This document provides an overview of microscopes, including: - A brief history of microscope development from the late 1500s to modern electron microscopes. - Key terms like resolution, numerical aperture, refractive index, and aberration. - The main types of light microscopes - brightfield, darkfield, phase contrast, and fluorescence - and their basic workings. - Transmission and scanning electron microscopes and how they produce higher resolution images than light microscopes. - Basic techniques like micrometry, specimen preparation, and microscope care and maintenance.

2. CONTENTS
Introduction
Timeline Of Microscope
Terms & Definitions
Properties Of Microscope
Types Of Microscope
Micrometry
Care Of Microscope

3. INTRODUCTION
 A Microscope (from the Ancient Greek:, mikrós, "small"
and, skopeîn, "to look" or "see") is an instrument used
to see objects that are too small to be seen by the
naked eye.
 Faber coined the name Microscope for the instrument
use by Galileo in 1625 (Galileo had called it the
"occhiolino" or "little eye")
 Since then it continue to play a important role in every
Brach of science.
 The only scientific equipment ,Charels Darwin took with
him on the Voyage Of Beagle, was a simple microscope.

4. TIMELINE OF MICROSCOPE
• 1590 :- Two Dutch eye
glass makers,
Zaccharias Janssen and
Hans Janssen
constructed the first
compound microscope.
• 1609 - Galileo Galilei
developed a compound
microscope with convex and
a concave lens.

5. Timeline…contd
• 1676 :- Antonie Philips Van Leeuwenhoek
built a simple microscope.
.

6. Timeline…contd
• 1931 :- Ernst Ruska developed the
Electron Microscope for
which he won the Nobel
Prize in Physics in 1986.
• 1981 :- Gerd and Heinrich invented
the Scanning Tunnelling
Microscope
Electron microscope
constructed by Ernst
Ruska in 1931

7. TERMS AND DEFINITIONS
RESOLUTION
• It is Also called Revolving Power.
• It is refer to ability to produce separate image of closely place objects,so
that they can distinguished as two separate entities.
• It is calculated by Abbé equation.
NUMERICALAPERTURE
• It is the light gathering capacity of a lens.
NA = n X Sin θ
n – Refractive index of the medium between
the object & the objective lens
Θ – ½ of angular aperture of objective

8. Terms & Definition…. contd
REFRACTIVE INDEX (RI)
RI = Sin i/Sin r
i = angle of incidence
r = angle of refraction.
Higher the density of the medium, greater the RI.
IMMERSION FLUID
• This is any liquid that occupy the space between
the object and objective lens.
• It can also use between the condenser and
the microscope slide.
• Use of immersion fluid produce a homogeneous
optical path way which minimize the refraction &
maximize the NA and improve the resolution.

9. WORKING DISTANCE
• It is the distance between the objective &
the objective slide.
• The working distance decreases with increasing
magnification.
ABERRATION
• Aberration are unwanted artefacts in the microscopic image
that are caused by elements in optical path.
• Spherical Aberration
• Chromatic Aberration
.
Terms & Definition…. contd

10. Terms & Definition…. contd

11. PROPERTIES OF MICROSCOPE
A good microscope should have at least three properties
Good Resolution
• The resolution power of Human eye - 0.2 mm
Light microscope - 0.2µm.
Electron microscope - 0.5nm.
Good Contrast
• This can be improved by staining the specimen.
Good Magnification
• It can achieve by lens
Ocular Lens, Objective Lens
• The total magnification of microscope
Power of the objective lens X Power of ocular lens

12. TYPES OF MICROSCOPE
LIGHT MICROSCOPE
Uses sun light or artificial light as source of illumination.
1. Bright Field Microscope
2. Dark Field Microscope
3. Phase Contrast Microscope
4. Fluorescence Microscope
ELECTRON MICROSCOPE
• Uses accelerated electron as source of illumination.
1. Transmission Electron microscope
2. Scanning Electron Microscope

13. -The bright field or light microscope forms a dark image against a bright
background ,hens the name is called so.
COMPONENTS
The parts of bright field microscope divided in to 3 parts
Mechanical Parts
Base , C-Shaped Arm, Mechanical Stage
Magnifying Part
Ocular Lens/Eyepiece, Objective lens
Illumination Part
Condenser, Iris Diaphragm, Light Source, Fine and Coarse adjustment
knob
BRIGHT-FIELD or LIGHT MICROSCOPE

14. Terms &Definition…..
.

15. Bright-field microscope contd…
WORKING PRINCIPLE
The image from in the microscope in two stage.
• First image (i1) from by the objective lens(L1),Which is magnified,
inverted & real.
• The second image (i2) is from by ocular lens (L2) which is more
magnified and virtual.

16. Bright-field microscope contd…
agnifying Part :- All these have 2 types of
lens system for magnification
THE LIGHT PATHWAY OF A MICROSCOPE

17. Bright-field microscope contd…
PROPERTY LOW POWER HIGH POWER OIL IMMERSION
Magnification
of objective
10x 40-45x 90-100x
Magnification of
eyepiece
10x 10x 10x
Total magnification 100x 400 – 450x 900 – 1000x
Iris diaphragm Partially closed Partially opened Fully opened
Position of condenser Lowest Slightly raised Fully raised

18. Bright-field microscope contd…

19. Bright-field microscope contd…
APPLICATION
• Used to visualize and study the morphologies, Identification of bacteria,
parasites etc.
ADVANTAGE
• It is simple to use with few adjustments involved while viewing the
image.
• It can be used to view both stained and unstained.
• The optics of the microscope do not alter the color of the specimen.
• The microscope can be modified to other type microscope
DISADVANTAGE
• The heat produce by the light source may damage the specimen.

20. • Dark-field microscope is similar to the
ordinary light microscope; however,
the condenser system is modified.
• In this type of microscope a Star
Diaphragm is used to produce dark
field.
• The diaphragm blocks all light
entering to the objective lens except
peripheral light that is reflected from
the specimen .
• This result , the specimen appear bright
against a dark back ground.
DARK-FIELD MICROSCOPE
The insertion of a star diaphragm into the
filter slot of the condenser will produce a dark field

21. Dark-field microscope contd…
APPLICATION
• It identify the living,unstain cells.
• t is also useful for the
demonstration of the motility of
flagellated bacteria and protozoa.
• This can rapidly demonstrate
Treponema pallidum in clinical
specimens.
• It is more useful in examining
external details, such as outlines,
edges, grain boundaries and surface
defects than internal structure.
Darkfield image of Treponema pallidum,

22. BRIGHT-FIELD vs DARK-FIELD

23. PHASE CONTRAST MICROSCOPE
• Phase-contrast microscope was first described by Dutch
physicist Frits Zernike.
• 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
wavelength, which can be observed as differences in image
contrast.
• COMPONETS
• Annulus condenser
• Phase Plate

24. Phase contrast microscope contd…

25. Phase contrast microscope contd…

26. Phase contrast microscope contd…
APPLICATION
• To see the microbial motility.
• Determine the shape of living cell.
ADVANTAGE
• Living cells can be observed in their natural state.
• No special preparation of fixation or staining etc. is needed to study.
DISADVANTAGE
• Phase-contrast condensers and objective lenses add considerable
cost to a microscope.
• To use phase-contrast the light path must be aligned.

27. FLUORESCENCE MICROSCOPE
• Refers to any microscope that uses fluorescence in addition
to, reflection and absorption to generate an image.
• It utilizes a powerful mercury vapor arc lamp for its light source.
• A darkfield condenser is usually used in place of the conventional
bright-field condenser
• COMPONETS
• Light Source
• Excitation Filter
• Dichroic mirror
• Emission/Barrier Filter

28. Fluorescence microscope contd…
WORKING PRINCIPLE
• Most cellular components are colourless and cannot be clearly
distinguished under a microscope. The basic premise of fluorescence
microscopy is to stain the components with dyes. Such dye are called
FLUOROCHROMES or FLUOROPHORES
• Fluorochromes, are molecules that absorb excitation light at a given
wavelength (generally UV), and after a short delay emit light at a longer
wavelength.
• The emission light then be filtered through the dichromatic mirror &
barrier filter to reveal the location of the fluorophores.
• The image produced is based on the emission light wavelength of the
fluorescent species .
• This result image consist of brightly shine area against the dark
background.

29. Fluorescence microscope

30. Fluorescence microscope contd…
ity

31. Fluorescence microscope contd…
FLUOROCHROMES EXCITATION
WAVELENGTH
EMISSION
WAVELENGTH
Acridine Orange 500 526
Auramine O, 460 550
Calcofluor white 440 505-520
Ethidium Bromide 545 605
Evans blue 550 610
FITC 490 525
TRITC 555 580

32. APPLICATION
• It is used to study some microbes directly under UV lams which are auto
fluorescence.
• Some microbes visible under this microscope by staining them with
fluorochromes. e.g :- Plasmodium, mycobacterium
ADVANTAGE
• Slides can be examined at a lower magnification, thus allowing the
examination of a much larger area per unit of time
DISADVANTAGE
• Fluorophores lose their ability to fluoresce as they are illuminated in a
process called photobleaching
• The mercury vapour lamp tends to loose their potency with time.
Fluorescence microscope contd…

33. ELECTRON MICROSCOPE
• The electron microscope uses accelerated
electrons as light source.
• As wavelength of electron 100,000 times
shorter than the visible light, so revolving
power is much better than a light
microscope
• The magnified image is visible on a
fluorescent screen and can be recorded on
a photographic film.
• Images produced by EM lack
color, always shades of black,
gray, and white.

34. EM Contd….
Transmission Electron Microscope (TEM)
• TME are the method of choice for viewing the detailed structure of cells and
viruses.
• This microscope produces its image by transmitting electrons through
the specimen.
• Because electrons cannot readily penetrate thick specimen, the specimen
must be sectioned into extremely thin slices (20–100 nm) thick
Specimen Preparation
To make a suitable specimen following steps are followed
1. Fixation
2. Dehydration
3. Embedding
4. Slicing

35. EM Contd….
Electron Pathway
• Electron generated by electron gun travellers in
vacuum path, with high speed, passes through
condenser lens bombard to specimen mount on slide.
• Scatters Objective Ocular Enlarge
electrons lens lens Image
• The thick area of specimen scatters more electron &
appears dark and thin area scatter less & appear bright.
APPLICATION
The important use of EM is for virus detection. It can
detect virus i) Direct from clinical specimen
ii)Tissue culture
iii)After adding specific antiviral antibody
to the specimen.

36. EM Contd….
Chlamydomonas

37. EM Contd….
Scanning Electron Microscope (SEM)
• SEM has been use for examine the surface of microorganism in
greater detail.
• It has resolution of 7nm or less.
I

38. LIGHT VS ELECTRON MICROSCOPE
FEATURES LIGHT MICROSCOPE ELECTRON
MICROSCOPE
Magnification 1000x – 1500x Over 100,000x
Resolution 0.2µm 0.5nm
Illumination source Visible light Electron beams
Medium of travel Air Vacuum
Specimen mount Glass slide Metal grid
Type of lens Glass Electromagnetic

39. MICROMETRY
• Micrometry refer to the measurement of dimensions of the desire
microorganisms under microscope.
• The most common procedure used in clinical laboratory is by using
a graduated scale (Reticles) located in one eyepiece.
• Reticles must be calibrated against a stage micrometre.

40. Micrometry contd….
CALIBRATION OF OCULAR MICROMETRY

41. CARE OF MICROSCOPE
TRANSPORT
• When carrying your microscope from one part
of the room to another, use both hands to hold
the instrument.
And, under no circumstances should one attempt
to carry two microscopes at one time.
WORKSTATION
• Should be placed in flat fixed area.
• Away from source of water.
• Should be uncluttered while doing
microscopy.
LENS CARE
• At the beginning of each use, make sure lenses
are clean.
• At the end of each lab session, wipe any
immersion oil from the immersion lens.
The microscope should be held firmly with
both hands while being carried.
When oculars are removed for cleaning,
cover the ocular opening with lens tissue. A blast from
an air syringe or gas canister removes dust and lint.

42. Care of microscope contd…
PUTTING IT AWAY
1. Remove the slide from the stage.
2. If immersion oil has been used, wipe it off the lens and stage .
3. Do not wipe oil off slides if wish to keep. Simply put them into a slide box
and let the oil drain off.
4. Rotate the low-power objective into position.
5. If the microscope has been inclined, return it to an erect position.
6. If the microscope has a built-in movable lamp, raise the lamp to its highest
position.
7. If the microscope has a long attached electric cord, wrap it around the
base.
8. Adjust the mechanical stage so that it does not project too far on either
side.
9. Pace the dust cover over the microscope at the end of use