Diffraction is the spreading out of waves as they pass through an aperture or around

objects. It occurs when the size of the aperture or obstacle is of the same order of
magnitude as the wavelength of the incident wave. For very small aperture sizes, the
vast majority of the wave is blocked.

D sintheta=nlambda

d = distance between slits

theta = diffraction angle

n = order number for the maximum

lambd = wavelength
a

Refraction
The change in direction of light, when it travels from one medium to another medium is
called refraction of light.

N1(sintheta1)=N2(sintheta 2)

Magnification

Magnification is the ability of a microscope to produce an image of an object at a scale

larger (or even smaller) than its actual size. T

slideject with the unaided

The two important properties of objective lenses are magnification and numerical aperture NA are
usually engraved on the barrel of objective lenses.

Magnification alone cannot assist in revealing detail unless the system also possesses resolving
power.

--------

Resolution :

slide
The resolution is defined as the distance between two closely spaced points in the
subject that can be made observable; thus, resolution is, expressed as a linear measure, is
better the smaller the distance between the points. Attainable resolution is established by the
properties of the objective lens, the wavelength of the illumination, and the index of refraction of
the medium between the objective lens and the specimen.

The degree of resolution is dependent upon the amount of diffraction information that can be
gathered by the objective lens. This factor is most conveniently expressed in terms of the
numerical aperture (N.A.) of the objective lens. The N.A. of an objective lens is given by NA= n sin
mu, where n is the index of refraction of the medium between the specimen and the lens (for air, n
= 1), and m is the half-angle of the cone of light capable of being accepted by the objective .

Resolution (R) is dependent upon the wavelength of light (l) illuminating the specimen,
and the N.A. of the objective lens: R= lambda/ 2 NA. The larger the numerical aperture
and/or the shorter the wavelength of light, the finer the detail that can be resolved. This
expression for resolution is used expressly for reflected- light microscopy, where the objective lens
carries light to the specimen, then acts again to collect light reflected from the specimen.

Depeth of focus:

Both slides.

Depth of focus is a property of an objective lens related to its numerical aperture. The depth of
focus is the dis- tance normal to the specimen surface that is within accep- table focus when the
microscope is precisely focused on the specimen surface. Depth of focus is of concern when the
specimen surface is not perfectly flat and perpendicular to the optic axis. In practice, the
preparation of specimens to obtain flatness, or the depth of etching, become impor- tant factors in
the utility of a given objective lens. Based on a criterion devised by Rayleigh, the depth of focus is
2
given by d nl=ðN:A:Þ , where d is the depth of focus, n is the index of refraction for the medium
occupying the space between the objective lens and the specimen, l is the wavelength of
illumination, and N.A. is the numerical aperture

Thus, for a 0.65 N.A. objective operating in air with illu- mination of 530-nm wavelength, the depth
of focus is 2.35 times the wavelength of light. This means that the surface of the specimen must be
flat to within 4.7 times the wavelength of light or, in this case, 2500 nm.
Simplified schematic diagrams of transmitted- and reflected-light microscopes are given in Figure
3.

the light path passes through the instrument starting with the source of illumination. In this
sequence Illumination of the specimen is accomplished by two or more components including the
lamp and a condenser or collector lens.

Sometimes, the aperture diaphragm, field diaphragm, heat-absorbing filter, narrow band-pass
filter, and neutral-density filter are also included as components of the illumination system.
 Proper cantering and focusing of the light from the illuminator is done which is essential to
obtaining satisfactory results with any optical microscope.

The image of the lamp filament or arc must be focused at the plane of the aperture diaphragm

(In some instruments, the light path is sufficiently open so that one can visually inspect the focus of
the lamp on a closed-aperture diaphragm. In closed systems, specific instructions for obtaining
proper focus are usually provided with the instrument.)

Cantering of the light source is accomplished by adjustments that laterally translate the lamp in
two directions.

A focusing adjustment moves the lamp closer to or further away from the condenser to focus the
lamp at the plane of the aperture diaphragm.

The aperture diaphragm must be properly adjusted to obtain the optimum resolution
commensurate with image contrast, for either a transmission- or reflected-light micro- scope.

The aperture diaphragm is inter- posed in the optical path to control the diameter of the pencil of
light so that it just fills the full circular area of the rear lens group of the objective lens.

In a transmitted-light microscope the aperture diaphragm is located in the condenser lens barrel,
while in a reflected-light microscope the aperture diaphragm is located in the optical path just
beyond the condenser (collector) lens of the light source. In both instruments, the image of the
diaphragm is focused in a plane coincident with the rear element of the objective lens. The
aperture diaphragm is always par- tially closed to reduce internal light scattering within the
microscope, a circumstance that degrades contrast in the image.

In practice, the method for obtaining the optimum setting of the aperture diaphragm is to open the
dia- phragm fully, bring the specimen into focus, and then close the diaphragm slowly until the
image begins to dim per- ceptibly. At this point the aperture is opened slightly to bring the image
back to full brightness.

The image of the field diaphragm is focused on a plane coincident with the plane of the specimen.
Its function is to reduce extraneous light scatter between the specimen sur- face and the front
element of the objective in order to enhance contrast.

To adjust the field diaphragm, one starts with it in the full-open position and gradually closes it
while viewing the focused specimen either through the eyepieces or on a photographic viewing
screen. When the image of the diaphragm is observed to just intrude into the field of view or into
the photographic field, it should be opened slightly.

Vertical illuminators are required for reflected-light microscopes, because the specimens are
opaque and the objective lens must act both to supply light to, and to col- lect reflected light from,
the specimen.

The light path must be passed to the specimen through the objective lens, then allow the reflected
image-forming light to pass back through the objective to the ocular. From Figure 3A, note that the
light path is diverted 908 from the horizontal to the specimen, which rests inverted on the stage
(inverted microscope).

This arrangement is of particular utility in using specimens that must have prepared sur- faces,
because it does not require that the specimen have a back surface exactly parallel to the prepared
surface.

Generally, vertical illuminators have no adjustment con- trols except for a provision for dark field
illumination, a topic that is discussed in REFLECTED-LIGHT OPTICAL MICRO- SCOPY.

The objective lens requires only two adjustments: cen- tering the optic axis of the lens with the
optic axis of the instrument and focusing the lens by moving it closer to or further away from the
specimen. Focusing the objective is as simple as moving the focusing adjustments while viewing
the specimen through the eyepieces and observing when the specimen appears to be sharp.

PRACTICAL ASPECTS OF THE METHOD Reflected-light Microscopes

The reflected-light microscope, shown by the light-path diagram in Figure 1, contains a vertical
illuminator, in this case employing a half-mirror to direct light to the

specimen and to allow light from the objective lens to pass to the ocular (eyepiece). In
arrangement of the speci- men stage, with respect to the axis of the horizontal incom- ing light
path, there are two types of microscopes—upright and inverted. Generally, the inverted instrument
is prefer- red because it ensures that a specimen surface is always normal to the optic axis of the
objective lens. Further, it allows for easier observation of large unmounted speci- mens and
specimens of irregular shape that have a prep- ared surface

Vertical illuminators have taken several forms during their evolution. For the most part, two types
of vertical illuminators are in use today; the plain-glass reflector and the half-mirror reflector. The
plain-glass reflector con- sists of a very thin, optically flat glass plate inclined at 458 with respect to
the path of the incoming illumination (Fig. 2). Part of the illumination is reflected at 908 through
the objective to illuminate the specimen. A portion of the light that returns from the specimen
through the objective lens passes through the glass plate on to the ocular. Note that the objective
first acts as an illuminating (condenser) lens and second as the image-forming lens.

When the plain-glass reflector is properly centered, plain axial illu- mination is the result and no
shadows of specimen surface relief are formed. While the loss of light intensity is signif- icant, this
type of reflector can be used with any objective. The half-mirror reflector is a totally reflecting
surface, usually silvered glass, with a thin clear coated glass area in its center. The clear central
area is elliptical in shape, so that when the reflecting plate is placed at 45 deg in the optical path a
round beam of light can be reflected .

An advantage of this type of reflector is that it is particu- larly well suited for dark-field illumination,
and it also works well with bright-field illumination and polarized light (see the discussion below
under ‘‘Illumination Modes and Image-Enhancement Techniques’’ for a description of these
illumination modes). Light rays reflected back through the objective lens from the specimen
surface pass through the central clear region of the reflector to form an aerial image that is further
magnified by the ocu- lar. Because the reflecting surface reflects all of the inci- dent light, there is
no loss of light intensity as with the plane glass reflector (Restivo, 1992). Figure 3 shows the light
path for the half-mirror reflector as it is used in dark-field illumination mode. Other vertical
illuminator reflector schemes have been used with some success; how- ever, they have fallen out
of favor because of limitations or expense in producing suitable reflectors.

number of variations in illumination modes and image enhancement techniques may be included
as bright-field illumination, oblique-light illumination, dark-field illumi- nation, polarized-light,
sensitive-tint, phase-contrast, and differential-interference-contrast.

Bright-field illumination is the most widely used mode of illumination for metal specimens that
have been prep- ared by suitable polishing and etching. The incident illu- mination to the specimen
surface is on axis with the optic axis through the objective lens, and is normal to the specimen
surface. As expected, those surfaces of the specimen that are normal or nearly so to the optic axis
reflect light back through the objective lens, giving rise to bright portions of the image. Those
features of the microstructure that have surfaces that are not normal to the optic axis appear dark
and thus give the image con- trast (Fig. 4). Image contrast also occurs in bright-field illumination
because of variations in reflectivity within a specimen.

For example, in properly prepared specimens of irons and steels that contain MnS (manganese
sulfide)

as a microconstituent, the MnS constituent appears some- what darker than the surrounding a-
ferrite iron, and pos- sibly lighter than Fe3C (cementite). The image contrast in this case is due to
the optical properties of the constituents that render their polished surfaces more or less reflective.
In fact, if the specimen preparation is very well done, the MnS appears to have a smooth dark-slate
color.

Dark-field illumination is a useful technique to enhance image contrast by making the normally
bright areas of an image dark while making dark features light. Dark-field illumination is effective
because of the perception charac- teristics of human vision. It has been demonstrated that a small
white disc on a black background is more easily perceived than a small black disc on a white
background. The size limit of the white disc on the black background depends entirely on its
reflected luminous flux, or the total light reflected per unit area (Gifkins, 1970).

Dark-field illumination occurs when the light incident on the specimen arrives at a low angle. If the
surface is fea- tureless, reflected light does not reenter the objective lens and the image is dark.
However, any feature on the surface that is inclined a relatively small angle can reflect light back
through the objective lens to form an image. To effect dark-field illumination, a small circular stop
is inserted in the light path ahead of the vertical illuminator to produce an annulus of light larger in
inside diameter than the back element of the objective lens. The objective lens is sur- rounded by a
‘‘shaped’’ or ‘‘figured’’ transparent tube that directs the incident light to the specimen at a
relatively low angle to the specimen surface. Reflected light from tilted features of the specimen
reflects light back into the objective lens (Fig. 6). Figures 7 A and B are bright-field and dark-field
photomicrographs, respectively, of a speci- men of low-carbon iron. Note that the grain boundaries
and inclusions are enhanced in dark-field.

--------------------------

1. Mention the factors that influence the resolution of optical

microscope.

Answer:
Numerical aperture
The numerical aperture (NA) is related to the refractive index (n) of a medium through
which light passes as well as the angular aperture (α) of a given objective (NA = n
sinα).
The resolution of an optical microscope is not solely dependent on the NA of an objective,
but the NA of the whole system

Abbe’s diffraction limit,

Abbe recognized that specimen images are composed of a multitude of overlapping, multi-
intensity, diffraction-limited points (or Airy discs).

In order to increase the resolution, d = λ/(2NA), the specimen must be viewed using either a
shorter wavelength (λ) of light or through an imaging medium with a relatively high refractive
index or

with optical components which have a high NA (or, indeed, a combination of all of these factors).

Airy discs
An Airy disc is the optimally focused point of light which can be determined by a circular
aperture in a perfectly aligned system limited by diffraction. Viewed from above (Figure
1), this appears as a bright point of light around which are concentric rings or ripples
(more correctly known as an Airy Pattern).
Rayleigh criterion
The Rayleigh criterion defines the limit of resolution in a diffraction-limited system, in
other words, when two points of light are distinguishable or resolved from each other. e
Rayleigh criterion.

Full Width at Half Maximum (FWHM)

The theoretical value for the FWHM is R FWHM = 0.51λ/(NA) which is approximately λ/(2NA).
So the FWHM as a resolution parameter is very close to Abbe’s diffraction limit

What is The Picture Element,

Picture element commonly known as a Pixel, is the smallest unit of

a digital image that can be displayed or manipulated. Pixels are
significant in digital imaging and display technologies because they

dDark e

Dark field illumination.

1. Dark-field illumination occurs when the light incident on the specimen arrives at a LOW
ANGLE
2. If the surface is featureless, reflected light does not reenter the objective lens and the
image is dark. However, any feature on the surface that is inclined at a relatively
small angle can reflect light back through the objective lens to form an image.
3. Dark-field illumination is a useful technique to enhance image contrast by making the
normally bright areas of an image dark while making dark features light.

Bright Field illumination

1. The incident illumination to the specimen surface is on axis with the optic axis
through the objective lens, and is normal to the specimen surface.
2. As expected, those surfaces of the specimen that are normal or nearly so to the optic axis
reflect light back through the objective lens, giving rise to bright portions of the image.
Those features of the microstructure that have surfaces that are not normal to the optic
axis appear dark and thus give the image contrast.

Polarization

A light wave that is vibrating in more than one plane is known as

unpolarized light. This electromagnetic vibrations can occur in numerous
planes plane polarized light consists of waves in which the direction of
vibration is the same for all waves.
The process of transforming unpolarized light into polarized light is known
as polarization. The devices like the polarizers you see are used for the
polarization of light.

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 (usually in culture),
microorganisms, thin tissue slices, lithographic patterns, fibres, latex dispersions, glass
fragments, and subcellular particles (including nuclei and other organelles).

Bri

Bb

f the