICROSCOPY

CHARACTERISATIONS OF MATERIALS

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.

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

Current Finding

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 "de-

magnification"

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.