This presentation is about the introduction of microscopy, its history, parts of a microscope and different types of microscopes along with a brief discussion of their working principles.

1. MICROSCOPY
HAFIZ MUHAMMAD MOAVIA ATIQUE
UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES ,LAHORE

2. DEFINITION
• Microscopy is the technical field of using microscopes to view
objects and areas of objects that cannot be seen with the naked
eye.
• The instrument used for this purpose is called a Microscope.

3. HISTORY
• ~710 BC - Nimrud lens
The Nimrud lens – a piece of rock crystal – may have been used as a
magnifying glass or as a burning-glass to start fires by concentrating
sunlight
• ~1000 AD - Reading stone
The first vision aid, called a reading stone, is invented. It is a glass
sphere placed on top of text, which it magnifies to aid readability.
• ~1021 AD - Book of Optics
Muslim scholar Ibn al-Haytham writes his Book of Optics. It eventually
transforms how light and vision are understood
• 1284 - First eye glasses
Salvino D’Armate is credited with inventing the first wearable eye

4. HISTORY
• 1590 - Early microscope
Zacharias Janssen and his son Hans place multiple lenses in a
tube. They observe that viewed objects in front of the tube
appear greatly enlarged
• 1609 - Compound microscope
Galileo Galilei develops a compound microscope with a convex
and a concave lens.
• 1625 - First use of term ‘microscope’
Giovanni Faber coins the name ‘microscope’ for Galileo Galilei’s
compound microscope.
• 1665 - First use of term ‘cells’

5. HISTORY
• 1673 - Living cells first seen
Antonie van leeuwenhoek- First observation of live
microorganisms
• 1830 - Spherical aberration solved
Joseph Jackson Lister reduces spherical aberration (which
produces imperfect images) good magnification without
blurring the image.
• 1931 - Transmission electron microscope
Ernst Ruska and Max Knoll.
• 1932 - Phase contrast microscope
Frits Zernike

6. HISTORY
• 1942 - Scanning electron microscope
Ernst Ruska builds the first scanning electron microscope
(SEM),
• 1978 - Confocal laser scanning microscope
Thomas and Christoph Cremer.
• 1981 - Scanning tunnelling microscope
Gerd Binnig and Heinrich Rohrer.
• 1986 - Nobel Prize for microscopy
The Nobel Prize in Physics is awarded jointly to Ernst Ruska (for
his work on the electron microscope) and to Gerd Binnig and
Rohrer (for the scanning tunnelling microscope).

7. TERMS RELATED TO MICROSCOPY
• Resolution:
It refers to the ability of the lenses to distinguish two points a
specified distance apart.
• Magnification power:
How much an image is magnified.
• Total magnification:
we can calculate the total magnification of a specimen by
multiplying the objective lens magnification (power) by
the ocular lens magnification (power).
• Refractive index:
The refractive index is a measure of the light-bending ability

8. PARTS OF A MICROSCOPES
• Illuminator (the light source)
• Condenser (condenses the llight towads the specimen)
• Diaphragm (manages the amount of light entering the
condenser)
• Stage (holds the microscopic slide in position)
• Objective lens (lens closest to the specimen, primarily
magnifies the image)
• Body tube ( have prism, transmits the image from objective lens
to ocular lens)
• Ocular lens (the image is viewed here)

9. COMPOUND LIGHT MICROSCOPE
Resolution power of a compound
microscope is 0.2 um.
the magnification achieved by best
compound light microscopes to
about 2000X.
For a clear image specimens must be
made to contrast sharply with their
medium. To attain such contrast, we must
change the refractive index of specimens
from that of their medium, by staining
them.
A general principle of microscopy is
that the shorter the wavelength of
light used in the instrument, the

10. DARK FIELD MICROSCOPE
Uses a darkfield condenser that contains an opaque disk.
Only light that is reflected off (turned away from) the specimen
enters the objective lens. Because there is no direct background
light, the specimen appears light against a black background-
the dark field.
To examine live microorganisms that are either:
• invisible in the ordinary light microscope,
• cannot be stained by standard methods
• distorted by staining.

12. PHASE CONTRAST MICROSCOPE
• The principle of phase-contrast microscopy is based on:
• the wave nature of light rays.
• light rays can be in phase (their peaks and valleys match) or out
of phase.
• The specimen is illuminated by light passing through an
annular (ring shaped) diaphragm. Direct light rays (unaltered by
the specimen) travel a different path than light rays that are
reflected or diffracted as they pass through the specimen.
These two sets of rays are combined at the eye. containing
areas that are relatively light (in phase), through shades of
gray, to black (out of phase.
• It permits detailed examination of internal structures in living
microorganisms. Not necessary to fix (attach the microbes to

13. PHASE CONTRAST
MICROSCOPE

14. DIFFERENTIAL
INTERFERENCE CONTRAST
MICROSCOPE
• It uses differences in refractive indexes.
• DlC microscope uses two beams of light
instead of one.
• Prisms split each light beam, adding
contrasting colors to the specimen. Therefore,
the resolution of a DIC microscope is higher
than that of a standard phase-contrast
microscope.
• The image is brightly colored and appears
nearly three-dimensional

15. FLUORESCENT MICROSCOPE
Fluorescence microscopy takes advantage of fluorescence
• Some organisms fluoresce naturally under ultraviolet light; if
the specimen to be viewed does not naturally fluoresce, it is
stained with one of a group of fluorescent dyes called
fluorochromes.
• When microorganisms stained with a fluorochrome are
examined under a fluorescence microscope with an ultraviolet
or near ultraviolet light source, they appear as luminescent,
bright objects against a dark background.
• The principal use of fluorescence microscopy is a diagnostic
technique called the fluorescent-antibody (FA) technique
• This technique can detect bacteria or other pathogenic

16. FLUORESCENT
MICROSCOPY

17. CONFOCAL MICROSCOPY
• Specimens are stained with fluorochromes so they will emit, or
return, light. One plane of a small region of a specimen is
illuminated with a short-wavelength (blue) light which passes
the returned light through an aperture aligned with the
illuminated region. Successive planes and regions are
illuminated until the entire specimen has been scanned.
• Exceptionally clear two-dimensional images can be obtained.
• Improved resolution of up to 40%.
• Most confocal microscopes are used in conjunction with
computers to construct three-dimensional images.
• Used to evaluate cellular physiology by monitoring the
distributions and concentrations of substances within the cell.

18. CONFOCAL
MICROSCOPY

19. TWO-
PHOTON
MICROSCOPY
• Specimens are stained with a
fluorochrome.
• Uses long-wavelength (red) light, and
therefore two pholons, are needed to
excite the fluorochrome to emit light.
• The longer wavelength allows imaging
of living cells in tissues up to 1mm
deep.
• Additionally, the longer wavelength is
less likely to generate singlet oxygen,
which damages cells
• Track the activity of cells in real time.
For example, cells of the immune
system have been observed

20. SCANNING
ACOUSTIC
MICROSCOPY
• Consists of interpreting the action of
a sound wave sent through a
specimen.
• A sound wave of a specific
frequency travels through the
specimen, and a portion of it is
reflected hack every time it hits an
interface within the material.
• The resolution is about 1um.
• SAM is used to study living cells
attached to another surface, such as
cancer cells, artery plaque, and
bacterial biofilms that foul
equipment.

21. ELECTRON MICROSCOPY
• A beam of electrons is used instead of light. free electrons
travel in waves.
• The better resolution of electron microscopes is due to the
shorter wavelengths of electrons; the wavelengths of electrons
arc about 100,000 times smaller than the wavelengths of
visible light.
• Objects smaller than about 0.2 u.m, such as viruses or the
internal structures of cells, must be examined with an electron
microscope.
• There are two types of electron microscopes:
Transmisssion electron microscope

22. TRANSMISSION ELECTRON
MICROSCOPE
• In a transmission electron microscope, electrons pass through
the specimen and are scattered. Magnetic lenses focus the
image onto a fluorescent screen or photographic plate.
• The internal structures present in the slice can be seen.
• Two dimensional appearance of cell.
• Resolution power is 2.5nm
• Magnification power is 10,000 to100000X.

23. TRANSMISSION ELECTRON MICROSCOPE

24. SCANNING ELECTRON MICROSCOPE
• In a scanning electron microscope. primary electrons sweep across
the specimen and knock electrons from its surface. These secondary
electrons are picked up by a collector, amplified and transmitted onto
a viewing screen or photographic plate.
• Three-dimensional appearance of the cell.
• This microscope is especially useful in studying the surface
structures of intact cells and viruses.
• Resolution power is10 nm,
• Magnification power is 1000 to 1O,000X.

25. SCANNING ELECTRON MICROSCOPE

26. SCANNED-PROBE MICROSCOPY
• They use various kinds of probes to examine the
surface of a specimen at very close range, and they do
so without modifying the specimen or exposing it to
damaging, high-energy radiation. Such microscopes
can be used to map atomic and molecular shapes, to
characterize magnetic and chemical properties, and to
determine temperature variations inside cells.
• There are two types of scanned-probe microscopes:
1) Scanning-tunneling microscopy
2) Atomic force microscopy

27. SCANNING-
TUNNELING
MICROSCOPY
• Uses a thin metal (tungsten) probe
that scans a specimen and
produces an image revealing the
bumps and depressions of the
atoms on the surface of the
specimen
• It can resolve features that are
only about 1/100 the size of an
atom
• Special preparations for specimens
are not needed.
• STMs arc used to provide
incredibly detailed views of

28. ATOMIC FORCE
MICROSCOPY
 A metal-and-diamond probe is
gently forced down onto a
specimen. As the probe moves
along the surface of the
specimen, its movements are
recorded, and a three-
dimensional image is
produced.
 AFM does not require special
specimen preparation.
 AFM is used to image both
biological substances and
molecular processes (such as

29. REFERENCES
• https://www.sciencelearn.org.nz/resources/1692-history-of-
microscopy-timeline
• https://www.microscopemaster.com/microscope-timeline.html
• Chapter no.3, Microbiology an introduction, by Tortora, Funke
and Case.