Principles, structure and apllications of bright field and dark field microscopy1 copy

SEMINAR ON PRINCIPLES, STRUCTURE AND
APPLICATION OF BRIGHT FIELD AND DARK
FIELD MICROSCOPY
Presented by
Selvaraj.p
Ph.D Scholar
Oct-2019 Batch Guide
Dr.Sasi.Vaithilingan
Professor Cum Vice-Principal
VMCON Pondicherry.

Define the bright field and dark field microscopy
List out the types of microscopy
Understand the structure of the bright field and dark field microscopy
Knows the principles of the bright field and dark field microscopy
Describe the application of the bright field and dark field microscopy
State the advantage and limitation of the bright field and dark field microscopy
Distinguishes between the bright field and dark field microscopy
Summarize usage and performance of the bright field and dark field microscopy
4/24/2020 VMRF(DU) NSG 19 OCT 07

INTRODUCTION
• A microscope is an instrument used to see objects
that are too small to be seen by the naked eye. .
• The magnifying power of a microscope is an
expression of the number of times the object
being examined appears to be enlarged and is a
dimensionless ratio
4/24/2020 VMRF(DU) NSG 19 OCT 07

DEFINITION
• The word “microscope” comes from the Latin
“microscopium,” which is derived from the Greek
words “mikros,” meaning “small,” and “skopein,”
meaning “to look at.”
• MICROSCOPE - An instrument for viewing objects that
are too small to be seen by the naked or unaided eye.
• MICROSCOPY - The science of investigating small
objects using such an instrument is called microscopy
4/24/2020
VMRF(DU) NSG 19 OCT 07

4/24/2020 VMRF(DU) NSG 19 OCT 07

CLASSIFICATIONS
Bright field or light Microscope
Dark field Microscope
Phase contrast Microscope
Fluorescence Microscope
Electron Microscope
Transmission electron microscope
Scanning electron microscope
4/24/2020 VMRF(DU) NSG 19 OCT 07

BRIGHT-FIELD MICROSCOPY
• Bright-field microscopy is the simplest of all
the optical microscopy illumination techniques.
• Bright-field microscopy is the simplest of a range of
techniques used for illumination of samples in light
microscopes,
• The typical appearance of a bright-field microscopy
image is a dark sample on a bright background.
4/24/2020 VMRF(DU) NSG 19 OCT 07

DEFINITION
• Bright field microscopy can define as the optical
microscopy, which is the simplest of all the illumination
techniques, wherein a smear, the stained or the dense
part appear darker with a white or brighter
background.
• It is a type of light microscopy, where a path of light is
very simple, which requires a light source like a halogen
lamp, condenser lens, objective lens and ocular lens.
4/24/2020 VMRF(DU) NSG 19 OCT 07

PRINCIPLES AND APPLICATION BRIGHT-
FIELD MICROSCOPY
• Bright field microscopy is the most elementary
form of microscope illumination techniques.
• The name "bright field" is derived from the fact
that the specimen is dark and contrasted by the
surrounding bright viewing field.
• Simple light microscopes are sometimes referred
to as bright field microscopes.
4/24/2020 VMRF(DU) NSG 19 OCT 07

STRUCTURE
4/24/2020 VMRF(DU) NSG 19 OCT 07

PERFORMANCE
4/24/2020 VMRF(DU) NSG 19 OCT 07

RESOLUTION
• It is the power of the optical lens, which
distinguishes between the two particular bodies
that held very close to each other.
• The resolution of the bright field microscope
depends upon the two factors:
1. Numerical aperture
2. Wavelength of light
4/24/2020 VMRF(DU) NSG 19 OCT 07

CONTRAST
• To obtain a clear, finely detailed image under a compound
microscope, specimen must be made to contrast sharply
• To attain such contrast, we must change the refractive index
of specimens from that of their medium.
• The refractive index is a measure of the light bending ability
of a medium.
• The refractive index of specimens can be changed by staining
the later.
• Light rays move in a straight line through a single medium.
4/24/2020 VMRF(DU) NSG 19 OCT 07

CON’T
• After staining when light rays pass through two
materials (the specimen and its medium) with different
refractive indexes, the rays changes direction (refract)
from a straight path by bending or changing an angle at
the boundary between the materials and increase the
image contrast between the specimen and the medium
as the light rays travel away from the specimen, they
spread out and enter the objective lens, and the image
is thereby magnified.
4/24/2020 VMRF(DU) NSG 19 OCT 07

STEPS OF BRIGHT FIELD MICROSCOPY
4/24/2020 VMRF(DU) NSG 19 OCT 07

USES OF BRIGHT FIELD MICROSCOPY
Bright field microscopy is best suited to viewing stained
or naturally pigmented specimens such as
1.stained prepared slides of tissue sections or living
photosynthetic organisms.
2. it's useless for living specimens of bacteria, and
inferior for non-photosynthetic protists or metazoans,
or unstained cell suspensions or tissue sections.
4/24/2020

CON’T
3.Prepared slides, stained - bacteria (1000x), thicktissue sections
(100x, 400x), thin sections with condensed chromosomes or
specially stained organelles (1000x), large protists or
metazoans(100x).
4.Smears, stained - blood (400x, 1000x), negative stained bacteria
(400x, 1000x)
5. Living preparations (wet mounts, unstained) – pond water (40x,
100x, 400x), living protists or metazoans(40x, 100x, 400x
occasionally), algae and othermicroscopic plant material (40x,
100x, 400x). Smaller specimens will be difficult to observe
without distortion, especially if they have no pigmentation
4/24/2020

An example bright-field microscopy. This image shows a
cross-section of the vascular tissue in a plant stem.
4/24/2020 VMRF(DU) NSG 19 OCT 07

Bright field microscopy
is very simple to use
with fewer adjustments
The optics used in the
bright field technique
don’t alter the color of
the specimen.
It is adaptable with new
technology and optional
pieces of equipment can
be implemented with
bright field illumination
to give versatility in the
tasks it can perform.
ADVANTAGES OF BRIGHT-FIELD MICROSCOPY
4/24/2020 VMRF(DU) NSG 19 OCT 07

DISADVANTAGES OF BRIGHT-FIELD
MICROSCOPY
Bright field microscopy can’t be used to observe living
specimens of bacteria, although when using fixed
specimens, bacteria have an optimum viewing
magnification of 1000x.
By using an aperture diaphragm for contrast, past a
certain point, greater contrast adds distortion.
4/24/2020

DARK FIELD MICROSCOPY
• Dark field optics is a low cost alternative to phase
contrast optics. The contrast and resolution
obtained with inexpensive dark field equipment.
• It is surprising that few manufacturers and vendors
promote the use of dark field optics.
4/24/2020 VMRF(DU) NSG 19 OCT 07

STRUCTURE
4/24/2020

PRINCIPLES AND APPLICATION OF DARK
FILED MICROSCOPY
• A dark field microscope is arranged so that the light
source is blocked off,
• This is ideal for making objects with refractive
values.
• When light hits an object, rays are scattered in all
azimuths or directions.
4/24/2020 VMRF(DU) NSG 19 OCT 07

CON’T
• The introduction of a condenser and/or stop below
the stage ensures that these light rays will hit the
specimen at different angles
• The result is a “cone of light” where rays are
diffracted, reflected and/or refracted off the object,
• The dark-ground microscopy makes use of the dark-
ground microscope, a special type of compound
light microscope
4/24/2020

CON’T
• The dark-field condenser with a central circular stop,
which illuminates the object with a cone of light
• This microscope uses reflected light instead of
transmitted light
• It prevents light from falling directly on the objective
lens.
• Light rays falling on the object are reflected or
scattered onto the objective lens with the result that
the microorganisms appear brightly4/24/2020

USE OF DARK FILED MICROSCOPE
• It is useful for the demonstration of very thin bacteria
not visible under ordinary illumination
• This is a frequently used method for rapid
demonstration
• It is also useful for the demonstration of the motility of
flagellated bacteria and protozoa.
• Dark field is used to study marine organisms such
as algae, plankton, diatoms, insects,
fibres, hairs, yeast and protozoa
4/24/2020 VMRF(DU) NSG 19 OCT 07

CON”T
• Determination of motility in cultures.
• Helpful in diagnosing of spirochete, spirochaete and spiroc
hete infection.
• Demonstration of live recent blood and its components-
ability to for operate and therefore the cellular resistance
of the leucocytes--- worth for immune disorders and
tumours.
4/24/2020 VMRF(DU) NSG 19 OCT 07

Con”t
• Therapeutic tests can even be allotted by adding the
medication on to the blood sample
and perceptive the reaction.
• The examination is very motivating for the patient
since he will witness the diagnostic findings
directly aboard the medical man
• Unstained, transparent and absorb little or no light.
4/24/2020 VMRF(DU) NSG 19 OCT 07

CON”T
• To study insects, fibers, hairs, yeast and protozoa as well as some
minerals and crystals, thin polymers and some ceramics.
• In the research studying of live bacterium, as well as mounted cells
and tissues e.g. Treponema pallidum, Leptospira, Vibrio cholera,
Campylobacter jejuni, Endospore
• It is more useful in examining external details, such as outlines,
edges, grain boundaries and surface defects than internal structure.
• Recently, dark field combined with other illumination techniques,
such as fluorescence, which widens its possible employment in
certain fields
4/24/2020 VMRF(DU) NSG 19 OCT 07

ADVANTAGES OF DARK FIELD MICROSCOPY
• Dark-field microscopy is a very simple yet effective
technique.
• It is well suited for uses involving live
and unstained biological samples, such as a smear from
a tissue culture or individual, water-borne, single-celled
organisms.
• Considering the simplicity of the setup, the quality of
images obtained from this technique is impressive.
4/24/2020 VMRF(DU) NSG 19 OCT 07

Con’t
• Dark-field microscopy techniques are almost
entirely free of artifacts, due to the nature of the
process.
• A researcher can achieve a dark field by making
modifications to his/her microscope.
4/24/2020 VMRF(DU) NSG 19 OCT 07

LIMITATION OF DARK FIELD MICROSCOPY
• The main limitation of dark-field microscopy is the
low light levels seen in the final image.
• The sample must be very strongly illuminated,
which can cause damage to the sample.
4/24/2020 VMRF(DU) NSG 19 OCT 07

DIFFERENT BETWEEN THE BRIGHT AND
DARK FIELD MICROSCOPY
BRIGHT FIELD MICROSCOPY DARK FIELD MICROSCOPY
Light from a plane-wave
source is focused through an
object by a condenser.
An opaque disc is put
between the source and the
condenser, blocking out the
middle of the beam.
Some light is blocked
(absorbed) by opaque parts of
the object, or reflected away
at boundaries between
components/materials of
different refractive indices.
The condenser focuses the
beam onto the sample.
4/24/2020

The remainder passes
through the objective lens, to
the observer
No light enters the objective
directly from the source. Light
from the beam is scattered by
the sample – some scattered
into the objective.
This produces a bright
background, with object
details appearing darker in the
image than their
surroundings. The brightness
of the brightest parts of the
image is determined by the
source brightness and block
size.
Only light scattered by the
object enters the objective.
This produces a dark
background, with sample
details appearing brighter
than surroundings. The
brightness of the brightest
parts of the image is
determined by the amount of
light scattered by the object.4/24/2020 VMRF(DU) NSG 19 OCT 07

This results in poorer contrast
compared to dark field, as the
dark areas are generally grey
rather than black
This results in superior
contrast to bright-field, as
dark areas may be completely
black, while increasing the
brightness of the light source
brightens the bright areas
4/24/2020 VMRF(DU) NSG 19 OCT 07

CONCLUSION
• The light microscope is a very powerful tool for understanding
the structure and function of tissues, and it is widely used in
biomedical science courses, as well as in research and
diagnostic laboratories. Understanding the capabilities and
limitations of the light. Microscope is important if one is to
get the best results from microscopy.
• When light hits an object, rays are scattered in all azimuths or
directions. The design of the dark field microscope is such
that it removes the dispersed light, or zeroth order, so that
only the scattered beams hit the sample
4/
24
/2
02
0
VMRF(DU) NSG 19 OCT
07

REFERENCE
1. Parija S.C. (2012). Textbook of Microbiology &
Immunology.(2 ed.). India: Elsevier India.
2.https://www.google.co.in/search?q=uses+of+dark+f
ield+microscopy&tbm.
3.https://www.britannica.com/technology/microscop
e
4.https://biologyreader.com/bright-field-
microscopy.html
5. https://microbenotes.com/darkfield-microscopy/
6.tps://www.google.co.in/search?q=uses+of+bright+fi
eld+microscopy&tbm=isch&source=iu&ictx
4/24/2020
VMRF(DU) NSG 19 OCT
07

Principles, structure and apllications of bright field and dark field microscopy1 copy

More Related Content

What's hot (20)

Similar to Principles, structure and apllications of bright field and dark field microscopy1 copy (20)

More from selvaraj227 (20)

Recently uploaded (20)

Principles, structure and apllications of bright field and dark field microscopy1 copy