optical microscopy

1. Presented by
Aqib Muzaffar
Research scholar
BSAU

2.  A microscope (Greek: micron = small and
scopos = aim)
 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
2

3.  Study of how light passes through thin sections –
rock cut and polished to about 0.3 mm thickness
 Use properties of light absorption and
propagation through a mineral  affected by
atomic arrangement and composition
 Learn the properties of light associated with
techniques governing the use of a petrographic
microscope

4. MAGNIFICATION
 Degree of enlargement
 No of times the length,
breadth or diameter, of
an object is multiplied.
4
VARIABLES USED IN MICROSCOPY
RESOLUTION
‣ Ability to reveal closely
adjacent structural details
as separate and distinct
LIMIT OF RESOLUTION
‣ The min distance between
two visible bodies at which they
can be seen as separate and
not in contact with each other
NUMERICAL APERTURE
‣ Ratio of diameter of
lens to its focal length
‣ NA = n Sin θ/2
n = refractive index,
θ = angle of aperture

5.  Uses visible light as source of illumination
 The Shorter The Wavelength, The Greater
The Resolution (Blue Is The Best)
 Contrast comes from absorbance of light in
the sample, or from staining.
 When the diaphragm is wide open the image
is brighter and contrast is low.

6.  Sources of illumination: Lamp on the base
 Types of image produced: Relatively large
internal structures and outline can be seen
 Total Magnification: (if 10x ocular
magnification is used)
Range: 10x-1000x
 Resolution: Up to 200nm (white light)

7. Image of a bright field microscope

8. Advantage:
Simplicity of setup with only basic equipment
required.
Disadvantage:
1. Very low contrast of most biological samples.
2. Low apparent optical resolution due to the blur of
out of focus material.
3. Samples that are naturally colorless and
transparent cannot be seen well (mammalian cells).
stained before viewing.
Advantages and disadvantages

9.  Type of microscopy which is the exact opposite
of a bright field microscope
 Dark background/field with the specimen being
the only one illuminated.
 Used in observing unstained specimens
 Most microscopes have the potential to do dark
field microscopy such as compound or
stereomicroscopes.

10.  Light source: Light bulb from a microscope
 Condenser type: Specialized to block most light from the
source
 Contains an annular/patch stop which disperses the light in
various directions, resulting to a “cone of light”
 Image formed: Dark background with illuminated specimen;
may be inverted or not depending on microscope used
 Total Magnification: Can range from those of compound
microscopes (10x to 1000x

11.  For viewing objects - unstained, transparent
and absorb little or no light.
 Useful in examining external details
(outlines, edges, grain boundaries and
surface defects than internal structure).
 To study marine organisms ( algae and
plankton), diatoms, insects, fibers, hairs,
yeast and protozoa as well as some minerals
and crystals, thin polymers and some
ceramics.

12.  Images are prone to degradation, distortion
and inaccuracies.
 Sample not thin enough or its density differs
across the slide, may appear to have artifacts
throughout the image.
 Special care that the slide, stage, nose and
light source are free from small particles like
dust - appear as part of the image.
 Not a reliable tool to obtain accurate
measurements of specimens.

13.  Phase contrast microscopy imparts contrast to unstained biological
material by transforming phase differences of light caused by
differences in refractive index between cellular components into
differences in amplitude of light, i.e., light and dark areas.
 Type of light microscope
 Enhances contrast in micrographs by converting phase shifts of light
waves into brightness
◦ Offers more contrast than bright field microscopy
 Does not require the use of staining procedures which usually kill
cells
◦ Especially useful for examining living, un pigmented cells

14.  Source of illumination: Visible light from an
illuminator
 Total Magnification: Phase contrast objective
lenses (Nikon) come in 4x, 10x, 20x, and
40x powers,
 So the total magnification for phase
contrast microscopes range from 40x to
400x

15.  Differences in density (Campbell et. al., 1999) or refractive index
(Tortora et. al., 2007) within the specimen or cell causes light
waves to be diffracted at different degrees
 Diffraction of light waves implies a change in the phase of their
wavelength
 A unique part of the phase-contrast microscope, called the phase-
plate, amplifies this change in phase to one-half wavelength
 When both the direct (undiffracted) and reflected (diffracted) types
of light waves converge at the ocular lens, constructive and
destructive interference occurs

16. Phase plate-
A transparent plate of doubly
refracting material that
changes the relative phase of
the components of polarized
light

17.  The end result is a magnified and highly
contrasted view of a living, unstained,
normally transparent specimen

18.  DIC is an optical microscopy technique used to enhance the contrast
in unstained, transparent samples.
 DIC works on the principle of interferometry to gain information
about the optical path length of the sample, to see otherwise
invisible features.
 The technique was developed by Georges Nomarski
 Differential Interference Contrast (DIC) microscopy converts phase
shift gradients across different parts of a specimen into intensity
differences.
 Doesn’t suffer from some artifacts seen in phase contrast

19. Sample
Objective
Tube lens
Projection Eyepiece
Camera
Imaging
path
Aperture iris
Field iris
Light source
Illumination
path
Collector
Condenser lens
Field lens
Wollaston
Wollaston
Polarizer (analyzer)
Polarizer

21.  DIC works by separating a polarized light source into
two orthogonally polarized mutually coherent parts
which are spatially displaced (sheared) at the sample
plane, and recombined before observation.
 The interference of the two parts at recombination is
sensitive to their optical path difference (i.e. the
product of refractive index and geometric path
length).
 The contrast is proportional to the path length
gradient along the shear direction giving the
appearance of a three-dimensional physical relief
corresponding to the variation of optical density of
the sample, emphasising lines and edges though not
providing a topographically accurate image.

23. Principle of Fluorescence
1. Energy is absorbed by the atom which
becomes excited.
2. The electron jumps to a higher energy
level.
3. Soon, the electron drops back to the
ground state, emitting a photon (or a
packet of light) - the atom is fluorescing.

24. It was developed by Haitinger and coons
It utilizes a powerful mercury vapour arc lamp for its light source.
A dark field condenser is usually used in place of the conventional Abbé
bright field condenser.
It employs three sets of filters to alter the light that passes up through
the instrument to the eye.
Microbiological specimen that is to be studied must be coated with special
compounds that possess the quality of fluorescence. Such compounds are
called fluorochromes.
AuramineO, acridine orange, and fluorescein are well-known fluorochromes.

25. 25
 PRINCIPLE
UV light
Fluorochrome
Visible
radiation
 UV rays passes through exciter filter
 Dark ground condenser
 Micro organisms stained with fluorescent dye, when
examined under microscope with ultraviolet light
are seen as bright object against dark background

26. Darkfield
Brightfield
Darkfield
Fluorescence
Comparison of optical microscopy
DIC IMAGE
PHASE CONTRAST IMAGE

27. Thank
You