Dark field
Dark field microscopy is a technique for improving the contrast of unstained, transparent specimens. Dark field illumination uses a carefully aligned light source to minimize the quantity of directly-transmitted (unscattered) light entering the image plane, collecting only the light scattered by the sample. Dark field can dramatically improve image contrast – especially of transparent objects – while requiring little equipment setup or sample preparation. However, the technique suffers from low light intensity in final image of many biological samples, and continues to be affected by low apparent resolution.
Rheinberg illumination is a special variant of dark field illumination in which transparent, colored filters are inserted just before the condenser so that light rays at high aperture are differently colored than those at low aperture (i.e. the background to the specimen may be blue while the object appears self-luminous red). Other color combinations are possible but their effectiveness is quite variable
A diatom under Rheinberg illumination
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. 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 field as opposed to bright field viewing.
You don't need sophisticated equipment to get a dark field effect, although the effect is most dramatic when the occulting disk is built into the condenser itself. You do need a higher intensity light, since you are seeing only reflected light. At low magnification (up to 100x) any decent optical instrument can be set up so that light is reflected toward the viewer rather than passing through the object directly toward the viewer.
When to use dark field illumination
Dark field illumination is most readily set up at low magnifications (up to 100x), although it can be used with any dry objective lens. Any time you wish to view everything in a liquid sample, debris and all, dark field is best. Even tiny dust particles are obvious. Dark field is especially useful for finding cells in suspension. Dark field makes it easy to obtain the correct focal plane at low magnification for small, low contrast specimens. Use dark field for
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).
Initial survey and observation at low powers of pond water samples, hay or soil infusions, purchased protist or metazoan cultures.
Examination of lightly stained prepared slides. ? Initial location of any specimen of very small size for later viewing at higher power.
Determination of motility in cultures
Phase contrast microscopy
Phase
contrast image of a cheek epithelial cell
It is an optical microscopy illumination technique that convertsphase shifts in light passing through a transparent specimen to brightness changes in the image. The phase shifts themselves are invisible to the human eye, but become visible when they are shown as brightness changes.
Phase contrast microscopy is particularly important in biology, as it reveals many biological structures that are not visible with a simpler bright field microscope. These structures were often made visible to earlier microscopists by staining the slide. This requires additional preparation and it also kills the cell. Phase contrast microscopy of live cells without staining allowed for the in vivo study of the cell cycle.
When light travels through a medium other than vacuum, interaction with this medium causes itsamplitude and phase to change in a manner dependent on properties of the medium. Changes in amplitude arise from absorption of light, which is often wavelength dependent and may give rise to colours. The human eye measures only the energy of light arriving on the retina, so changes in phase are not easily observed under optimal bright field illumination, yet often these changes in phase carry much important information.
T
he
same situation applies in a typical microscope with "Köhler" bright
field illumination,
i.e., although the phase variations introduced by the sample are
preserved by the instrument (at least within the instrumental limits
of imaging perfection) this information is lost in the process of
image recording, which measures only light intensity. In order to
make phase variations observable, it is necessary to combine the
light passing through the sample with a reference so that the
resulting interference reveals the phase structure of the sample.
This problem was first appreciated by Frits Zernike during his study of diffraction gratings. During the course of his work he realised that it is necessary both to achieve interference with a reference beam, and (for maximizing the contrast achieved with the technique) to introduce a phase shift to this reference beam so that the no-phase-change condition gives rise to completely destructive interference.
He later realized that the same technique can be applied to optical microscopy. The necessary phase shift is introduced by rings etched accurately onto glass plates so that they introduce the required phase shift when inserted into the optical path of the microscope. When in use, this technique allows the phase of the light passing through the object under study to be inferred from the intensity of the image produced by the microscope. This methodology is known as the phase-contrast technique.
In optical microscopy many biological objects such as cell components in protozoans, bacteria and sperm tails are fully transparent unless stained. (Staining is a difficult and time-consuming procedure which can destroy or alter the specimen structure). The difference in densities and composition within the imaged objects however often give rise to changes in the phase of light passing through them, hence they are sometimes called "phase objects". Using the phase-contrast technique makes these structures visible and allows their study in living specimens.
This phase contrast technique proved to be such an advancement in microscopy that Zernike was awarded the Nobel prize (physics) in 1953.
1. Condenser annulus 2. Object plane 3. Phase plate 4. Primary image plane