MICROSCOPE
DR. FERADICA D
LALOO
SENIOR RESIDENT
JGMCH
LEARNING OBJECTIVES
• Properties of microscope
• Parts of microscope
• Types of microscope
• How to use a microscope
• Microscope care
WHAT IS A MICROSCOPE ??
• Microscope is an instrument for viewing objects that are too small ( bacteria , fungi , parasites in
micrometers and like viruses in nanometers) or viewing objects that cannot be seen with naked
eye
HISTORY
• ANTONIE PHILIPS VAN
LEEUWENHOEK (1676 )
• First scientist to observe bacteria and other
microorganisms using a single lens
microscope constructed by him
• He named those small organisms as Little
animalcules
PROPERTIES OF MICROSCOPE
• Resolution: resolution power represent the capacity of optical system to produce separate
image of very closely placed object so that they can be distinguish as two separate entities
• The resolution power of :
unaided human eye is 0.2mm
light microscope is 0.2 µm
electron microscope is 0.5 nm
• Resolution depends on refractive index of medium
• Oil has a higher refractive index than air , hence use of oil enhances the resolution power of
a microscope
• Magnification : magnification power of microscope is the degree of image enlargement
• It depends upon
1) length of optical tube
2) magnifying power of objective lens: scanning (4X) , low power (10X) ,high power
( 40X) , oil immersion (100X)
3) magnifying power of ocular lens 10X
• Magnification power of microscope = Magnifying power of objective lens X Magnifying
power of ocular lens
• Scanning field = 40x
• Low power field = 100 x
• High power field = 400 x
• Oil immersion field =1000 x
TYPES OF MICROSCOPE
• Light microscope
• Dark field microscope
• Phase contrast microscope
• Fluorescence microscope
• Electron microscope
LIGHT MICROSCOPE
• Simple microscope : uses a single lens for
magnification , such as magnifying glass
• Principle : when a tiny object is placed within its
focus , a virtual , erect and a magnified image of
object is formed at the least distance of vision from
the eye held close to the lens
• Uses : to study sample of algae , fungi , biological
specimen
• Compound microscope : 2 types
• Monocular
• Binocular
Monocular Binocular
PARTS OF MICROSCOPE
Parts of a microscope
Mechanical Illuminating Magnifying
parts Parts Parts
MECHANICAL PARTS
• Base: holds various parts of the microscope
• C-shaped arm: holds the microscope, connects - ocular lens to objective lens
• Mechanical stage: hold the slides.
• MAGNIFYING PARTS
• Ocular lens : the arm contains eyepiece that bears ocular lens of 10X
• Objective lens: the arm also contains a revolving nose piece that bears 3- 4 objective lens with
magnifying power of ( 4x , 10x , 40 x , 100 x)
• ILLUMINATING PARTS
• Condenser: focuses light on the slide (beneath the stage)
• Iris diaphragm: controls the light that passes through the condenser
• Light source: mirror or an electric bulb
• Fine and coarse adjustment knobs: sharpen the image.
HOW TO FOCUS
• 10 X
• 40 X
• 100X
BRIGHT FIELD OR LIGHT MICROSCOPE
• Produces a dark image against a brighter background
• Uses to view live or stained cells
• Resolution is limited to 200 nm , viruses cant be visualized
• Objective lens form an enlarged real image
• Eyepiece ( ocular) lens further magnifies this primary image into the final virtual
image
IMAGES UNDER LIGHT MICROSCOPE
GRAM STAINING AFB STAINING
KOH mount
OPTICS OF BRIGHT AND DARK FIELD MICROSCOPE
DARK FIELD MICROSCOPE
• Produce bright image against dark background
• A special dark field condenser is used, which has a central opaque area
Principle –
special condenser doesn't allow light to pass directly through the specimen
It directs the light to hit the specimen at oblique angle
Only light which hits the specimen will be deflected upwards into
objective lens for visualization
All other light that pass through the specimen will miss the objective
Making the background dark field
PHASE CONTRAST MICROSCOPE
• Visualize the unstained living cell by
creating difference in contrast
between cells and water
• Useful for studying -
• Microbial motility
• Detecting bacterial component
such as endospores and inclusion The light rays go through → condenser → specimen
bodies (e.g. bacteria) → phase ring → objective lens → ocular
lens
• Determining shape of living cells
Principle of Phase Contrast Microscope
• PRINCIPLE OF PHASE CONTRAST MICROSCOPE
Condenser has an opaque central area . Cone of light passes through a cell
Some light rays are bent due to variations in density and refractive index within the specimen and
are retarded by 1/4th of a wavelength
Undeviated light rays strike a phase ring
Deviated rays miss the ring and pass through the rest of the plate
Background is bright, while the unstained object appears dark
FLUORESCENCE MICROSCOPE
Fluorescent dyes - exposed to UV rays, become excited and fluoresce
i.e. They convert short wavelength rays(invisible) into longer
wavelengths( visible light )
Mercury lamp emits rays that pass through an excitation filter
Excitation filter allows only short wavelength UV light to pass
through
Exciting rays get reflected by a dichromatic mirror - fall on the
specimen stained by fluorescent dye.
The fluorescent dye absorbs the exciting rays of short wavelength ,
activated and in turn emits rays of higher wavelength
FLUORESCENCE MICROSCOPE – USES
▫ Use fluorescence property to generate image
▫ Auto fluorescence: some microbes directly fluoresce when placed under UV lamp
eg :cyclospora(protozoan parasite)
▫ Microbes coated with fluorescent dye:
▫ Acridine orange; used for detection of plasmodium and nematode parasite by qbc examination
▫ AURAMINE phenol; used for detection of TB bacilli
▫ Immuno fluorescence: dye tagged immunoglobulins or antibodies to detect cell surface antigens
▫ Direct
▫ Indirect
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ELECTRON MICROSCOPE
• An electron microscope is a microscope that uses a beam of electron
as a source of illumination
• It gives powerful magnification and resolution
• Use : detection of viruses
• 2 types:-
• Transmission electron microscope (TEM)
• Scanning electron microscope(Sem)
• PRINCIPLE OF ELECTRON
MICROSCOPE
• Electrons - generated by electron gun-travel in
high speed.
• Medium of travel - fully vacuum path
Provides 2D image only Provides 3D image of the surface of the
sample
MICROSCOPE CARE
• Handle with care
• Keep lenses clear of slides
• While using oil immersion , clean lenses with special lens paper or wipes
• Cover when not in use
• Store in clean, dry place
THANKYOU
