OPTICAL MICROSCOPY

INTRODUCTION:
An instrument used to see small objects with naked eye is known as a microscope. There
are small things which we cannot directly see with our naked eye and in that case we use the
help of this microscope to see them.There are many types of microscopes like electron
microscope etc; , among them optical microscope is one of the types.

OPTICAL MICROSCOPE also known as light microscope is a type of microscope which uses
visible light and a system of lenses to generate image of small objects.This is the oldest type of
microscope (we are using this microscope in compound form from almost 17th century).

TYPES IN OPTICAL MICROSCOPE:

There are two types of microscopes: 1) simple, 2) compound.

Simple microscope: It has only one lens, used to obtain small magnifications.

Shown above is a simple microscope. Main parts are biconvex lens, stand slide and
plano-concave mirror.It magnifies around 10 times.A simple microscope is used to obtain small
magnifications. It is usually used for study of microscopic algae, fungi and biological specimens.
Working Principle:Light from a light source (mirror) passes through a thin transparent object
(Figure 4.2). A biconvex lens magnifies the size of the object to get an enlarged virtual image
which is viewed from the other side.

Ray diagram of a simple microscope

Compound microscope: It consists of a system of lenses used to magnify the image.It can
magnify an image upto 1000 times.
 The picture shown above is a compound microscope.
Components of a compound microscope are divided into two parts
1) Mechanical
2) Optical
Mechanical parts:
1) Base or metal stand
2) Pillars
3) Inclination joints
4) Curved arm
5) Body tube
6) Draw tube
7) Coarse adjustment
8) Fine adjustment
9) Stage
10) Mechanical stage
 11) Revolving nosepiece

Optical parts:
1) Light source
2) Diaphragm
3) Condenser
4) Objective
5) Eye piece

Working Principle:
The most commonly used microscope for general purposes is the standard compound
microscope. It magnifies the size of the object by a complex system of lens arrangement. It has
a series of two lenses; (i) the objective lens close to the object to be observed and (ii) the ocular
lens or eyepiece, through which the image is viewed by eye. Light from a light source (mirror or
electric lamp) passes through a thin transparent object .

Shown above is the ray diagram of a compound microscope

The objective lens produces a magnified ‘real image’ (first image) of the object. This image is
again magnified by the ocular lens (eyepiece) to obtain a magnified ‘virtual image’ (final image),
which can be seen by eye through the eyepiece.[ As light passes directly from the source to the
eye through the two lenses, the field of vision is brightly illuminated. That is why; it is a
bright-field microscope.]
Magnification, Resolution and their importance:
➔Magnification:

Magnification is the action of magnifying something or the process of being magnified.

Magnification of a simple microscope i.e., a thin lens,
m=hi/ho= -di/do
Magnification of a compound microscope will be the product of magnification caused by the
objective lens and the ocular(eyepiece).
𝑚𝑓𝑖𝑛𝑎𝑙 = 𝑚𝑜𝑏𝑗𝑒𝑐𝑡𝑖𝑣𝑒 × 𝑚𝑜𝑐𝑢𝑙𝑎r
Determining magnification is vital when comparing the sizes of different objects being viewed
with a microscope.
➔Resolution:
The process or capability of making distinguishable the individual parts of an object, closely
adjacent optical images, or sources of light is called resolution. It is the ability to distinguish
(resolve) two close together points as separate. The resolution of a microscope is as shown in
the figure:

Where,
dmin = minimum distance between point objects that can be resolved
λ = wavelength of the light source used
n = refractive index of the medium between the objective lens and the specimen
α = half of the objective angular aperture as shown in the figure
 NA = numerical aperture = nsinα
As it is clear from the definition of resolution, lower dmin implies higher resolution.
Resolution of a light microscope operating at the blue end of the visible spectrum will therefore
be higher than the operating at the red end, assuming all other parameters remain same. It is
evident that the resolution can be increased if the wavelength of the source radiation is reduced.
The higher the resolution, the larger an image file will be and on a screen, an image will
look sharper and clearer as there are more pixels to represent objects on the screen and it is
harder for your eyes to depict individual pixels (eventually impossible) which will make the
image look much better

Bright Field microscopy:

In a bright-field microscope, both diffracted (diffracted by the specimen) and undiffracted

(light that transmits through the sample undeviated) lights are collected by the objective lens.
The image of the specimen is therefore generated against a bright background; hence its name
is bright-field microscopy. Most biological samples are intrinsically transparent to the light
resulting in poor contrast. To increase the contrast of the image, the specimens are therefore
generally stained with the dyes. However, intrinsically colored samples such as erythrocytes can
directly be observed using bright-field microscopy.

The name “brightfield” is derived from the fact that the specimen is dark and contrasted
by the surrounding bright viewing field. Simple light microscopes are sometimes referred to as
brightfield microscopes.

It is best suited to viewing stained or naturally pigmented specimens such as stained

prepared slides of tissue sections or living photosynthetic organisms. It is useless for living
specimens of bacteria, and inferior for non-photosynthetic protists or metazoans, or unstained
cell suspensions or tissue sections. In bright field microscopy, the sample illumination is
transmitted white light and contrast in the sample is caused by absorbance of some of the
transmitted light in dense areas of the sample. It typically has low contrast with most biological
samples as few absorb light to a great extent. Staining is often required to increase contrast. It
is useful for samples which have an intrinsic colour, for example chloroplast in plant cell.
The light path consists of : a) a transillumination light source, commonly a halogen lamp in the
microscope stand; b) a condenser lens which focuses light from the light source onto the
sample and c) objective lens which collects light from the sample and magnifies it d) Oculars
and/or a camera to see the sample image
→How it works?:
In brightfield microscopy, a specimen is placed on the stage of the microscope and
incandescent light from the microscope’s light source is aimed at a lens beneath the specimen.
This lens is called a condenser. The condenser usually contains an aperture diaphragm to
control and focus light on the specimen; light passes through the specimen and then is collected
by an objective lens situated in a turret above the stage. The objective magnifies the light and
transmits it to an oracular lens or eyepiece and into the user’s eyes. Some of the light is
absorbed by stains, pigmentation or dense areas of the sample and this contrast allows you to
see the specimen. For good results with this microscopic technique, the microscope should
have a light source that can provide intense illumination necessary at high magnifications and
lower light levels for lower magnifications.

This technique can be used to view fixed specimens or live cells, Since many organic
specimens are transparent or opaque, staining is required to cause the contrast that allows
them to be visible under the microscope. Different stains and staining techniques are used
depending upon the type of specimen and cell structure being examined.
For example:
Fuchsin is used to stain smooth muscle cells
Methylene blue is used to stain cell nuclei
Gram-stain is used on bacteria and gives rise to the name gram-negative or gram-positive
bacteria based on the reaction of the bacterial to the stain. In fact, many scientific journals will
not accept microbiological research for publication that is not supported by gram staining and
brightfield illumination methodology. Most routine medical microscopic examination of blood and
tissue is performed using this illumination technique.

Advantages:

● Simplicity of setup with only basic equipment required.

● Living cells can be seen with bright-field microscopes.
● It is very simple to use with fewer adjustments needed to be made to view
specimens.
● Some specimens can be viewed without staining and the optics used in the
brightfield technique do not alter the colour of the specimen.
● It is adaptable with the new technology and optional pieces of equipment can be
implemented with brightfield illumination to give versatility in the tasks it can perform.
Disadvantages:

● Very low contrast of most biological samples.

 ● By using an aperture diagram for contrast, past a certain point, greater contrast adds
distortion. However, employing an iris diaphragm will help compensate for this
problem.
● Brightfield microscopy can’t be used to observe living specimens of bacteria,
although when using fixed specimens of bacteria, although when using fixed
specimens, bacteria have an optimum viewing magnification of 1000x.
● The practical limit to magnification with a light microscope is around 1300X. Although
higher magnifications are possible, it becomes increasingly difficult to maintain image
clarity as the magnification increases.
● Low apparent optical resolution due to the blur of out-of-focus material.
● Samples that are naturally colorless and transparent cannot be seen well, e.g. many
types of mammalian cells. These samples often have to be stained before viewing.
Samples that do have their own color can be seen without preparation, e.g. the
observation of cytoplasmic streaming in Chara cells.

Dark Field Microscopy:

Dark-field microscopy increases the contrast of the image by eliminating the

undiffracted light. The specimen is illuminated by the light coming from a ring at an
oblique angle. If there is no specimen in the optics path, no light is collected by the objective
lens. Presence of specimen results in the diffraction of light; the objective lens collects the
diffracted light generating a bright image against a dark background.
 condenser annulus

→Uses of darkfield microscopy:

• Diagnosis of Syphilis
• Viewing blood cells
• Viewing bacteria
• Viewing different types of algae
• Viewing hairline metal fracture
• Viewing diamonds and other precious stones •
Viewing shrimp or other invertebrates

Advantages:
● A dark field microscope is ideal for viewing unstained object, transparent and absorb
little or no light.
● These specimens often have similar refractive indices as their surroundings, making
them hard to distinguish with other illumination techniques.
 ● Dark field microscopes are used in research of live bacterium, as well as mounted cells
and tissues.
● It is useful in examining external details, such as outlined, edges and defects that
internal structures.
● It is used to study marine organisms such as algae, plankton, diatoms, insects, as well
as some minerals and crystals, thin polymers and some ceramics.
● It gas regained its popularity when combined with other illumination techniques, such as
fluorescence, which widens its possible employment in certain fields.

Disadvantages:

● A specimen that is not thin enough or its density differs across the slide, may appear to
have artefacts throughout the image.
● The preparation and quality of the slides can grossly affect the contrast and accuracy of
a dark field image.
● We need to take special care that the slide, stage, nose and light source are free from
small particles such as dust, as these will appear as part of the image.
● Also, if we need to use oil or water on the condenser or slide, it is impossible to avoid air
bubbles. These liquid bubbles will cause image degradation and distortion.

Applications:
Optical microscopy is used extensively in microelectronics, nanophysics, biotechnology,

pharmaceutical research, mineralogy and microbiology. Optical microscopy is used for medical
diagnosis, in the field termed histopathology where they deal with tissues, or in smear tests on
free cells or tissue fragments

Advantages and disadvantages(optical microscopy):

Advantages:

1. Light microscopes are by far less costly than atomic force and electron
microscopes.
2. In most cases, they are easier to use and need less training.
3. Most microscopic organisms are transparent to light.
 4. Fluorescence makes light microscopy very powerful: by staining different parts
of cells with different colors, one can study structure and function (for instance
if a protein is being expressed or not, as well as where it is localized).
Fluorescence has also enabled novel techniques like single-molecule
microscopy.

5. After centuries of development, variants of light microscopy have appeared that

fit most applications.

Disadvantages:

1. Diffraction imposes a limit on the maximal resolution that a microscope can

achieve. For light microscopes, that is roughly half of the wavelength that is
being detected. Suppose you are working with 500nm blue-greenish light, your
best achievable resolution will be of 250 nm. Many sub-cellular structures, as
well as viruses, proteins, and DNA strands are below this limit, thus may not be
studied with light microscopy (super resolution microscopy techniques have
overcome this issue impressively, but they are costly and complicated).
2. Light microscopes have even worse axial resolution, i.e. they are bad at
differentiating depths. Atomic force microscopy is amazingly better at doing
this, so it is used when topographies or volume-maps are needed.
3. Minerals and metals are usually opaque to light.

4. Typical light microscopes cannot measure properties of materials, they are

used just to image them. Atomic force microscopy, for example, can measure
rigidity in soft materials by pulling and pushing them.

Question and Answers:

Q.1)
The derivation for the thin lens equation is as below:
Let the focal length of the thin lens is f. The object distance BC is u and the image distance B’C
is v.
Now, from the image we can say that triangles ABC and A’B’C are similar. So,

Similarly, triangles OCF and A’B’F are similar.

So,

Because, OC = AB.
Now, we know that ℎ ′ = ℎ𝑣 /𝑢 , putting this in the above equation,
Which is the equation of the thin lens.
➔ If we want to increase the magnification of the image, and if the object and the
eyepiece are fixed, we should move the lens closer to the eyepiece or eye. But the condition is
that the distance between the object and the lens should always be lesser then the focal length
of the lens otherwise the image will not be visible.

Q.2)

Resolution means the shortest distance between two individual points that can be
distinguished.

We know that resolution (r) = 0. 61λ/𝑁𝐴

⇒ 0. 61 × 400 ×10-9÷𝑠𝑖𝑛(α)

⇒ 2 × 0. 61 ×400×10-9 = 0.345µm

dmin= 0.345µm

If the distance between two features is 0.5 micron, we cannot distinguish between them with a
microscope that has a resolution 0.75 micron. 0.75 micron resolution means it is the shortest
distance it can distinguish, it cannot distinguish between features less than 0.75. To distinguish
between features that are 0.5 micron apart the microscope should be a better resolver with
resolution less than or equal to 0.5 micron. Then only we can distinguish between them.

Q.3)

a) To identify the features on the specimen of alloy using optical microscope, we should
use reflection as a mode of imaging. Generally, the metal and their alloys are opaque. In
those cases, transmission of light is not possible through the specimen and transmitted
light microscope will typically be of little use to anyone wanting to examine the structure
of metallic samples, the surface of ceramics, integrated circuits, or printed paper
documents. As a result, the reflected light microscope has been developed for these
purposes.

b) Due to limitation in magnification of the optical microscope, if we can’t see the

features of the grain structures the we can use UV-microscopy, X-rays microscopy or
else electron microscopy which is very effective in case of resolution. This is because
the wavelength of these rays is lesser than the light rays, so the dmin will decrease and
so the resolution will increase as we go from UV-microscopy to electron microscopy.

c) Manufacturers of optical microscopes: kern, Keyence, OSAW, Nikon, Leica, Zeiss,

Olumpus, Omax, AmScope