SHRI SHANKARACHARYA MAHAVIDYALAYA

JUNWANI, BHILAT.

MICROSCOPY

Dr. RACHNA CHOUDHARY

 MICROSCOPY

INTRODUCTION
HISTORY
PRINCIPLE
FACTORS OF MICROSCOPY
TYPES OF MICROSCOPY
>» LIGHT MICROSCOPY
A. BRIGHT-FIELD MICROSCOPY
B. DARK-FIELD MICROSCOPY
C. FLUORESCENCE MICROSCOPY
D. PHASE-CONTRAST MICROSCOPY
» ELECTRON MICROSCOPY
A. TRANSMISSION MICROSCOPY
B. SCANNING MICROSCOPY
APPLICATIONS
CONCLUSION
REFERENCE
 MICROSCOPY

Microscopy is the science which deals “with the use of microscope

instrument that magnifies the size of the object & interpretation of
their magnified images”.

Microscope is the technology of making very small things visible to

the human eye. Therefore, microscope is a major tool of the
microbiologist & biotechnologist.

At the magnification of 1,000x most of the microorganisms, e.g.:-

Fungi, Bacteria, Mycoplasma, algae, & protozoa can be viewed &
this can be achieved with a light microscope.
 Antony Van Leeuwenhoek (1676)
discovered the microbial world through
the use of a single microscope containing
a single biconvex lens of shorter focal
length.
Anthony van Leeuwenhoek
Ne

Robert Hooke point the ‘cell’ built 1632-1723

microscopes with two lenses called
compound microscope.
COA

The Dutch spectacle-maker, Zaccharias

Jansen, is also. credited with the
development of compound light
KA

microscope.

Zacharias Jansen
In 1830, many improvements were made 1588-1631
by Joseph Lister which resulted in the
development of many types’ of
microscopes that are being used now a
days.
 MICROSCOPY

¢ Microscopy is the most important instrument of any biological

laboratory. Used to magnifying the size of the object.

¢ By the help of resolution provided by this instrument very small

organisms or substances are magnified and made visible through
the naked eyes.

¢ Thus, microscopy is the major tool for microbiologist &

biotechnologist.
 MICROSCOPY
Conti..

1. Magnification
* The primary of a microscope is magnification that is the power of
enlargement of image of an object.
* Its is the ratio of size of image to the size of object.
Image distance
Magnification= _—_--------------------
object distance

* Three types of objective lens of different magnification are used:

a. Low power- 10x
b. High power- 45x
c. Oil emulsion- 100x

2. Contrast
* It refers to difference in light intensity in order to the perceived to the
microscope, an object must possess a certain degree of contrast with its
surrounding medium.
 MICROSCOPY
! . esoluto0on TLLLLLLAL

¢ The resolving power of a microscope is the to distinguish two adjacent

point as separate and distinct rather then a blurred image.

¢ The greater the resolving power of the microscope the more detailed
can be see in the specimen.

* Resolving power of microscope is determined by three factors:

1. Size of the objective lens.

li. Wavelength of light passing through the specimen.

iii. The refractive index of the material between the objective lens
and the specimen.
 MICROSCOPY

1. LIGHT MICROSCOPY

Bright-Field light microscopy

0a > Dark-Field light microscopy
Fluorescence light microscopy
Phase-contrast light microscopy

2. ELECTRON MICROSCOPY

A. Transmission electron microscopy

B. Scanning electron microscopy
 MICROSCOPY
(A)

LIGHT MICROSCOPY

eyepiece
(ocular) |
Light as source of (B)
illumination

Glass lenses
objective |

Limited resolution specimen

a

(loses __ resolving condenser

power at \VVi/
light
source /jj1\\

Fig-1 light microscope

 MICROSCOPY
A. BRIGHT FIELD MICROSCOPY

PRINCIPLE
* Bright field use the visible light as the source of illumination.

* Light microscope with a single lens are called simple microscope.

* Compound microscope with the two lens system the objective lens place near specimen
and the occular lens or eye piece located next to the eye.
Eye
Per part of Bright-field microscope fap
an itis bn
A broad base \ 7
= Curved arm X
* Adjustable light source or mirror
= Fine and core adjustable nose =>— Sa ae
= Body tube Kf
= Stage or platform See
* Diaphragm \
= Condenser Sp Condenser lens
* Ocular lens \ /
= Eye piece VY
» There are three types of eye piece Light
a) Huggensian eye piece .

c) Compensating
eye piece

10
 A. BRIGHT FIELD MICROSCOPY Conti..

ie
Y » The object to be viewed with the compound light microscope is normally
P placed on a glass slide and illuminated with a light source. The specimen is
E focused by moving the ocular lens & objective lens together relative to the
S specimen until the image is clear.
O
F » When the specimen has been focused the objective lens magnifies the
M specimen & produces earlier image.
I
C » The real image is projected to the microscope to the ocular lens which
R magnifies the real image and produces an image seen by the observer and
O called as virtual image.
S
C » The resolving power of the lens system is important in microscopy because it
P indicates the size of the smallest object that can be seen clearly.
Y
» Resolving power varies for each objective lens and depends on-
1. Wavelength of light used in a optical system,
2. Numerical aperture.
11
 MICROSCOPY
A. BRIGHT FIELD MICROSCOPY Conti...

NUMERICAL APPARATURE

« It is the light gathering capacity of the objective lens.

«= It is a measurement of the angle of maximum cone of light that enter the objective.
NA = n Sin@
where, N = refractive index of medium
sin 8 = one and half angle created by light passing through condenser and specimen
& transmitted to object.
o Incase of dry air
NA= |
o Incase of oi] medium
NA= 1.33
« The greater the NA the greater is the resolving power.

LIMIT OF RESOLUTION

«= The smallest distance by which two points can be separated & distinguished as two separate
objects.
Resolution = A

2NA
* Higher resolution obtained with shorter wavelength & maximum NA.
12
 MICROSCOPY
B. DARK-FIELDMICROSCOPY

* In dark field microscopy the background |

remains dark & only the objects Deu C>
illuminated. It is opposite to that of - -
Bright-field microscopy in which
specimen appears darker against light
background. fy
Objective lens )/~ Only light reflected
= Dark-field microscopy operates on the 4 the specimenis
principle of scattering which means a ray Specimen , ea hey
of light changes direction or scatters i
when it strikes & bounces off a small _ \Unrefiected light
object. Condenser lens ——<_>
’ 4
7?
Opaque
disc
* In this a special kind of condenser with an 1
opaque disc or “dark field stop” is of
provided. Thus, the light rays reach the Light
object in the form of the hollow cone.
Fig-3 dark-field microscopy

13
 MICROSCOPY
Conti...

B. DARK-FIELD MICROSCOPY
« The disc block the light that could enter the objective directly & redirects the light
beam so that it goes to the specimen but misses the objective lens.

« The only light rays that enters objective lens & reach the eyes are those that have been
scattered by striking the specimen.

* In tis way specimens appears bright against a dark microscopic field.

APPLICATION

= Helps in examining movement of motile cells, live microorganisms that are

either invisible in the ordinary light microscope

= In diagnostic of microorganisms.

14
 MICROSCOPY
C. FLUORESCENCE MICROSCOPY

= The fluorescence microscopy differs from the Bright-field microscopy as

it uses a mercury vapour arc lamp or a halogen instead of the
incandescent lamp.

PRINCIPLE
# The principle of fluorescence microscopy is a diagnostic technique
called the fluorescent-antibody. Antibodies are natural defense
molecule that are produced by humans and many animals in
reaction to a foreign substance or antigen.

TYPES OF FLUORESCENCE

1. Auto fluorescence - collagen fibers (blue green light).

1. Secondary fluorescence - commonly used dye Congo red, eosin.

1. Induced fluorescence — some substances on treatment with some

chemical shows fluorescence.
15
 MICROSCOPY
COMPONENTS & Conti...
INSTRUMENTATION re

1. LIGHT SOURCE — Mercury arc lamp,

ultraviolet light of — shorter ioe emission fiter
wavelength.
————s
2. HEAT FILTER — Removes infrared dichroic miror,
rays. 3
lignt source

3. EXITER FILTER —- Allow only - saa

required wavelength to pass through =
and block others. Ty

4. DICHRONIC MIRROR - Divide & . ,

divert the beam, reflect light of specimen
certain wavelength but transmit other. ‘7
Fig-3 fluorescence microscopy
5. CONDENSOR -— Dark field condenser
is provide black background against
which the fluorescent object glow. 16
 MICROSCOPY
Conti...

C. FLUORESCENCE MICROSCOPY
6. BARRIER FILTER — Remove exited wavelength

APPLICATION

Detection various material e.g. protein can be detected by staining

with rod amine.

Banding pattern of chromosome.

Fluorescent antibody technique or Immuno-fluorescent.

17
 MICROSCOPY

D. PHASE CONTRAST MICROSCOPY

Phase-contrast microscopy is based on the principle that rays of light

move at different speed through materials of different refractive index.

The phase contrast microscope amplifies the slight difference in

refractive index of the cell and that of its aqueous environment and
converts it to a difference in contrast.

The phase-contrast microscope consists of special condensers and

objectives that enable one to increase the contrast between the
transparent components in the cell by exploiting differences in their
densities.

>» PRINCIPLE

= Phase contrast microscopy is used for studying living cell to

convert the invisible small phase changes caused by cell
component into visible intensity changes.
18
 MICROSCOPY
Conti...
D. PHASE-CONTRAST MICROSOPY E
ye
= When light rays passed through the living f\ Ocular lens
cells they undergo phase changes due to -
different refractive indices & thickness of ¥ Diffraction plate
cell organelles \| light

= When light rays are passed through cell }\ (unaltered

by specimen)
organelle, they are transmitted at a Objective lens
velocity inversely proportional — to ¥ Refracted or diffracted
refractive index of the cell organelle. Cell J light (altered by
organelle are of different refractive specimen)
indices. j
{7}~ ’ Specimen
= The light rays emerging would show » 4. ‘Condenser lens
variable phase changes. — "a!
\ i

« This invisible phase changes re converted ¥ Annular diaphragm

into visible intensity changes by phase- Light
contrast microscopy.
Fig4-phase-contrast microscopy

19
 MICROSCOPY
Conti...
D. PHASE-CONTRAST MICROSCOPY

= The more the refractive index & thickness the more will be the
change in the phase.

" The cells and their component show phase changes value of
phase change is one- fourth of light. This phase change is
imperceptible to the human eye.

« The principle behind the phase-contrast microscopy is to convert

the imperceptible phase change.

CONSTRUCTION

= It is specially designed light microscope with annular

diaphragm and annular phase plate fitted into it.
 MICROSCOPY
Conti...
D. PHASE-CONTRAST MICROSCOPY

They unable to increase the contrast between the transparent into

the ell by exploiting differences in their density.

Annular diaphragm or annular stock is a disc with a thin

transparent wing at a lower focal plane of the condenser.

It consist of a circular disc with a circular grove through where

light rays are allowed to pass.

WORKING

Light rays pass through the annular group of the annular

diaphragm.

This rays are focused on the on the object.

From the object two types of rays immersed out. one is refracted
rays which under goes a phase change. And other is central rays
which does not under goes any phase —change.

21
 MICROSCOPY
Conti...
D. PHASE-CONTRAST MICROSCOPY

= The refracted rays are band due to refraction in density and refractive
index with in the specimen and get refracted by about one-fourth
wavelength.

IMAGE FORMATION

= Depending on the type of phase plate used image formation takes

place is of two types-

Image formation by positive contrast

¢ Formed by subtractive super position of central & diffracted rays.

* The object appears dark against the light background also called as
dark positive contrast.
 MICROSCOPY

D. PHASE-CONTRAST MICROSCOPY Cont

Image formation by negative contrast

* Formed by the super position of the central & diffracted rays.

* The specimen appear bright against dark background that may

called as bright phase contrast.

* In this negative plate is used.

ADVANTAGES

= We can see living cells and there is no need for staining.

= Highly transparent material can be seen.

* Intracellular component can be observed, e.g. endospores.

 MICROSCOPY

ELECRTON MICROSCOPY

= The electron microscope is an optical instrument which utilizes electrons as

a source of illumination for observing objects at a great magnification.

= It can achieve a very high power of resolution because it uses electrons of

much shorter wavelength.

= Use electromagnetic lenses to focus a beam of electrons onto a

specimen.

» This required 10,000x plus magnification.

 MICROSCOPY
Conti..
ELECTRON MICROSCOPY
PROPERTIES OF ELECTRON BEAM
* The electron get absorb by the atoms of specimen instead low energy
electron are emitted which are called secondary electron. Useful for
forming image in scanning electron microscope.

= Electrons are emitted when electric current is passed through the tungsten
filament. The wavelength of electron is based on the voltage of electric
current. It can be calculated by de-Broglie formulae:

N=/ 4123
Angstroms
Vv
= According to this formulae if
v = 50,000v then A of electron would be 0.053 A
At 10,000 v wavelength will be 0.035A.
 MICROSCOPY
TRANSMISSION ELECTRON MICROSCOPY

> PRINCIPLE

= Higher magnification and higher resolution.

= The wavelength of electron one lakh time shorter then that of light
rays.

= Image is produced on fluorescence on photographic plate.

= Instead of mounting the specimen on a glass slide it held on a

proper way.

= Instead of using light to that absorb lig ht, to increase contrast

tungsten which absorbs electron are used.
 MICROSCOPY
CONSTRUCTION Conti...

1 ELECTRON GUN TRANSMISSION ELECTRON

G 5 MICROSCOPE
« It consist of a hot tungsten filament. It is the source
of electrons forming the beam.
Ptectronr aun

2. ELECTROMAGNETIC LENS
* The electromagnetic lens corresponds to the
condenser, objective lens and ocular lens. Condenser

3. MICROSCOPE COLUMN . |
* Itconsist of an evacuated metal tube. Objectve “ens

4. FLUORESCENCE SCREEN fF |
* Since electron are harmful to our eyes magnified CW i Projector “ions”
image observed from fluorescence screen. Nees 5

5. VACUUM PUMP
* Electron are reflected by collision air molecule.

6. TRANSFORMERS Viewng binoculars _ Flunrescem screen

* It provide high voltage from 220v to 50-100 kV.

7. WATER COOLING SYSTEM Fig 5- transmission electron microscopy

= Required to prevent over heating of different part 4
of microscope. 27
 MICROSCOPY
TRANSMISSION ELECTRON MICROSCOPY Conti...

WORKING

= The electron gun generate electron beam , thin tungsten filament.

= Electrons are in the form of collimated beam passes to the condenser coil & fall
on the object.
= They get scattered & transmitted to the object & pass through the objective coil
which magnifies the image of the object.
= The projector coil further magnify the image & thus final image is formed on the
fluorescence screen.
= Dense region in the specimen scatter is more and therefore appear darken in the
image where as in contrast, electron transparent regions are brighter.

MAGNIFICATION

# 1,60,000x - 10,00,000x

APPLICATION
# It provides sufficient magnification and resolution to view viruses and the
internal structures of all organisms.
 MICROSCOPY
SCANNING ELECTRON MICROSCOPY

= This microscopes gives a typical three-

dimensional appearance.

= The illuminating system of SEM is similar

to transmission electron microscopy.

> PRINCIPLE

= It differs from TEM introducing an image

from electron emitted by object surface
rather than from transmitted electron
microscopy.

= It consist of an electron gun which

produces a finely focus beam of electron
called the primary electron beam.

=" This electron passes through Fig 6- scanning electron microscopy

electromagnetic lens & rapidly scan the
surface of specimen.
 MICROSCOPY
B. SCANNING ELECTRON MICROSCOPY Conti...
= When the beam of electron strikes the specimen secondary electrons are released
and transmitted to the electron collector.

= Secondary electrons are collected and use to generate a signal that produces an
image on cathode screen.

= It has a resolution of about 50A.

scanned lamp on the screen

magnification can be given by Se

scanned lamp on the specimen surface

APPLICATION

= The scanning electron microscope has a wide scope in biology for

the study of small specimens, surface scanning of the cells, tissues
and membrane.
 MICROSCOPY

In medical microbiology for detecting pathogenic bacteria.

For the detection of various types of products of microorganisms.

It helps in examining the movement of motile cells.

Shows greater differentiation of internal structure and clearly shows

the pellicle.

By the use of electron microscopy structures smaller than 0.2

micrometer can be resolved.

By the use of fluorescence microscopy rapidly detection and

identification of microbes can be done.
 MICROSCOPY

The microorganisms are so small that their study requires appropriate

methods for observing and culturing them.

Microscopy is the technology of making very small things visible to

the human eye.

Therefore, microscope is a major tool of the microbiologist &

biotechnologist

Historically, it was the microscope that first revealed the secrets of

microbia | structure, even today, it remains a powerful tool in
microbiological studies.
 MICROSCOPY
BOOK AUTHORS bara EDITION

THE BIOLOGY OF 2010

MICROORGANISM
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ATEXT BOOKOF R.C DUBEY 2008

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