MICROSCOPY-PRINCIPLES AND TYPES

MICROSCOPE
• Microscope is an optical instrument consisting of one
or more lenses to magnify images of minute objects

• By ‘magnification’ it provides us to see microorganisms

and structures invisible to the naked eyes

• The magnification attainable by using microscope

range from100x to 400,000x
 Relative size of the microorganisms and their visibility

Man can see about 0.5 mm sized object whereas,

•Light microscopes can be used to visualize up to 1 m.
•Electron microscopes can be used to view 1 nm sized
objects.
 PRINCIPLES OF LIGHT MICROSCOPY

• The magnification is obtained by a system of

optical lenses using light waves
• Magnification refers the number of times a
specimen is appeared to be larger than its
original size.
 MAGNIFICATION
• It is the product of the magnifications of objective lens
and eyepiece.

Objectives : Low power, high dry and oil immersion

Eye piece : 5X,10X,15X

• Magnification of about 1500x is the upper limit of

compound microscopes. This limit is set because of the
resolution.
 RESOLUTION
• Resolution is dictated by the physical properties of the
light
• It is the ability to distinguish two adjacent points as
distinct and clear
Limit of resolution
• Smallest distance by which two objects can be separated
and still distinguishable as two separate objects
• RP =  / 2NA
• NA is Numerical aperture
•  is wave length of light used
 RESOLVING POWER

• Basic limitation of the bright field microscope

• Magnification without resolution is not
beneficial, since the image obtained may be
unclear or fuzzy
• RP of the microscope is the function of wave
length of light used and the numerical aperture
of the lens system
 NUMERICAL APERTURE
• Measure of light gathering ability of the lenses
NA = n sin 
Where,
 - angle between the optical axis and the outermost
rays covered by the object ie., half aperture angle. If
‘’ is increased, oil immersion lens is used
n - refractive Index of the medium filling the space
between front lens and cover slip
• Resolution increases when numerical aperture
increases
 ANGULAR APERTURE AND RESOLUTION

=32 =60

n=1.0 n=1.2

Immersion oil

Cover slip
Specimen

Substage condenser

Numerical aperture of oil immersion objective lens

Numerical aperture of dry objective lens
NA = 0.529 NA = 1.03
 REASON FOR INCREASED NUMERICAL APERTURE
WHILE USING OIL IMMERSION OBJECTIVE LENS

• The light passed the glass slide and specimen and

reached to objectives.

• The medium between the specimen and

objective lens is air. Since, the refractive index of
air is lower than glass slide, the light passes from
slide to objective may bend or refract, which may
reduce the resolution.
• When oil (immersion oil / cedar wood oil) is placed
between slide and objective, the refraction of light can be
reduced. (The refractive index of oil is equal to glass
slides, of about 1.5).

• Hence, the numerical aperture of oil immersion objectives

are more than dry objectives, leads to good resolution.

• The sub-stage condenser (located below the specimen

stage of the microscope) increased the angle of light cone
too. Theoretical limit of R.P. for light scope is 0.2
micrometers.
 INCREASE OF RESOLVING POWER
Two ways,
• Decrease in wavelength of light used
• Increase in numerical aperture
1. Reducing the 

• Blue – 400 nm; red – 700 nm; green – 550 nm

• Resolution is greatest with blue light
2. Increasing NA (n sin)
• Numerical aperture measures how much light
cone spreads out between condenser &
specimen.
• More spread of light gives better resolution.
• The numerical aperture depends on the
objective lens of the microscope.
a. Dry objective lens
b. Oil-immersion objective lens
• The oil immersion objective lens has more NA
than dry objective lens.
Optical Instrument Resolving Power RP in Angstroms
(RP)

Human eye 0.2 mm 2,000,000 A

Light microscope 0.20 µm 2000 A

Scanning electron 5-10 nm 50-100 A

microscope (SEM)

Transmission 0.5 nm 5A
electron
microscope (TEM)
QUALITY PARAMETERS OF MICROSCOPIC IMAGES

1.Focus:
– It refers whether the image is well defined or
blurred (out of focus).
– The focus can be adjusted through course and
fine adjustment knobs of the microscope which
will adjust the focal length to get clear image.
– The thickness of specimen, slide and coverslip
also decide the focus of the image. (Thin
specimens will have good focus).
2. Brightness:
– It refers how light or the dark the image is.
– Brightness of the image depends on the
illumination system and can be adjusted by
changing the voltage of the lamp and by
condenser diaphragm.
3. Resolution:
– It refers the ability to distinguish two objects
close to each other.
– The resolution depends on the resolving
power, which refers minimum distance
between the two objects which can be
distinguishable
4. Contrast:
– It refers how best the specimen is
differentiated from the background or the
adjacent area of microscopic field.
– More the contrast will give good images.
– It depends on the brightness of illumination
and colour of the specimen.
– The contrast can be achieved by adjusting
illumination and diaphragm and by adding
colour to the specimen.
 TWO MAIN CATEGORIES OF MICROSCOPE

• Depending upon the principles on which

magnification is based,

a. Light Microscope - used to look at intact cell

b. Electron Microscope - used to look at
internal structure or details of cell surfaces

• In all microscopes specific lenses magnify the

images of the objects
a) Light Microscope: Magnification is obtained
by a system of optical lenses using light
waves.

It includes,
(i) Bright field
(ii) Dark field
(iii) Fluorescence
(iv) Phase contrast and
(v) UV Microscope.
b. Electron Microscope: A system of
electromagnetic lenses and a short beam of
electrons are used to obtain magnification. It
includes,
(i) Transmission electron microscope
(TEM)
(ii) Scanning electron microscope (SEM)
 LIGHT MICROSCOPY

Broadly grouped into two categories. They are,

(a) Simple microscope: It consists of only one bi-

convex lens along with a stage to keep the
specimen.
(b) Compound microscope: It employs two separate
lens systems namely,
- objective lens and
- ocular (eye piece)
 BRIGHT FIELD MICROSCOPY

• The compound student microscope is a bright

field microscope

• Microscopic field is brightly lighted and

microorganisms appear dark since they absorb
some of the light when they are stained.

• Generally they produce a magnification of about

1000x to 2000x
BRIGHT FIELD MICROSCOPY
 DARK FIELD MICROSCOPE
Principle:
•In a dark-field microscope, the object is brilliantly
illuminated against a dark background This is
accomplished by equipping a light microscope
with a special kind of condenser.
•Condenser with a dark-field stop, which is an
opaque disc obstructing the path of light from the
light source centrally, but allowing a peripheral
ring of light.
 Thus, the condenser transmits a hollow cone of
light from the light source.
 This cone of light converges on the object and
diverges from there again as an inverted hollow
cone.
 Thus, no light enters into the objective, as it
remains in the dark cone and the field essentially
appears dark in absence of any object
 However, if some objects such as microbial cells
are present, some of the light rays are scattered
(diffracted) by them.
• These diffracted rays enter into the objective
and reach the eye.
• Thus, the object (microbial cells) appears
bright in an otherwise dark microscopic field.
DARK FIELD MICROSCOPE
DARK FIELD MICROSCOPE
BRIGHT FIELD MICROSCOPY

DARK FIELD MICROSCOPE

 USES OF LIGHT MICROSCOPY

1. Study the morphology and motility of

microorganisms.
2. Dark field - Living, unstained cells and
organisms, useful for finding cells in suspension
3. Initial survey and observation at low powers of
pond water samples, hay or soil infusions,
purchased protist or metazoan cultures.
4. Bright field : dead, stained cells and organisms
 PHASE CONTRAST MICROSCOPE
• In the 1930’s Frederick Zernike devised a method of
converting phase changes into differences in light
intensity.
• Converts slight differences in refractive index and cell
density into easily detectable variations in light intensity
• This invention leads to the development of phase contrast
microscope.
• It has a special type of
• condenser,
• objective and
• a special magnifier.
• Light passing from one material into another of
slightly different refractive index will undergo a
change in the phase.
• This change in the phase of the light will in-
turn increase the contrast.
• A system of rings in the condenser and
objective separate the diffracted rays from the
specimen.
• These diffracted rays from the specimen and
the undiffracted rays combine and the phase
difference is converted into difference in light
intensity.
• Uses:
1.Internal contrast of various parts of a
specimen against its surroundings is
increased.
2.Unstained/living organisms can be examined.
3.It has a resolving power of 0.1 – 0.2 µm.
4.Bacterial motility, bacterial shape, inclusion
bodies and bacterial endospores
5.Widely used to study the eukaryotic cells.
PHASE CONTRAST MICROSCOPE
Use:
Observation of unstained cells because of its
ability to generate images that reveals the
internal cell structures that are less apparent
by bright field techniques.
three-dimensional structures
Structures such as cell walls, endospores,
granules, vacuoles, and eucaryotic nuclei are
clearly visible
Bright field, Phase contrast and Dark Field images
 UV MICROSCOPE

• Resolution of a microscope depends upon the

wavelength of light used.

• If, longer the wavelength of light used, lower

will be the resolving power while shorter the
wavelength, more will be the resolution.

• With this principle, UV rays of shorter

wavelength are used as light source.
• Since UV rays can’t penetrate the glass, quartz
lenses are used.
• Since the UV rays are invisible, photographic
plates should be used to record the image or
special type of filters should be used to
eliminate the UV rays from reaching the
eyepiece.
• This is used in conjunction with fluorescent
microscopy.
• Upon illumination with UV light, certain
fluorescent dyes emit light in visible range,
which can be directly viewed.
UV MICROSCOPE
 FLOURESCENCE MICROSCOPE

• A high intensity mercury lamp is used as the light

source, which emits white light.

• The exciter filter transmits only blue light to the

specimen and blocks out all the colours. The blue
light is reflected downward to the specimen by the
dichroic mirror.

• The specimen is stained with fluorescent dye

(acridine orange). Only certain portions of the
specimen retain the dye, others do not.
• The stained portion of the specimen absorb
blue light and emit green light, which passes
upwards, penetrate the dichoric mirror and
reaches the barrier filter.

• This filter allows only green light to pass

through and the eye receives only green light
emitted from the specimen
FLOURESCENCE MICROSCOPE
• Also, ultraviolet light is used to excite molecules so
that they release light of a different wavelength.

Uses:
• This technique is especially important in immunology
in which the reactions of antigens and antibodies are
studied in great detail.
• Fluorescent antibody staining is now widely used in
diagnostic procedures to determine whether an
antigen is present.
• Not all bacteria get stained with fluorescent chemicals.
FLOURESCENCE MICROSCOPE
Limitation of light Microscope
•500x - 1000x magnification

•Resolution = 0.2 micrometers.

•Detailed internal structure of larger

microorganisms also cannot be effectively
studied by light microscopy
 ELECTRON MICROSCOPE
• In electron microscope, short beam of electrons
and magnetic condenser lenses are employed to
produce the image.

• The electrons have short wavelength, which helps

in better resolution.

• It is possible to resolve objects as small as 5A or

0.5nm, which is 1000 times more than that of light
microscope. It can magnify object up to 200,000X.
• Developed by Max Knoll and Ernst Ruska in
Germany in 1931.
• Scanning Electron Microscope (SEM) debuted
in 1942
• In electron microscope, a hot tungsten
filament forms the source of electrons.
• The object is placed in the path of moving
electrons.
• Since electrons move only in the vaccum, the
entire path of electrons should be kept under
vaccum.

• The magnetic condenser lens causes the

primary magnification.

• A second magnetic lens amplifies the primary

image and this image is viewed on a
fluorescent screen or captured on
photographic plates
 TYPES OF ELECTRON MICROSCOPE

There are two types of electron microscope

1. Transmission electron microscope (TEM)

2. Scanning electron microscope (SEM)
 TRANSMISSION ELECTRON MICROSCOPE

A high voltage established between the filament and the

anode accelerates the electrons from the hot tungsten
filament.
• The electron beam is focused on the specimen with an
electro magnetic condenser.
• Ultra thin sections of the specimen(20 to 100 nm) must be
prepared since electrons can penetrate matter only a
short distance. This is done by embedding or freezing the
specimen and sectioning it with a diamond or a glass
Knife.
• The sections are floated in water and collected in
a copper grid. They are stained with heavy metals
such as gold or palladium and kept within the
evacuated column of the electron microscope.
• TEM has a projector lens that project the image
onto a fluorescent viewing screen or film plate,
because the beam cannot be viewed directly.
• With TEM greater resolution and higher
magnifications than light and Scanning Electron
Microscope can be obtained.
• In TEM, the differential scattering of electrons by
the specimen makes the contrast. Since most of
the atoms of the biological material are of low
mass, the contrast of the specimen is low.
• Staining with heavy metals such as platinum,
uranium or tungsten can increase the contrast.
They are of various types.
• Positive staining: The heavy metals are fixed
on the specimen.
• Negative staining: It is used to increase the
electron opacity of the surrounding area.
TRANSMISSION ELECTRON MICROSCOPE
 Techniques commonly employed for the
observation of biological specimen

Metal shadowing: The dried specimen is exposed

at an acute angle to a stream of heavy metals
like platinum, palladium or gold and thereby
producing an image, that reveals the three-
dimensional structure of the object.
• The penetrating power of electrons is low;
hence the ultra thin sections of the specimens
should be used.
• Freeze fracturing: The frozen specimen is
fractured with a knife, and the exposed
surface is coated with a heavy metal (Gold) at
an acute angle. A supporting layer of carbon is
evaporated on the metal surface. Then the
specimen is destroyed and the replica is
examined. This method is used for studying
cell wall and cell membrane.
SPECIMEN HOLDER GRID
 SCANNING ELECTRON MICROSCOPE

• A scanning electron microscope (SEM) is a type

of electron microscope that produces images
of a sample by scanning it with a focused beam
of electrons
• SEM can achieve resolution better than 7
nanometer
• Specimens can be observed in high vacuum, in
low vacuum, in wet conditions (in
environmental SEM), and at a wide range of
cryogenic or elevated temperatures.
• The specimen is coated with a thin layer of
heavy metal and the specimen is subjected to
a narrow beam of electrons, which rapidly
moves and scans the surface of the specimen.
• The irradiated specimen depending upon its
physical and chemical composition will release
secondary electrons
• These secondary electrons are then collected
by anode detector, which generates an
electronic signal
• Then the electronic signal is scanned in TV
system to produce an image on a cathode ray
tube. Magnification on SEM is about 75,000 to
1,00,000 times.
SCANNING ELECTRON MICROSCOPE
LICHENS

Root Nodules & Rhizobium

Advantages:
• The use of electrons rather than light provides
a nearly 1000 fold increase in resolving power
(i.e., ability to focus fine details) over light.
• Yield information about the topography,
morphology, composition and
crystallographic information (how the atoms
are arranged in the object)
 LIMITATIONS

• Specimen is kept under high vacuum on the path of

electron beam. So, living cells can’t be examined.
• Electrons have low penetration capacity, hence
ultra thin section and staining should be done
which is time consuming and also sometimes alter
or distort the structures of microorganisms.
• High cost and specialized techniques prevent its
use in all microbiology labs in spite of greater
magnification and resolution.
 ATOMIC FORCE MICROSCOPY

• Three dimensional imaging of the biological

structures
• It has an tiny stylus (cantilever) silicon or silicon
nitrate - that is extremely positioned close to
the specimen such that weak repulsive atomic
forces are established between probe and the
specimen, when laser light is passed
• When the specimen is scanned, the stylus
ridges up and down the hills and valleys of the
specimen surface and recorded it’s interaction
• Detectors feed the digital information into the
computer, that generates the image
Uses:
• Allows living and hydrated specimens to be
viewed, something that is not possible with
electron microscope
• Specimen preparation is similar to light
microscopy
• to study biological macromolecules and
even living organisms
• provide higher resolution than SEM
High-resolution AFM image of Zn2SnO4/glass, where grains smaller
than 100 Å can be observed.
Disadvantages
• Single scan image size, which is of the order of
150x150 micrometers, compared with
millimeters for a scanning electron
microscope.
• Relatively slow scan time, which can lead to
thermal drift on the sample.
 CONFOCAL SCANNING LASER MICROSCOPY
• Computerized microscope that couples a laser light
source to a light microscope

• Precisely illuminates only a single plane of the

specimen, illuminating intensity drops off rapidly
above and below the plane of focus

• So the stray light from other plane of focus is

minimized

• In biofilms, cells in various layers can also be

observed in one plane of focus
• This technique allows for the generation of
three-dimensional digital images of
microorganisms and other biological
specimens

Use:
• Used mostly in microbial ecology, especially
for identifying phylogenetically distinct
populations of cells present in a microbial
habitat
CONFOCAL LASER MICROSCOPE