A SEMINAR

ON

SPECIAL MICROSCOPY
TECHNIQUES
PRESENTED BY:

Dr. K. Lalith Prakash Chandra

Post Graduate,
Dept. Of Oral & Maxillofacial Patholo
SIBAR Institute Of Dental Sciences
Guntur

INTRODUCTION:
A majority of the subjects examined or
photographed through a conventional
microscope
appear either dark or colored, against a light
background.
Dark - is due to light absorption within their
elements.
Colored - is usually because they have been
treated with biological stains, to produce
color
contrast - a result of differential absorption of
various stains by the elements of the
specimen.

 Many unstained specimens exhibit little or no

contrast, when viewed against a bright
background.
They are
transparent.

colorless

and

comparatively

Hence - Are practically invisible.

When staining a specimen is either impossible
or
undesirable, a conventional Bright-field
microscope
cannot be used for photo-micrography.
Alternative microscopic technique is to be
selected.

DARK-FIELD MICROSCOPY:
Allows the low contrast samples to be
examined.
Dark field optics are a low cost alternative to
phase
contrast optics.

The contrast and resolution - with dark field

equipment are superior to the phase contrast
equipment.

THEORY OF DARK-FIELD MICROSCOPY:

Many transparent and semi-transparent
specimens
are not readily visible in a bright-field
microscope.
Their visibility can be improved greatly by the
Darkfield illumination method
The specimen is seen as a bright object against
a
dark or even-black background.
Specimens are visible in a microscope because
of
1. Their ability to alter the refractive index

 To see a specimen in a brightfield microscope,

the
light rays passing through it must be changed
sufficiently to be able to interfere with each
other
this produces contrast (differences in light
intensities) and, thereby, build an image.
If the specimen has a refractive index that is
too
similar to the surrounding medium (between
the
microscope stage and the objective lens), it
will not
be seen.
The dark-field microscope allows the viewer to

LIGHT PATH IN A DARK-FIELD MICROSCOPE:

The cone of light
normally
illuminating the
specimen
must not enter the
microscope objective.

The
condenser
is
designed
to form a hollow cone
of
light, rather than
illuminating the
sample
with a filled cone of
light.

 As this light moves past

the specimen plane, it
spreads again into a
hollow cone.
The objective lens sits
in
the dark hollow of this
cone.
Though the light travels
around and past the
objective lens, no rays
enter it.


The
entire
field
appears
dark when there is
no
sample on the
microscope stage;
thus
the name darkfield
microscopy.
When a sample is on
the
stage, the light at
the
apex of the cone
strikes
it.

 The image appears bright against the dark

background.

Dark field optics rely on scattered light

hence, we
generally need the maximum illumination
intensity view of the specimen
Dark-field
Bacteria in a
possible.

Diagram to illustrate the difference between the

Dark-field & Bright-field illumination

The darkfield effect can be achieved in a

brightfield microscope, by the addition of :
a. Dark-field diaphragm stops
(or)
b. Special dark-field substage condensers like

1.Abbe condenser
2.Paraboloid condenser
3.Cardiod condensers
a. The stop - a piece of opaque material placed
below
the substage condenser.
It blocks out the center of the beam of light
coming

1.ABBE CONDENSER:
This is used with a low-power objective or
objects which do not need high magnification.
It could be used either with the stop or by
unscrewing the top element and replacing it
with a dark-field element.

2. PARABOLOID CONDENSER:
It is used with high-power oil-immersion
objectives
and an intense source of light.
The oil is placed between the bottom of the
glass
slide and the top of the condenser.
Care must be taken that bubbles are not
introduced
either in the oil under the oil-immersion
objective or
in the oil between the slide and the condenser.
Special dark-field condensers are to be used
with oil
- particular care must be taken with regard to
the

3. CARDIOID CONDENSER:
This is the most refined type of dark-field
illuminator.
It is used in the examination of objects like
colloidal
solutions, (particles less than 0.25mm).
A strong arc lamp, and preferably the fused
quartz
object slides and cover slips, are used with
this
condenser.
For darkfield at higher NA, special cardioid
darkfield condensers using mirror surfaces are
manufactured.
These are oiled to the slide (instead of oil,

 Darkfield condensers have two NAs that should

be
considered:
a. The inner NA (i.e. the portion of the light
that
is blocked) and
b. The outer NA (which for technical
reasons is
limited to at most 1.4).
So - cannot be used with objectives of NA
Inner &1.2
outer NA of a cardioid condenser
A Zeiss Cardiod condenser
above
or higher.

ADVANTAGES OF DARK-FIELD MICROSCOPY:

1.Contrast
in
dark-field
is
extreme
resolution is as
high as the objective can yield.

and

2.It is spectacular for observations of live

protozoa or bacteria (cilia or flagella are
visible) and for diatoms.
3.Dark-field illumination is most readily set up
at low magnifications (up to 100x); although it
can be used with any dry objective lens.
4.It is the best method to view everything in a
liquid sample, and debris.
5.Even tiny dust particles are obvious.

6. It is especially useful for finding cells in

suspension, such as yeast, bacteria, small
protists, or
cell and tissue fractions including cheek
epithelial
cells, chloroplasts, mitochondria, even blood
cells.
7. Dark field makes it easy to obtain the correct
focal
plane at low magnification for small, low
contrast
specimens.
8. It is also used for initial survey and
observation at
low powers of pond water samples, hay or soil

9. Determination of motility in cultures.

10. In marine biology, a dark-field microscope, at
very
low power, is used for recording sea life such
as
LIMITATIONS
OF DARK-FIELD
algae and plankton.

MICROSCOPY:

1.Thick or extended objects and even dense

strews are unsuitable (glare).
2.Residual errors in objectives (notably spherical
aberration) become maximally visible.
3.All specks of dirt or traces of grease also
become maximally visible.

PHASE-CONTRAST MICROSCOPY:
Invented by Frits Zernike.

The phase contrast microscope is the most

outstanding contribution to the field of
microscopy.
Staining is not required to view the slide.

This microscope made it possible to study the

cell
cycle.

THEORY OF PHASE CONTRACT MICROSCOPY

The human eye is only sensitive to differences in
amplitude (brightness) or wavelength (color).
Differences in phase are not visible to human
eye.
Many microscopic objects do not cause
appreciable
changes in the intensity or color of the light
falling
through them, but only changes in phase.
Such objects are called phase-objects. e.g.
Living
cells and diatoms

Difference in phase

Differences in phase result from:

1. Differences in optical path length (e.g.,
differences in thickness in a piece of
glass) and
2. Differences in refractive index (n).
When phase changes are very large the human
eye
perceives them as differences in brightness -

PRINCIPLE OF PHASE CONTRAST MICROSCOP

It can be
diffraction.

explained

with

the

theory

of

In the following figure, two light waves are

drawn,
where1. The B wave passes outside the phaseobject
(i.e. the background illumination).
2. The O wave passes through the object,
which as drawn, has the same
amplitude as
B (i.e. theres no
absorption), but it is
delayed
Effect
of phase-object
(phase-difference).

A graphic plot of the difference between B and

O - is another wave D that represents the
difference between the background and the
object.

The diffraction theory has shown that D

actually is:
the diffracted light generated by the object.
In the image plane, B and D interfere,
which
amounts to a simple addition of their graphs.

 For small phase differences, D always differs

in
phase from B by about th the wavelength.
The phase difference is said to be .
If D is artificially shifted by another and
then
interferes with B, a new wave O results,
which
has smaller amplitude than B.

The object becomes darker than the background

Additional delay
Dw
the invisible phase-difference
has of
been
converted to
a visible amplitude difference.

 This effect can be further enhanced when B

is
attenuated (shown in the following figure)
Now, the amplitude of B is the same as that
of D,
(but of opposite value).
The wave O is then extinguished to zero,
which
equates to maximum dark.

Attenuation of B-wave

RAY PATHS IN A PHASE CONTRAST MICROSCO

The above principle is used in practice by the
following method:
a. Adjust illumination.
b. With Khler illumination the field stop can be
left
open because it is not very effective here.
c. Set the condenser iris wide open.
d. Select correct phase annulus for objective
(10x, 20x
etc. as indicated on the rotating plate of the
condenser).
e. Insert centering telescope.
f. Center the annulus.

g.
The
phase-contrast
condenser
is mostly of the
uncorrected
type and any condenser
can
be used to make our own
phase-annulus for each
objective.
h. The annular stop (ring) is
placed in the front focal
plane
of the condenser.
i. This is imaged at infinity:
parallel rays exit the

j. The light coming from the condenser is split

into two
bundles by the object - the undeviated and the
deviated light.
k. The annular rings in the objective lens and the
condenser separate the light.

l. The light that passes through the central part

of the
light path is recombined with the light that
travels
around the periphery of the specimen.
j. The interference produced by these two paths
produces images in which the dense structures
appear darker than the background.

Ray paths in a phase contrast microscope

Ray paths in a phase contrast microscope

Imaging of undeviated light (left hand of

Fig.):
The undeviated light B continues its
parallel
path and is therefore imaged at the rear
focal
plane of the objective.
Imaging of deviated light (right hand of Fig.):

 As these two images are so widely separated,

B
can be tinkered with while leaving D alone.
The objective has a special ring-shaped phaseplate
in its rear focal plane, which corresponds in
size to
the image of the phase annulus in the
condenser.
In the phase-plate, B is given the extra
phaseshift
and also attenuated
(the
A phase-contrast
image of
a ring in the
phase-plate
Glial cell cultured from a
can
seen if you look through the objective).
ratbe
brain
This technique is best for looking at living
specimens, such as cultured cells.

LIMITATIONS:
a.Its only suitable for phase-objects, that is:
thin
objects that do not cause major changes in
absorption.
b.Phase-contrast may not cause a major
reduction of resolution.
c. The phase-annulus in the condenser limits the
angle of admittance to less than the
maximum angle the objective handles.
d.The ring in the phase-plate has to be smaller
than the aperture of the objective.

e. For the 10x, 20x and 40x objectives in a

phasecontrast set, this reduction is mostly
acceptable, but
for some inexplicable reason the ring in the
phaseplate of the 100x objective (where the
highest
resolving power is necessary) is too small in
many
routine sets.
f. 100x phase-contrast objectives with a ring
corresponding to NA 0.9 approximately, yield
both

g. Such 100x objectives do not resolve a diatom

like
Surirella gemma, which one can easily
resolve in
brightfield (without phase-contrast), even
with
objectives of lower NA.

h. If the ring of the phase-plate has a diameter

less
than half that of the lens, the design is not
good
enough.

CONTROLLING CONTRAST:
In a phase-contrast work, contrast can be
controlled by selection of the medium in
which the
specimen is mounted.
If the refractive index of the medium is too
close to
that of the specimen, very little contrast will
result.
Hence, the medium should differ sufficiently in
the
refractive index to provide adequate contrast
for

 A medium such as glycerine - temporary

mounts or
diaphragm, and white kard syrup, hardened
at
room temperature - for permanent mounts,
will
provide much better contrast.
The refractive index of balsam is - 1.53
Both, diaphragm and glycerine have a low
index
(about 1.47).
If the refractive index of the specimen is less
than
that of the mounting medium - bright

 If it is greater than that of the mounting

medium
dark (positive) contrast will result.
Thus, a subject appears either bright against a
dark
background or dark against a bright
background.
Some phase-contrast objectives are termed
darkcontrast - When the index of the mount is
less than
that of the specimen, the effect is positive
contrast,
that is, the subject is dark against a bright
background.

POLARIZED LIGHT
It MICROSCOPY
provides all the benefits

of brightfield
microscopy.
It is superior to any other optical microscopy
technique.
It can distinguish
anisotropic
materials.

between

isotropic

and

It provides information on absorption of color

and
boundaries between minerals of differing
refractive
indices.
The technique exploits optical properties of

 Isotropic materials are

unstressed
glasses and cubic crystals.

gases,

liquids,

They demonstrate the same optical properties

in all
directions.
They have only one refractive index and no
restriction on the vibration direction of light
passing
through them.

 Anisotropic materials - include 90 percent of all

solid
substances.
They have optical properties that vary with the
orientation of incident light with the
crystallographic
axes.
They demonstrate a range of refractive indices
depending both on the propagation direction
of light
through the substance and on the vibrational
plane
coordinates.
More importantly, anisotropic materials act as
beam

A POLARIZED LIGHT MICROSCOPE

PRINCIPLE OF POLARIZED LIGHT MICROSC

The technique of polarizing microscopy exploits
the
interference of the split light rays, as they are
reunited along the same optical path, to extract
information about these materials.
Polarized Light:
In a "common light - light waves vibrate at
right
angles to the direction of travel of light, with
all
vibration directions being equally probable.
In plane-polarized light, there is only one
vibration

 The most widely used material is Polaroid TM

film.
It was invented by Land in 1932.
It consists of long chain polymers, treated with
light
absorbing dyes, and stretched so that the
chains are
aligned.
Light vibrating parallel with the chains is
absorbed
while light perpendicular to the chains is
transmitted.

There are two polarizing filters in a polarizing

microscope
1.

The polarizer: It is situated below the

specimen
stage, usually with its permitted vibration
direction
fixed in the left-to-right, East-West direction,
and
this is usually rotatable through 3600.

2. The analyzer: It is usually aligned North-South

but is
rotatable on some microscopes.

It is situated above the objectives and can be

moved
in and out of the light path as required.

PRINCIPLE OF POLARIZATION LIGHT MICROSC

RAY PATHS IN A POLARIZED LIGHT MICROSCOP

The polarizer and analyzer are the essential

components of the polarizing microscope.
Other desirable features include:
A rotating specimen stage - to facilitate
orientation of the objectives and stage with the
microscope optical axis, to make the center of
rotation coincide with the center of the field of
view.
Strain free objectives stress in assembly can
produce optical effects under polarized light, a
factor that could complicate observations.
A Bertrand lens to enable easy examination of
the rear focal plane of the objective, to allow
accurate
adjustment
of
the
illuminating
aperture diaphragm and to view interference
figures.

An eyepiece fitted with a cross wire graticule to mark the center of the field of view. Often,
the cross wire graticule is substituted for a
photomicrography graticule that assists in
focusing the specimen and composing images,
either digitally or onto film.
A
slot
to
allow
the
insertion
of
compensators/retardation plates between the
polarizers - which are used to enhance optical
path differences in the specimen.
Polarizing microscopy can be used both with
reflected and transmitted light.
Reflected light is useful for the study of
opaque
materials such as mineral oxides and
sulphides,