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Dark field microscopy

Principle- 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. Everything is visible regardless of
color, usually bright white against a dark background.
Pigmented objects are often seen in "false colors," that is,
the reflected light is of a color different than the color of the
object. Better resolution can be obtained using dark as
opposed to bright field viewing.

The apparatus (Dark Field Microscope)-It has all the


same parts of a simple bright field microscope which are-

1.Base - Supporting structure that usually contains an


electrical light source or illuminator
2.Objective lens(es)- Magnify the image
3.Oculars - Magnify the image from the objective lens. A
microscope with one ocular lens is often called a monocular,
a microscope with two oculars is called a binocular.
4.Arm - The support structure that connects the lens systems
to the base.
5.Body tube - Sends light to the ocular lens.
6.Condenser lens - Directs light to pass through the
specimen.
7.Stage - Platform that allows mechanical movement of a
microscope slide. Adjustment.
8..knobs - Course and fine focus adjustment

Except one additional An annular stop is used underneath to


condenser to create a cone of oblique illumination to reflect
light.

Working method-

Ray diagram showing position of an annular disc in


between source & condenser in dark field
microscope
1.Light enters the microscope for illumination of the sample.
2.The condenser lens focuses the light towards the sample.
3. A specially sized disc, the "patch stop" blocks some light
from the light source, leaving an outer ring of illumination.
4. The light enters the sample. Most is directly transmitted,
while some is scattered from the sample.
5.The scattered light enters the objective lens, while the
directly transmitted light simply misses the lens and is not
collected.
6. Only the scattered light goes on to produce the image,
while the directly transmitted light is omitted.

Application-

1. Initial examination of suspensions of cells such as yeast,


bacteria, small protists, or cell and tissue fractions including
cheek epithelial cells, chloroplasts, mitochondria, even blood
cells (small diameter of pigmented cells makes it tricky to
find them sometimes despite the color).

2. Initial survey and observation at low powers of pond water


samples, hay or soil infusions, purchased protist or metazoan
cultures.

3. Examination of lightly stained prepared slides,


determination of motility in cultures.

Advantages and disadvantages-

Dark field microscopy is a very simple yet effective


technique and 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 are impressive.

The main limitation of dark field microscopy is the low light


levels seen in the final image. This means the sample must
be very strongly illuminated, and can cause damage to the
sample.

Phase Contrast Microscopy

The technique was invented by Frits Zernike in the 1930s for


which he received the Nobel prize in physics in 1953. Phase-
contrast microscopy is a mode available on most advanced
light microscopes and is most commonly used to provide
contrast of transparent specimens such as living cells or
small organisms

Principle- Phase-contrast microscopy is an optical


microscopy illumination technique in which small phase
shifts in the light passing through a transparent specimen
(unstained) are converted into amplitude or contrast
changes in the image.
The Apparatus (Phase Contrast Microscope)-

Basically microscope contain all parts which are


characteristics of a bright field microscope except two t
additional changes are required to make a Phase Contrast
which are-

A phase ring (located in a conjugated aperture plane


somewhere behind the front lens element of the objective)
and a matching annular ring, which is located in the primary
aperture plane (location of the condenser's aperture).

Working Method-

1.The light emanating from the phase annulus is captured by


the condenser and emerges as light with only parallel
wavefronts from the condenser.

2.When these plane waves (parallel wave fronts) hit the


phase specimen some of this light is diffracted (and/or
refracted) while moving through the specimen. Assuming
that the specimen does not significantly alter the amplitudes
of the incoming wavefronts but mainly changes phase. now
two types of waves, the surround wave(s) and the diffracted
wave (d) which have a relative phase-shift of 90 (/4).

Ray diagram showing position of phase annulus


& phase ring in pc

3.The objective focuses the D-wave inside the primary image


plane , while it focuses the S-wave inside the back focal
plane. The phase plate 'P' reduces the amplitude of all light
rays traveling through the phase annulus (mainly S-waves)
by 70 to 90% and advances the phase by yet another 90
(/4). Hence the recombination of these two waves (D + S) in
the primary image plane (labeled '4') results in a significant
amplitude(contrast).

Apllication-

In studying living sample such as cell cycle& colorless object


as Amoeba ,Flagella

Limitations-

The images are usually surrounded by "halos" around


the outlines of details. These are optical artifacts, which
may obscure the boundaries of details.
There may be a reduction in the image resolution.

Phase contrast does not work well with thick specimens


because of shifts in phase occur from areas slightly
below or slightly above the plane that is in focus. Such
phase shifts confuse the image and distort image detail.

Fluorescence microscope

A fluorescence microscope is a light microscope used to


study properties of organic or inorganic substances using the
phenomena of fluorescence and phosphorescence instead of,
or in addition to, reflection and absorption.

In most cases, a component of interest in the specimen is


specifically labeled with a fluorescent molecule called a
fluorophore (such as Green fluorescent protein, fluorescein or
DyLight 488). The specimen is illuminated with light of a
specific wavelength (or wavelengths) which is absorbed by
the fluorophores, causing them to emit longer wavelengths
of light (of a different color than the absorbed light). The
illumination light is separated from the much weaker emitted
fluorescence through the use of an emission filter. Typical
components of a fluorescence microscope are the light
source (Xenon or Mercury arc-discharge lamp), the excitation
filter, the dichroic mirror (or dichromatic beamsplitter), and
the emission filter (see figure below). The filters and the
dichroic are chosen to match the spectral excitation and
emission characteristics of the fluorophore used to label the
specimen.

Most fluorescence microscopes in use are epi-fluorescence


microscopes (ie : excitation and observation of the
fluorescence are from above (epi) the specimen). These
microscopes have become an important part in the field of
biology, opening the doors for more advanced microscope
designs, such as the confocal laser scanning microscope and
the total internal reflection fluorescence microscope (TIRF).

Fluorophores lose their ability to fluoresce as they are


illuminated in a process called photobleaching. Special care
must be taken to prevent photobleaching through the use of
more robust fluorophores or by minimizing illumination.

Epifluorescence microscopy

Epifluorescence microscopy is a method of fluorescence


microscopy that is widely used in Life Sciences. In this
process, instead of transmitting the excitatory light through
the specimen it is passed through the objective onto the
specimen. Since only reflected excitatory light filters
through, the transmitted light is filtered out, giving a much
higher intensity. Fluorescent or fluorochrome stains are
applied to the specimen to provide an estimated count.

It can be used to find routine direct total counts of bacteria


in water samples