MICROSCOPES- Principles

and Types of microscopes

Dr. Priyanka Saini

Introduction:
• Microscopy refers to the use of a specialized instrument called
“microscope” to view objects and area of objects that cannot be seen with
naked eye so that their characteristics are readily observable.

 It makes enlarged images of minute objects, sub cellular structure which

can’t be seen by naked human eyes.
History:
 1590: Hans Janssen developed first microscope.

 1609:
• Galileo Galilei worked on the principle
of lens.
 1665 - Robert Hooke's book called Micrographia officially
documented a wide range of observations through the
microscope.
His microscope had maximum magnification of 200x.
 1676:
• Anton Van Leeuwenhoek used a single lens microscope
constructed by him.

• He was the 1st scientist who observed bacteria & other

micro-organisms with single lens microscope.

• He is the “father of microscopy”.

VARIABLES USED IN
MICROSCOPY
Magnification:
Expressed as the number of times the length, breadth or diameter of the object is
multiplied.

TOTAL MAGNIFICATION = magnification of the eyepiece

X
magnification of the objective

Objective Power Eye Piece Power Total Magnification

4X 10X 40X

10X 10X 100X

40X 10X 400X

100X 10X 1000X

RESOLUTION
• Ability to reveal closely adjacent structural details as separate
and distinct.
• LIMIT OF RESOLUTION (LR) - The minimum distance
between two visible bodies at which they can be seen as
separate and not in contact with each other.
• LR=
W= wavelength, NA- Numerical aperture

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 NUMERICAL APERTURE(NA)

• Ratio of diameter of lens to its focal length

• NA = n Sin θ
• n = refractive index,
• θ = half of angle between object and lens
• n of air = 1
• n of oil = 1.5
Effect of Immersion Oil on
Resolution
WORKING DISTANCE
Distance between the front surface of lens and surface of cover
glass or specimen.
The working distance decreases with increasing magnification .
Properties of Microscope:
A good microscope should have following properties:
I. Good Resolution:
• The resolution power of;
o Unaided human eye -0.2mm (200µm)
o Light microscope- 0.2µm
o Darkfield microscope- 0.1µm
o Electron microscope- 0.5nm

• Resolution depends on refractive index of the medium.

• Oil has higher refractive index ,it enhances the resolution power of microscope.
II. Good Contrast:
• Improved by staining of the specimen.

• When the stain binds to the cells, the contrast is increased.

III. Good Magnification:

• Achieved by use of lenses.

• There are two types of convex lenses which is used.

i. Ocular lens with a magnification power of 10X.

ii. Objective lenses: Scanning(4x), Low power(10x), High power(40x) and Oil
immersion(100x)
TYPES OF MICROSCOPE
1.Optical or light microscope
2.Phase contrast microscope
3.Dark field / dark ground microscope
4.Fluorescent microscope
5.Electron microscope
6. Confocal microscope
Bright field
or Light
microscope
• It forms a dark image
against a brighter
background.
STRUCTURE
1. Mechanical Parts 2. Magnifying Parts 3. illuminating Parts
• Base • Ocular lens (10x ) • Condenser- focus cone of
• C- shaped arm • Objective lens (4x, 10x, light on slide.
• Mechanical stage 40x , 100x) • Iris diaphragm – control
light that pass through
condenser
• Light source- mirror or
electric bulb.
• Fine and coarse
adjustment knobs-
sharpen the image
 Light microscope….
Principle :
• The rays emitted from the light source pass
through the iris diaphragm and fall on the
specimen.

• The light rays passing through the

specimen are gathered by the objective and
a magnified images formed.

• This images is further magnified by ocular

lens to produce the final magnified virtual
image.
Light microscope….
 Light microscope….

Gram-positive bacteria observed under oil Gram-negative bacteria observed under oil
immersion appear purple. immersion appear pink.
 Light microscope….

 ADVANTAGES:
• Used to view live or stained cells

• Simple setup

 DISADVANTAGES:
• Staining may destroy the specimen or introduce artifacts.

• Resolution is limited to 200nm or 0.2 micron.

DARK FIELD MICROSCOPE
• Produces a bright image of the object against a dark background.

• Enhances the contrast of unstained object.

• Principle :
• It has condenser with central opaque area that blocks light
from entering the objective lens directly & has a
peripheral annular hollow area which allow the light pass
through and focus on the specimen obliquely.

• Only the light which is reflected by specimen enters the

objective lens whereas unreflected light does not enter the
objective as a result specimen is brightly illuminated but
the background appears dark.
 USES OF DARK GROUND
MICROSCOPY:
Useful in demonstrating
• Treponema pallidum
• Leptospira
• Campylobacter jejuni
• Endospore

Treponema pallidum in Dark-field microscope

 DARK FIELD MICROSCOPE….

 ADVANTAGES:
• Used to identify the living unstained cells and thin bacteria like spirochetes
which are not visualized by light microscope.

 DISADVANTAGES:
• Specimen needs to be strongly illuminated which can damage delicate
samples.
PHASE CONTRAST MICROSCOPE
• First described in 1934 by Dutch physicist Frits Zernike.
• This improves the contrast and make structures evident within
cells that differ in thickness or refractive index.
• The differences in refractive index between bacterial cell and
surrounding medium make them clearly visible.
 This microscope visualizes the unstained living cells by creating
difference in contrast between cell and water.

 Principle:
• Use of Condenser – consists of an opaque central area with a thin
transparent ring, which produce hollow cone of light.
• Light passes through a cell, some light rays are bent due to
variation in density & refractive index within the specimen and
retard by about one- fourth of wavelength.
• The undeviated light rays strike a phase ring in the phase plate (due to
which its wavelength advance by one fourth of wavelength) while deviated
rays miss the ring and pass through the rest of the plate.

• The deviated and undeviated waves will be about half wavelength out of
phase and will cancel each other and form an image.

• The background formed by undeviated light is bright while the unstained

object appears dark and well defined.
 PHASE CONTRAST MICROSCOPE…

 Advantages: Useful for studying

• Microbial motility
• Determining the measurement of living cells
• Detecting microbial internal cellular components

 Disadvantages :
• Phase ring limit the aperture which decrease the resolution.
• Not ideal for thick organism and particles .
MICOORGANISM SEEN UNDER PHASE CONTRAST
MICROSCOPE
Fluorescence microscope
 It refers to microscope that uses fluorescence property to generate an
image.
 In this microscope, fluorescent dyes are exposed to UV rays, dyes are
excited and make this invisible rays (shorter wavelength) in to visible rays
(longer wavelength).

 Principle :
• Light from source are passed through the excitation filter
• Excitation filter allows only rays of shorter wavelength like UV light
(about 400nm, exciting rays), and blocking all other long wavelength
rays.
 Fluorescence microscope…

• Exciting rays then get reflected by

dichromatic mirror so that they fall on the
specimen stained by fluorescent dyes.

• Fluorescent dyes absorbs the exciting rays,

gets activated and emits rays of higher
wavelength (visible rays).

• A barrier filter, after objective lens removes

any remaining UV rays which could
otherwise damage the viewer’s eyes.
 Fluorescence microscope…

• Applications:
a) Auto fluorescence:
• Certain microbes directly get fluoresce when
placed under UV lamp e.g. Cyclospora

CYCLOSPORA oocysts on stool examination. The oocyst wall is

autofluorescent.

b) Microbes coated with fluorescent dye:

• Acridine orange dye – Plasmodium / filarial
nematodes
• Auramine phenol- tubercle bacilli

Auramine O stain sputum smear showing TB

BACILLI
 Fluorescence microscope…

c) Immunofluorescence:
• It uses fluorescent dye tagged immunoglobins to detect cell surface antigens or
antibodies bound to cell surface.

• two types: Direct and Indirect immunofluorescence test.

• It is similar to ELISA, but differs by some important features;

 Fluorescent dye is used instead of enzyme for labeling of antibody.
 It detects cell surface antigens.
 Used to detect antibodies bound to cell surface antigens, unlike ELISA which detects free
antigen or antibody.
 Fluorescence microscope…

 Principle:
• Absorbing high energy- shorter wavelength uv light rays by the fluorescent
compounds and in turn emitting visible light rays with a low energy-longer
wavelength.

• Fluorescent dye is used to conjugate the antibody and such labeled antibody can
be used to detect the antigens or antigen-antibody complexes on the cell surface.

• Fluorescein isothiocyanate is commonly used.

 Fluorescence microscope…

 Applications

• Detection of autoantibodies(e.g. antinuclear antibody) in autoimmune

diseases.

• Detecting microbial antigens, e.g. rabies antigen in corneal smear.

• Detection of viral antigens in cell lines inoculated with the specimens.

Confocal Laser Scanning Microscope
 It is an advanced design of fluorescence microscope.
 It uses a laser beam to illuminate a specimen whose image is then digitally
enhanced for viewing on a computer monitor.
 It uses point illumination and a pinhole in an optically conjugate plane to
eliminate out-of-focus signal and to get a better resolution of the
fluorescent image.
Confocal Microscope
 Uses of confocal microscope:
• Observing cellular morphology in multilayered specimen.
• Diagnosing Ca cervix.
• Evaluation & diagnosis of basal cell carcinoma of skin.

What is the advantage of using a confocal microscope?

 By having a confocal pinhole, the microscope is really efficient
at rejecting out of focus fluorescent light so that very thin
section of a sample can be analyzed.
By scanning many thin sections through a sample, one can
build up a very clean three-dimensional image.
Electron microscope (EM)
 It was invented by Ernst Ruska in 1931.

 It uses accelerated electrons as a source of illumination.

 EM has a much better resolving power than a light microscope as wavelength of

electrons can be up to 100,000 times shorter than that of visible light photons.

 It can reveal the details of flagella, fimbriae and intracellular structures of a cell.
Differences between light microscope and electron microscope

Features Light microscope Electron microscope

o Highest magnification About 1000-1500 Over 100,000

o Best resolution 0.2 0.5nm

o Radiation source Visible light Electron beam

o Medium of travel Air High vacuum

o Specimen mount Glass slide Metal grid

o Type of lens Glass Electromagnet

Principle
• Electrons (electron gun) pass through a magnetic
condenser & then bombard on thin sliced specimen
mounted on copper slide.

• Specimen scatters electrons passing through it then

electron beam is focused by magnetic lenses to form an
enlarged, visible image on a fluorescent screen.

• A denser region in the specimen scatters more electrons &

therefore appears darker in the image since fewer electrons
strike that area of the screen.
Transmission electron microscope :
(TEM)
• Most common electron microscope
• Specimen preparation:
• Fixation : cells are fixed by using glutaraldehyde or osmium tetroxide for
stabilization.
• Dehydration: specimen is then dehydrated with organic solvents (e.g. acetone
or ethanol)
• Embedding: specimen is embedded in plastic polymer and then is hardened
to form a solid block.
• Slicing: specimen is then cut into thin slices by an ultra microtome knife.
• Thin slices of the specimen are mounted on a metal slide(copper)
SCANNING ELECTRON
MICROSCOPE
• This is used to create images of the surfaces of specimens
• Resolution- 7nm or less, giving magnification up to approximately
50,000X.
• It gives three dimensional views of the exterior of cells
• It uses electron beam and electromagnetic lenses
• It is very expensive
USES
Observation of exterior surfaces of cells in great detail.
 FEATURES TEM SEM

o Electron beam Broad, static beam Beam focused to fine point;

Transmitted electron Sample is scanned line by line,
Scattered electron
o Voltage needed Ranges from 60-30,000 volts Accelerating voltage much lower

o Interaction of beam electrons Specimen must be very thin Wide ranges of specimen allowed

o Details Internal details Surface/ structural details

o Dimensional 2D 3D
Applications of EM:
 Virus detection.
 The contrast of EM - increased by —
 Negative staining with heavy metals (phosphotungstic acid)
 Shadowing.
 Freeze-etching technique - alternative method for specimen
preparation which help to disclose shape of organelles within
microorganisms.
 Metal Shadowing or shadow casting

Coat - thin film of platinum / other heavy metals at 45 angle –

metal strikes only on 1 surface
Area coated – scatter - light in photograph
Uncoated and shadow – dark
Use-Studying virus morphology , flagella , plasmids
Freeze fracturing and freeze etching
• In freeze fracturing a • In freeze etching, water is
specimen is frozen in a evaporated directly from
block of ice and broken the ice and frozen
apart with very sharp cytoplasm of a fractured
knife. The fracture reveals specimen ,uncovering
the interiors of cellular additional surfaces for
structures observation.
 Applications of microscopy in diagnostic microbiology

• Rapid preliminary organism identification by direct

visualization in patient specimens.

• Detection of different organisms present in the same

specimen.

• Detection of organisms not easily cultivated in the laboratory.

• Pre-culture information about which organisms might be expected to grow so
that appropriate cultivation techniques are used.

• Evaluation of patient specimens for the presence of cells indicative of

inflammation (i.e., phagocytes) or contamination (i.e., squamous epithelial cells).

• Determination of which tests and methods should be used for identification and
characterization of cultivated organisms.
 Microscope care:
Proper care and maintenance of microscope is important.

 Handle with care

 Keep lenses clear of slides
 Clean lenses
 Care of the bulb
 Cover
References
1. Murray R. Patrick, Baron Ellen Jo, P faller Michael A., Manual of Clinical
Microbiology, seventh edition, American Society for Microbiology,1999

2. Sastry Apurba S, Essential of Medical Microbiology, third edition , Jaypee

brothers medical publishers. 2022

3. Ananthanarayan R. Ananthanarayan and Paniker's textbook of

microbiology. Orient Blackswan; 2006.

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 Thank
you…