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