Microscopy-I

Dr.Minal Trivedi
Principal,
HVHP Institute of Post Graduate Studies and Research,
Kadi Sarva Vishwavidyalaya
Kadi – Gujarat
 CONTENTS

Basics of Microscopy
History
Bright Field Microscopy

Dark Field Microscopy

 Learning Outcomes

Identify and describe the function of the primary

parts of a binocular microscope.
Calculate total magnification with each objective.
Demonstrate basic skills of light microscopy:
locating and bringing into focus, using the correct
procedure, an object under low and high power.
Understand theoretical knowledge about
microscopy .
Enhance microscopic skills
 History
Antony Van Leeuwenhoek

 First to observe living microbes.

 His single-lens magnified 50-

300X magnification.
 Introduction
• Microscopy
• Use of microscope to view objects that are too small to be visible with naked eye.

• Microscopes
• Are basic tools employed by microbiologist for observation of microorganisms.

• The size of microorganism or the microbial structure determines the degree of magnification
needed to see it.
• 1000x :
• Bacteria and larger microorganisms,
• Using light microscope.
• 10000x to 100000x
• viruses and other internal structures of bacterial cells
• Use of electron microscope.
 Light Microscopy
 Simple microscope
Consist of single magnifying lens
Shortcoming- difficulty in focusing the field and development of colour
defect.
 Compound microscope
Consist of more than one lenses.
 produce higher magnification and overcome problem of color defects.
Two main lenses
Objective lens: produces primary magnification
Eyepiece or occular : that enlarges image produced by objective lens
Modern microscopes

• Modern microscopes are all compound microscopes.

• That is, the magnified image formed by the objective lens

is further enlarged by one or more additional lenses.
MODERN MICROSCOPY

MICROSCOPY

LIGHT ELECTRON

PHASE
BRIGHT FIELD DARK FIELD FLOURESCENCE
CONTRAST
 BRIGHT FIELD MICROSCOPY

 The ordinary microscope is called a bright-field microscope because it

forms a dark image against a brighter background.
 Bright field microscopy
 Components of Compound microscope
 Arm/ Handle
Light source
Focusing knobs
Condenser
Objective stage
Objectives
Body tube
Eye piece
Magnification
Supporting stand
 Arm/ Handle

• The microscope consists of a sturdy metal body or stand composed

of a base and an arm to which the remaining parts are attached.

Light Source
• A mirror or electric bulb/ illuminator is the source of light.
• It is located at the base .
• Normally light passes through a blue filter, which absorbs the
longer wavelength and allows shorter wavelength.
• This improves resolution.
Focusing knobs
 Two focusing knobs.
 They are fitted to the arm of microscope
 Coarse adjustment
• It rapidly changes the distance between the objective
and the specimen
• Used to locate the specimen.
 Fine adjustment
• Slowly changes the distance between the objective
and the specimen.
• Used to bring object in to sharp focus.
 Stage

It is fitted half way above the arm and above the condenser
The stage has an aperture(1-1 ½ inches) through which light may
pass.
 it is a platform for holding the slide. It has metal spring clips.
 The stage supports glass slide bearing the specimen and therefore
should be sturdy and perpendicular to the optical path.
 In order to hold the slide firmly and to move the slide easily and
smoothly, a mechanical stage is either attached or built in.
 A mechanical stage allows the operator to move a slide around
smoothly during viewing by use of stage control knobs.
 Body tube and the body assembly

 It is a barrel like part that is attached to the curved part of the arm.
 It forms the assembly.
The nose piece and
One or more eyepieces or ocular lenses.
 To the nose piece are attached objective lenses
 there can be three to five objective lenses of varying magnification.
 They can be rotated to position any objective lens below the body
assembly.
Condenser
 It is a system of lenses that collect and
converge the beam of light and focus on the
object.
 It is fitted below the stage so also called sub
stage condenser.
 Its position may be fixed or can be adjusted
vertically in advanced microscopes.

Iris diaphragm
 It is attached to the condenser on the lower
side.
 Used to control the amount of light that
reaches the specimen.
 Eyepiece/ Occular
 It is attached to topmost part of body tube.

 Normal microscope has single eyepiece (monocular microscope).

 Advanced microscopes has two eyepiece for two eyes.

 Their function is to magnify the image formed by the objective

within the body tube, and present the eye with a virtual image.
 Objective lens

It is the most important part of lens of the microscope.

It produces primary image.
Its quality affects the quality of image.
It collects the rays of light from the object and produces
the image within the body tube.
An objective lens is a combination of several concave and
convex lenses.
Different types of objectives possess certain features.
There are generally three types of objectives
 low power : 10X
High power : 40X
Oil immersion : 100X
 The image one sees when viewing a specimen with a compound
microscope is created by the objective and ocular lenses working
together.

 Light from the illuminated specimen is focused by the objective

lens, creating an enlarged image within the microscope.

 The ocular lens further magnifies this primary image.

 The total magnification is calculated by multiplying the objective

and eyepiece magnifications together.
 Magnification
 Magnification is defined as an ability of microscope lens to enlarge the
size of image as compared to that of object.
 The total magnification of microscope is the multiplication product of
magnifying power of objective lens and that of eye piece lens.
 The image one sees when viewing a specimen with a compound microscope
is created by the objective and ocular lenses working together.
 Light from the illuminated specimen is focused by the objective lens,
creating an enlarged image within the microscope.
 The ocular lens further magnifies this primary image.
 The total magnification is calculated by multiplying the objective and
eyepiece magnifications together.
 For example, if a 45X objective is used with a 10X eyepiece, the overall
magnification of the specimen will be 450X.
 Path of light
 The ray of light beam originating from the source
converges on the specimen through the condenser
and than passes to the objective.

 The objective lens produces an enlarged real

image (primary image) in the body tube of
microscope.

 The ocular lens further magnifies this primary

image.

 The image produced by the ocular lens is virtual,

which is produced at about 25cms from the stage.
 Resolving power

The main function of microscope is to produce a clear as well

as magnified image of the object.

Thus the resolution of microscope is extremely important.

Resolution denotes the ability of lens to see two nearby

objects as separate and discrete units.

Resolving power is the numerical measure of the resolution

that can be obtained by the lens.
 Two factors influence the resolving power.

 Wavelength ()of light

Used to illuminate the object

 Numerical aperture of the objective lens

Which is the light gathering capacity of lens.

 Resolution is described by Ernst Abbes equation

 Smaller the value of d , greater will be the resolution and finer
details will be seen in the specimen.

 The d becomes smaller

as the wavelength of light() used decrease.
And as numerical aperture ( nsin) increases.

 Thus the greatest resolution is obtained

using a lens with the largest possible NA and
light of the shortest wavelength, light at the blue end of the visible
spectrum (in the range of 450 to 500 nm)
 Highest resolution obtainable with compound light microscope is
about 0.2m, which is about half of wavelength of light.
 Numerical aperture (NA)
NA of lens is used to measure the resolving power.

It is a mathematical expression that explains amount of light

concentrated by condenser and collected by the objective.

The NA is defined by two components

NA= nsin
n= the refractive index of medium in which lens works.
= the ½ half angle of cone of light entering in to the objective.
 When the cone has a narrow angle,
it does not spread out much after leaving the slide and therefore does not
adequately separate images of closely packed objects.

 If the cone has a wide angle,

the light spreads out rapidly after passing through the specimen, closely
packed objects appear widely separated and are resolved.

 The angle of cone of light that can enter a lens depends on refractive index
of the medium in which lens works as well as objective itself.
 sin =1 ( the maximum value of angle  is 90o so
sin 90 will be one ).

 The refractive index of air is 1.00.

 No lens working in air will have NA greater

than1.

 The value of NA can increase only with increase in

n.

 n can be increased , when light rays are passed

through a medium of higher refractive index e.g
immersion oil.

 n of glass and oil are same , so amount of light rays

that can enter the will be much more than the dry
lens. This will increase NA.
 Total resolution
Total resolution i.e d microscope can be calculated by.
d microscope = 
NA(objective)+ NA(condenser)
Most microscopes have NA of condenser 1.2 to 1.4.
However NA of condenser will not be much above 0.9.
Unless the top of condenser is oiled which is usually
not done.
So normally only NA of objective is considered while
calculating resolution of microscope.
 d= (0.5) (530nm) = 2.12nm or 0.2m
1.25
 Thus maximum theoretical resolving power of microscope
with oil immersion objective and blue green light is
0.2m.
 At best a bright field microscope can distinguish between
dots 0.2m apart( size of very small bacterium)
Dark Field Microscopy
Effect produced by dark field
microscopy
 Dark back ground against which objects
are brilliantly illuminated.
Uses a special type of condenser.
It transmits a hollow cone of light from
the source of illumination.
Most of the light transmitted from the
condenser does not enter the objective.
The field is essentially dark.
Some of the light rays will be scattered
(diffracted) if the transparent medium
contains cells.
 The diffracted light will enter the
objective and reach the eye.
 Thus the object or the cell will
appear bright in otherwise dark
field.
 Devices in microscope for dark field
 A dark field stop: an opaque disc ,
which is fitted in to the condenser to
prevent the passage of direct rays of
light.
 An iris diaphragm : in to front of
objective. It is used to decrease the
numerical aperture.
Advantage
Produces high resolution
Objects not visible by bright field can be seen by this
technique.
Significance
 Used to observe living, unstained cells and organisms.
Used to study motility of organisms.
Allows identification of thin bacteria
e.g Treponema pallidium.
Useful in study of morphology of bacteria.
Used to observe antigen antibody reaction.
 Reference :

Prescott, Harley, and Klein’s Microbiology (Seventh

Edition)

Microbiology by Pelczar MJ, Chan ECS and Krieg

NR(1993)