The document provides an overview of microscopes including their history, key inventors and developments. It discusses light microscopes and electron microscopes, describing their basic components and working principles. Key points covered include the earliest microscopes in the 13th century, Anton van Leeuwenhoek's contributions, the invention of the first electron microscope in 1932, and differences between transmission electron microscopes and scanning electron microscopes.

2. Greek: ‘mikro’ – small, ‘scopia’ – observation
Microscope: It is an instrument consisting
essentially of a lens or a combination of lenses,
designed to magnify very small objects such as
microorganisms, to look larger so that they can be
seen and studied.

3. 13th century
Leonardo da Vinci
Filimicroscopes (fili – insects)
Anton van Leeuwenhoek (1674):
 Father of microbiology
 300X
 247 microscopes
Robert Hooke (1665): Compound microscope
 Abbe: first modern light microscope

4. When light rays travelling in a medium enter into another
medium of a different optical density, they deviate slightly
from their path.

5. An image is a visual representation of an object

6. Magnification(size): ratio of the size of the focused image
produced by a lens, to the actual size of an object.
Resolving power(clarity): capability of the lens to show 2
different points/lines lying very close to each other as separate.
Contrast: difference between the brightness of various details in
the object, and the difference as compared with the background.
Sharpness: distinct, realistic image detail and contrast. A sharp
image requires minimal effort to interpret & examine.

7. R= 2 x n.sinα
λ
Where, R - resolution
λ - illumination wavelength
n - imaging medium refractive index
α - semi-angle of cone of light falling on the
. objective lens

8.  Discovered by Thompson (1897)
 Wave properties predicted by Louis-Victor de Brogile(1924)
 They are negatively charged subatomic particles
 Used as a source of illumination
 Wavelength = 0.5 A (EM)
 When electricity of high voltage is used to heat atoms of a metal,
electron velocity is accelerated, & electrons leave the orbit.

10. Optical instrument which utilizes electrons as a source of
illumination for observing objects at a great magnification.
The first EM was designed by M. Knoll & E. Ruska
(Germany, 1932). Later, Prebus & Miller of Belgium, made
some improvements to it.
In the year 1939, Siemens Halske, a Germany company,
manufactured EM for marketing. In 1941, Radio
Corporation of America, started manufacturing EM on a
large scale.

11. Electron gun
Microscope column
Electromagnetic lenses/coils
Fluorescent screen
Vacuum pumps
Water cooling system

12. It consists of an anode & cathode
Cathode: ‘V’ shaped tungsten filament. Used for Thermionic
emission. Temp. is directly proportional to emission.
Tungsten: 3000 C; Electricity: 40 to 100kV
It is covered with Wehnalt cylinder. It prevents dissipation
of electrons.
Anode: present a little away from Wehnalt cap. Kept at zero
volt current. The difference in voltage leads to acceleration
of electrons & is known as accelerating voltage.

14. Electron lens consists of a coil, consisting of a few
thousand turns of wire, with a current of about 1 amp
passing through it. It produces a radially symmetrical
magnetic field.
It is encased in a soft iron casing, which helps in
concentrating the magnetic field produced.
Magnetic field forces the electron to spiral around a
central axis. The electron beam passes through the
microscope column & gets deflected by a variable degree.
The strength of the electric current can be varied and the
focal length depends on it.

16. Lens system in EM:
1. Condenser lens system
2. Objective lens
3. Intermediate lens
4. Projector lens

17. As electrons are harmful to human eye, the magnified
image is formed on the fluorescent screen.
The screen is coated with a chemical which by its
excitation forms the image.

18.  Electrons cannot travel far in air (it collides with gas mol. in air).
Therefore, its entire path must be evacuated for which vacuum is
required and it is achieved with help of vacuum pumps.
 Standard rotary pump: used to develop an initial low vacuum
 Oil diffusion pump: used to create high vacuum for later operations
 Coldfinger: It consists of a metal that is cooled by liq. nitrogen.
 Air-lock

20. Image formation occurs by electron scattering. Electrons strike
the atomic nuclei & get dispersed, these dispersed electrons
form an image which is projected on the fluorescent screen.
Procedure:
 Electrons in the form of a collimated beam pass through the condenser
coil and strike the object.
 They get scattered and transmitted through the object & pass through
the objective coil, which magnifies the object.
 Projector coil further magnifies the image & projects it on the
fluorescent screen.

21. Image formation occurs when energy of electrons is
transformed into visible light through excitation of chemical
coating.
Electrons which reach the fluorescent screen form the bright
spots while the areas where electrons do not reach the screen
they form the dark spots. Areas which scatter electron are
known as electron dense.
Varying degree of intensity of electrons forms an image with
varying degrees of grey.

22. Objective & projector coils help in magnifying the image,
additionally an intermediate coil can also be fitted
between them to achieve maximum magnification
Ex: if magnification of objective coil is 100 & that of
projector is 200, a magnification of 20,000 can be achieved.
But with the help of intermediate coil a magnification of
1,60,000 can be achieved.
During microphotography this magnification can be
further increased upto 1,000,000 times without loss of
sharpness

23. Electrons have a much shorter wavelength as compared to
light, this concept led to the invention of EM.
At 100kV voltage wavelength of an electron will be
0.0037A, and resolution should be half of the wavelength
(theoretically) but in practice 4-10A resolving power is
achieved due to aberrations.

24. A defect in the image formation due to defective
construction of the microscope, faulty techniques, is called
an artefact.
Electromagnetic lenses
1. Spherical aberration: slight variation in magnetic field
2. Chromatic aberration: variation in voltage or magnetic
field leads to variation in wavelength
Electron beam: heating of section
Focusing: failure of focusing

25. Diamonds/glass knives
Ultramicrotome:
 sensitive instrument
 Binocular microscope
 Manually/semi-automatic operation
 Fluid reservoir (acetone/water)
 Sections are floated on water, picked on a perforated Cu grid (3mm
diameter). Grids are coated with a film (10-40nm thick). It is made
of parlodion (nitrocellulose), formvar (polyvinyl formal), &
polymerized plastic. These films are supported by a thin film of
carbon (10nm thick), layered by vacuum evaporation.

27. Transmission electron microscope (TEM)
Scanning electron microscope (SEM)

28. It is an imaging technique whereby a beam of electrons is
focused onto the specimen & the diffraction pattern caused by
different internal regions of specimen creates an enlarged
image on the fluorescent screen.
Here, electrons are allowed to be transmitted through the
object.
Albert Prebus & James Hilllier (University of Toronto, 1938)
JEOL, Hitachi, FEI Co., Philips and Carl Zeiss

29. Here, the electron beam is directed to scan the surface
features of the specimen so that the diffraction pattern
caused by the surface features creates the image.
C.W. Oatley (1965)
Used for studying surface structure of thick specimens.
Electron beam is compressed with the help of condenser
coils, forming a narrow electron probe.
Pri. Electrons in the form of beam strike the surface of the
specimen and sec. electrons are emitted from the surface
(emission depends on topography of surface)

30. Sec. electrons are first collected, amplified and then used
for formation of image on the phosphor screen of CRT.
SEM has at least 2 CRTs, one for visual observation &
other for photography.
Magnification: ratio of the scan length on the screen
(constant) to the scan length on the specimen surface
(variable).
It is a combination of electron microscopy & television
electronics.
3D image of the surface is obtained.
resolution (50A)

31. Techniques in Microscopy and Cell Biology
by V. K. Sharma
Microscopy and Microtechnique
by R. Marimuthu
Images from the internet