Download (direct link):
Nomarski images of a cell undergoing division. The condensed chromosomes and other cell organelles can easily be seen using the Nomarski optics in these photographs, since this cell remains rather flattened during division.
Care and Handling of the Phase Contrast Microscope
Adjusting the phase contrast is a rather simple technique that often is assiduously avoided for no good reason. Conversely, there are the uninitiated among us who will, with the best of intentions, badly misalign the phase rings (phase annular diaphragm) and fail to center the condenser, thus rendering the principal function of the microscope useless (Fig. 6.7). For those readers who have an interest and no manual is readily available, we offer the following steps for keeping the microscope in pristine alignment. This is illustrated using the Nikon inverted microscope, so placement of the adjusting screws will differ depending on the microscope used, but the theory should be similar.
1. Place a dish of cells on the microscope stage and focus using the 10x objective.
2. Narrow down the field aperture diaphragm until the entire diaphragm can be seen in the field of view (Fig. 6.8).
3. Rotate the condenser knob until the aperture edge is in focus.
4. Adjust the condenser centering screws until the field aperture diaphragm image is in the center of the field of view.
5. Open up the field aperture diaphragm.
6. Set the turret assembly (on the eyepiece tube) to "B". If you are using the older Diaphot, remove an eyepiece and replace it with a centering telescope.
7. You will see a phase plate image (dark) and an annular diaphragm image (illuminated) (Fig. 6.8). The idea is to center those two images.
a. Diaphot 300. This has two centering screws that you adjust by inserting two 1-mm Allen hex wrenches or the Allen hex wrenches that Nikon provides with their microscope (and that sooner or later everyone misplaces) into the hexago-
(Left) In-phase and (right) out-of-phase image of a cell culture. For a phase contrast microscope to be useful, the phase must be kept well adjusted.
is fully open. nal screws on the rotating condenser turret. Make sure the condenser diaphragm
b. Diaphot TMD. This condenser has two knurled centering screws located near the rear of the condenser turret (not to be confused with the two knurled knobs on the front of the turret for
_centering the condenser). Adjust these as above to center the two images. In theory, these things
should rarely have to be done (unless there is a compulsive knob-twister in the laboratory). But in this imperfect world you will find that a practical knowledge of these things can save a lot of marriages, friendships, and professional collaborations.
As mentioned earlier, an inverted phase contrast microscope can be fitted with epifluorescent (derived from the term episcopic, or incident-light) illumination, which will greatly expand the capabilities of the microscope. If a great deal of fluorescence microscopy is anticipated for visualization of fixed cells, an upright microscope, dedicated to fluorescence, is desirable. The upright epifluorescent microscope can also use oil immersion, high-magnification objectives if very high magnification is needed for detecting microorganisms such as mycoplasma. Immunofluorescence has been the most common application of fluorescence microscopy, in that it combines the specificity and spatial resolution of fluorescence microscopy with the selective binding of antibodies to their respective epitopes. By choosing fluorophores of different colors, multiple sites can be localized on the same cell. Moreover, fluorescent probes can be incorporated into living cells to localize, visualize, and measure a wide variety of physiological changes.
The components of an epifluorescent system consist of a light source, usually mercury, a filter block containing an excitation filter, a barrier filter and a dichroic mirror, and suitable objectives for fluorescence. The excitation filter is a color filter that transmits only those wavelengths of the illumination light that excites a specific dye. The emission or barrier filter is a color filter that attenuates all the light transmitted by the excitation filter and transmits any fluorescence emitted by the cell. The dichroic mirror is set at a 45ja angle to the optical path of the microscope. Its coating has the ability to reflect the excitation light and transmit the fluorescence.
Both organic and nonorganic substances can exhibit some fluorescence, known as autofluorescence. Autofluorescence is often a major source of unwanted light in the observed fluorescent image and can be minimized in fixed cells by paying careful attention to the fixation method to be used. For example, glutaraldehyde will fluoresce at wavelengths well into the UV range, as will a number of natural peptides (Robertson and Schaltze, 1970). Careful selection of the wavelength and bandwidth of the excitation and detection systems can optimize signal-to-noise and aid in minimizing unwanted background (Chroma, 1994). Some fluorochromes are considerably brighter than others when localizing multiple epitopes, so that it is important to adjust the filtration to compensate when using multiple fluorochromes. The use of narrow band-pass filters can help to eliminate this problem (Fig. 6.9).