MICROSCOPY

What do you mean by microscopy?

Microscopy is the technical field of using microscopes to

view samples & objects that cannot be seen with the
unaided eye (objects that are not within the resolution
range of the normal eye).
 What is microscopy and its types?

The most familiar type of microscope is the optical or

light microscope, in which glass lenses are used to form
the image.
Other types of microscopes use the wave nature of
various physical processes. The most important is the
electron microscope, which uses a beam of electrons in
its image formation.
 What are the principles of microscopy?

To use the microscope efficiently, should

understand the basic principles of
microscopy: magnification, resolution, numerical
aperture, illumination, and focusing.
 What is the importance of microscopy in
microbiology?

Light (or optical) microscopy is an important tool

used by biologists.
It enables them to study specimens that are too
small to see with the naked eye.
Light (natural or artificial) is transmitted through,
or reflected from, the specimen and then passed
through a system of lenses that produce a
magnified image.
 Who uses microscope?

Some of the major jobs or careers that are known for

their frequent use of the microscope are forensic
scientists, jewelers, gemologists, botanists, and
microbiologists. An example of a career emphasis that
would predominantly use microscopes are researchers
for science and public health.
- A microscope (Greek: mikron = small and
scopeos = to look).

- MICROSCOPE: Is 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.
Anton van Leeuwenhoek
(1632-1723)

Robert Hooke
(1635-1703)
 1.MAGNIFICATION
• Degree of enlargement.
• No of times the length, breadth or diameter, of
an object is multiplied.

• It depends upon – Optical tube length

Focal length of objective
Magnifying power of eye piece

• TOTAL MAGNIFICATION:
magnification of the eyepiece x magnification of
the objective lens.
 2.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 = 0.61 x W = Wavelength
W NA = Numerical aperature
NA
3.NUMERICAL APERTURE(NA)
- Ratio of diameter of lens to its focal length
C B D
- NA = n Sin θ/2
n = refractive index, θ/2
θ = angle of aperture (CAD)

4.DEFINITION
Capacity of an objective to render outline of
the image of an object clear and distinct.
 5.
ABERRATION
SPHERICAL CHROMATIC

Focus of axial rays

Focus of marginal rays

 - Chromatic aberration

Incident
light

Blue focus
Red focus
6.WORKING DISTANCE
Distance between the front surface of
lens and surface of cover glass or
specimen.
 7.CONTRAST
• Differences in intensity between two objects, or
between an object and background.
COMPOUND LIGHT MICROSCOPE
LIGHT MICROSCOPE : use sunlight or
artificial light.
A. Bright field microscope.
B. Dark field microscope.
C. Phase contrast microscope.
D. Fluorescence microscope.

ELECTRON MICROSCOPE : use of

electron.
1. Transmission electron microscope.
2. Scanning electron microscope.
 What is meant by bright field
microscope?
A piece of apparatus, consisting of an eyepiece, an
objective lens, a condenser lens, stage, and light source,
which collects electromagnetic radiation in the visible
range.
 How does bright-field microscopy
work?

In brightfield microscopy a specimen is placed on

the stage of the microscope and incandescent
light from the microscope's light source is aimed
at a lens beneath the specimen.
Some of the light is absorbed by stains,
pigmentation, or dense areas of the sample and
this contrast allows you to see the specimen.
 When would you use a bright field
microscopy?

Bright field microscopy is best suited to viewing stained

or naturally pigmented specimens such as stained
prepared slides of tissue sections or living photosynthetic
organisms.
Can viruses be viewed with a brightfield microscope?
Light microscopy
Standard light microscopes allow us to see our cells
clearly. However, these microscopes are limited by light
itself as they cannot show anything smaller than half the
wavelength of visible light – and viruses are much
smaller than this.
A. BRIGHT-FIELD MICROSCOPE

 Produces a dark image against a brighter

background.

 Has several objective lenses.

 2 types: Simple
Compound
SIMPLE
• Contain a single magnifying lens.

COMPOUND
• Series of lenses for magnification.

• Light passes through specimen into objective lens

• Oil immersion lens increases resolution.

• Have one or two ocular lenses.

• Resolution=200nm
THE EFFECTS OF IMMERSION
OIL ON RESOLUTION
OPTICS OF COMPOUND MICROSCOPE
ADVANTAGE
S
• Used to view live or stained cells.

• Simple setup with very little preparation required.

DISADVANTAGES
• Biological specimen are often of low contrast and
need to be stained.

• Staining may destroy or introduce artifacts.

• Resolution is limited to 200nm.

IMAGE FORMED BY BRIGHT FIELD
MICROSCOPY

(unstained)
 What is a dark field microscope?

Dark-field microscopy is ideally used to illuminate

unstained samples causing them to appear brightly lit
against a dark background. This type of microscope
contains a special condenser that scatters light and
causes it to reflect off the specimen at an angle.
 What is the principle of dark field
microscope?

Principle. To view a specimen in dark field, an

opaque disc is placed underneath the condenser
lens, so that only light that is scattered by objects
on the slide can reach the eye (figure 2). Instead
of coming up through the specimen, the light is
reflected by particles on the slide.
 What is dark field and bright field
microscope?

Darkfield illumination is a technique in optical

microscopy that eliminates scattered light from
the sample image. This yields an image with a
dark background around the specimen, and is
essentially the complete opposite of the
brightfield illumination technique.
 What are the advantages of dark field
microscope?

Darkfield microscopy has many advantages. Its dark

background offers a high degree of contrast, making it
easy to see samples on difficult backgrounds. This
technique is easily accessible since many brightfield lab
microscopes can be configured for darkfield illumination.
B.DARK FIELD MICROSCOPE

• Produces a bright image of the object against a

dark back ground.

• Optical system to enhance the contrast of

unstained bodies.

• Specimen appears gleaming bright against

dark background.
 REQUISITES FOR DARK FIELD
MICROSCOPY

- Dark ground
condenser

- High
intensity
lamp

- Funnel stop
32
OPTICS OF DARK FIELD
MICROSCOPY
 USES OF DARK GROUND
MICROSCOPY

Useful in demonstrating
-Treponema pallidum
- Leptospira
- Campylobacter jejuni
- Endospore

Treponema pallidum
ADVANTAGES
• Simple setup

• Provides contrast to unstained tissue,so

living cells can be viewed.

DISADVANTAGES
• Specimen needs to be strongly illuminated
which can damage delicate samples.
 What is phase contrast microscope?

Phase contrast is an optical contrast

technique for microscopy which makes
unstained structures in the cells of
biological specimens visible. Cell structures
that appear transparent with brightfield
illumination can be viewed in high contrast
and rich detail using phase contrast.
 What is the principle of phase contrast
microscope?

The phase contrast microscopy is based on the

principle that small phase changes in the light
rays, induced by differences in the thickness and
refractive index of the different parts of an
object, can be transformed into differences in
brightness or light intensity.
 What is a phase contrast microscope
used for?

Phase contrast is a light microscopy technique used to

enhance the contrast of images of transparent and
colourless specimens. It enables visualisation of cells and
cell components that would be difficult to see using an
ordinary light microscope.
 Why is phase contrast microscopy
advantages?

One of the major advantages of phase contrast

microscopy is that living cells can be examined in their
natural state without previously being killed, fixed, and
stained.
As a result, the dynamics of ongoing biological processes
can be observed and recorded in high contrast with sharp
clarity of minute specimen detail.
PHASE CONTRAST MICROSCOPE
• First described in 1934 by Dutch physicist Frits
Zernike

• Produces high-contrast images of

transparent specimens.

• Advantage - Living cells can be examined in their

natural state.
 PRINCIPLE OF PHASE CONTRAST
MICROSCOPY
• It is an optical illumination technique in which small phase
shifts in the light passing through a transparent specimen
are converted into contrast changes in the image.

• Light rays in phase produce brighter image.

• Light rays out of phase form darker image.

• Contrast is due to out of phase rays.

38
 REQUISITE FOR PHASE
CONTRAST MICROSCOPY

• Annular Diaphragm

• Phase Plate
 CONDENSER ANNULUS

• The condenser annulus or annular diaphragm

is opaque flat-black (light absorbing) plate with
a transparent annular ring.
• Produces hollow cone of light.
 PHASE PLATE
- Placed in back focal plane of objective.
- Function:
1. Enhances phase difference by
retarding diffracted wave front by one
quarter of wavelength .
2. Reduces intensity of direct rays and
equalizes it with diffracted rays intensity.
OPTICS OF PHASE CONTRAST
MICROSCOPE

42
IMAGES OF PHASE CONTRAST
MICROSCOPY
 ADVANTAGE
• SPhase contrast enables visualization of internal
cellular components.

• Diagnosis of tumor cells .

• Examination of growth, dynamics, and

behavior of a wide variety of living cells in cell
culture.

• Ideal for studying & interupting thin specimen.

DISADVANTAGE
S
• Annuli or ring limits the apperature to
some extents which causes decrease in
resolution.

• Not ideal for thick specimen.

• Shade off and Halo effect may occur.

 ELECTRON
MICROSCOPE
 Co-invented by Max knoll
and Ernst Ruska in
1931.
 Electron Microscopes uses
a beam of highly energetic
electrons to examine
objects on a very fine
scale.
 Magnification can upto
2million times while best
light microscope can 51
 TRANSMISSION ELECTRON
MICROSCOPE (TEM)
 Stream of electrons is formed.

 Accelerated using a positive electrical potential.

 Focused by metallic aperture and Electro

magnets.

 Interactions occur inside the irradiated

sample
which are detected and transformed into
an image.
-Projector Lens forms
image on Fluorescent
viewing screen

- 2D Image

- Magnification
10,000 X to 100,000 X
OPTICS OF
TEM
 SCANNING ELECTRON
MICROSCOPE
- Scan a gold-plated specimen to give a 3-D view
of the surface of an object which is black and
white.

- Used to study surface features of cells and

viruses.

- Scanning Electron microscope has resolution

1000 times better than Light microscope.
OPTICS OF
SEM
 SEM IMAGES

Vibrio cholerae with polar

Treponema pallidum flagella
 COMPARING SEM AND TEM
TEM SEM
Beam focused to fine point;
Electron Beam Broad, static beams
sample is scanned line by line

TEM voltage ranges from Accelerating voltage much lower; not

Voltages Needed
60-300,000 volts necessary to penetrate the specimen
Wide range of specimens allowed;
Interaction of the
Specimen must be very thin simplifies
beam electrons
sample preparation
Information needed is
Electrons must pass through and
Imaging collected near the surface
be transmitted by the
of the specimen
specimen
Transmitted electrons are
Image Rendering Beam is scanned along the surface of
collectively focused by the
the sample to
objective lens and magnified to
build up the image
create a real image
Micrometry
• PROPER STORAGE

• HANDLING

• CARE OF LENSES

• CARE OF OIL EMERSION OBJECTIVE

• CARE OF LAMP
• Helps in measurement of the size
of microscopic objects.

• Achieved by the use of-

1.STAGE MICRO METER
2.MICOMETER EYE PIECE
CALIBRATION OF THE
OCULAR MICROMETER
 Stage micrometer
(objective micrometer) Ocular micrometer with retaining ring inserted
into
has calibration lines or the eyepiece’s base

graduations that are

separated 0.01 mm (10
µm) apart.

 Calibration achieved by Stage micrometer positioning by centering

the small glass disk over the light source
superimposing the two
scales and determining
how many ocular
graduation coincide
with
graduations on stage
micrometer. After completed calibration, specimen’s slide is
positioned for measurement
 Questions ?

Ref: Slideshare
Microbiology by Prescott