MICROSCOPY

WHAT IS MICROSCOPY
Microscopy is an important component of diagnostic micro- biology.
Bacteria being very small cannot be visualized by the naked eye,
because the limit of resolution with the unaided eye is about 200
microns. So, the study of bacteria requires the use of microscopes. A
microscope is an instrument that uses one or more lenses to produce a
magnified image of an object that is invisible to the unaided eye.

MICROSCOPE

What is a compound microscope?

Real and magnified images of minuscule particles or objects can be
achieved using a combination of lenses. A compound microscope is
an intricate gathering of a combination of lenses that renders a highly
maximized and magnified image of microscopic living entities and
other complex details or tissues and cells.

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Diagram

Parts of Compound Microscope

The parts of the compound microscope can be categorized into:

 Mechanical parts
 Optical parts

(A) Mechanical Parts of a Compound Microscope

1. Foot or base
It is a U-shaped structure and supports the entire weight of the
compound microscope.
2. Pillar
It is a vertical projection. This stands by resting on the base and
supports the stage.

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3. Arm
The entire microscope is handled by a strong and curved structure
known as the arm.
4. Stage
The flat and rectangular plate that is connected to the arm’s lower end
is called the stage. The specimen is placed on the stage for studying
and examining the various features. The centre of the stage has a hole
through which light can pass.
5. Inclination joint
It is a joint, wherein the arm is fastened to the compound
microscope’s pillar. The microscope can be tilted using the inclination
joint.
6. Clips
The upper part of the stage is connected to two clips. The slide can be
held in its position with the help of the clips.
7. Diaphragm
The diaphragm is fastened below the stage. It controls and adjusts the
intensity of light that passes into the microscope. The diaphragm can
be of two types:

 Disc diaphragm
 Iris diaphragm

(B) Optical Parts of Compound Microscope

1. Eyepiece lens or Ocular
At the top of the body tube, a lens is planted which is known as the
eyepiece. On the rim of the eyepiece, there are certain markings such
as 5X, 10X, 15X, etc. These indicate the magnification power. The
object’s magnified image can be observed with the help of an
eyepiece.

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2. Mirror
A mirror is found attached wither to the pillar or the lower end of the
arm. It consists of a concave mirror on one side and a plain mirror on
the other side. It can be used for reflection of light rays into the
microscope.
3. Objective lenses
At the bottom of the body tube, there are two objective lenses, which
are connected to the revolving nose piece. The three objective lenses
are as follows:

 Oil immersion objective – 100X

 High power objective – 45X
 Low power objective – 10X

Working Mechanism of The Compound Microscope

 View into the eyepiece. Rearrange the mirror such that adequate
light passes into the microscope.
 The mirror, lenses, stage, and slides should be cleared of dust
and be clean.
 Place the slide in the middle of the stage.
 Firmly secure the slide with clips at two edges of the slide to
ensure that the slide cannot move.
 The nose piece is adjusted in such a way that the low power
objective is aligned with the object of focus placed on the slide.
 The coarse adjustment knob can be shifted upwards or
downwards such that the slide is well under focus.
 Turn the fine adjustment knob by moving upwards or
downwards to get a clear and sharp image of the object under
focus.
 All minute details of the object are observed under low power
objective. Necessary diagrams are sketched.
 The nose piece is now turned to bring the high-power objective
aligning with the object. The fine adjustment knob is tuned as
much as possible to get a bright and precise view of the object.

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Precautions

 The objective lenses and eyepiece should be cleaned with the

help of silk cloth and cleaning liquid before use.
 The microscope should not be tilted when working, using it.
 When an object needs to be studied, focus on the low power
objective first and then move to high power.
 The lower power needs to be left in place after all observations
are completed.
 When focusing, care needs to be taken to ensure that the
objective lens never strikes the stage or the slide.
 Only the fine adjustment knob should be used when the high-
power objective is employed.

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Dark Field Microscopy

Dark field microscopy is a type of microscopy technique that is used

in both light and electron microscopy, where only the specimen is lit
by a light or electron beam, and the rest of the specimen field is dark.

It’s also called dark ground microscopy, and it usually works as a

cheaper yet higher contrast and resolution alternative technique to
phase contrast microscopy, a type of optical microscopy technique
wherein the brightness of the specimen image is altered through phase
shifts.

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Bright Field Microscopy
It is defined as the optical microscopy, which is the simplest of all the
illumination techniques, wherein a smear (the stained or the dense
part appears darker with a white or brighter background). Bright-field
microscope is a compound light microscope, which illuminates the
background against a stained specimen. It is commonly used in the
practical labs to study organisms’ behaviour and characteristics like
size, shape and arrangement.

It is a type of light microscopy, where a path of light is very simple,

which requires a light source (like a halogen lamp), condenser lens,
objective lens and ocular lens. Bright-field microscopy can use either
critical or Koehler magnification or illumination system to add
contrast to the image.

Resolution

It is the optical lens’s power, which distinguishes between the two

particular bodies that held very close to each other. The highly
magnified image will not give better results as the picture becomes
gauzy. The resolving power increases, as the lines per unit area,
appears as distinctive lines.

When there is a small distance between the two distinct objects, the
resolving power can be best known. The resolution power can give
1000-1500 times magnified image. Resolving power of the
microscope decides the quality of the picture by the objective lens.
Higher is the magnification power; higher will be the resolution of
the microscope. The bright field microscope’s resolution is
represented as ‘r’ which is equal to the half of the
light wavelength by the numerical aperture

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Phase Contrast Microscope
Unstained living cells absorb practically no light. Poor light
absorption results in extremely small differences in the intensity
distribution in the image. This makes the cells barely, or not at all,
visible in a brightfield microscope. Phase-contrast microscopy is an
optical microscopy technique that converts phase shifts in the light
passing through a transparent specimen to brightness changes in the
image. It was first described in 1934 by Dutch physicist Frits Zernike.

Camera Captures the Impossible at a Trillion Frames Per Second

When light passes through cells, small phase shifts occur, which
are invisible to the human eye. In a phase-contrast microscope, these
phase shifts are converted into changes in amplitude, which can be
observed as differences in image contrast.

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Fluorescence Microscope
Fluorescence microscopy is a light microscope that works on the
principle of fluorescence. A substance is said to be fluorescent when
it absorbs the energy of invisible shorter wavelength radiation (such
as UV light) and emits longer wavelength radiation of visible light
(such as green or red light). This phenomenon, also called
fluorescence, is widely used in clinical and diagnostic settings to
detect microorganisms, antibodies, and many other substances rapidly.

Working mechanism of Fluorescence Microscope

Transmission Electron Microscope

The working principle of the Transmission Electron Microscope
(TEM) is similar to the light microscope. The major difference is that
light microscopes use light rays to focus and produce an image while
the TEM uses a beam of electrons to focus on the specimen, to
produce an image.

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Electrons have a shorter wavelength in comparison to light which has
a long wavelength. The mechanism of a light microscope is that an
increase in resolution power decreases the wavelength of the light, but
in the TEM, when the electron illuminates the specimen, the
resolution power increases increasing the wavelength of the electron
transmission. The wavelength of the electrons is about 0.005nm
which is 100,000X shorter than that of light, hence TEM has better
resolution than that of the light microscope, of about 1000times.
This can accurately be stated that the TEM can be used to detail the
internal structures of the smallest particles like a virion particle.

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