Possible Pitfalls

Reliable and accurate cytophotometry requires that the microscope be aligned properly to minimize glare and an adequate source of monochromatic light (e.g., 560 nm), near the absorption maximum of the Feulgen-DNA reaction product (560-570 nm). It also requires modulation of ordinary line current fluctuations for the light source and a vibration-free environment for the microscope and other electronic components (see Notes 11 and 12).

Careful attention must be paid to the optics and the alignment of the microscope used to obtain IOD values of the DNA-Feulgen dye complex bound to nuclei. Variations in transmission across the field of view may be the result of poor optical alignment or dirt on optical surfaces, which can contribute the total noise level of the system. Where the light path through a measured object is asymmetric, conditions of absorption may be changed and lens flare greatly increased. Figure 9 shows the major components reviewed in the following:

1. Check the symmetry of the light path through the microscope using a phase microscope focusing telescope and a low-power objective to visualize the filament of the light source, the field diaphragm, and the condenser iris diaphragm.

2. Note color fringes as you open and close the diaphragms. Are the red-blue shifts symmetrical at the edges of these apertures as you come up to and away from focus in the object plane?

3. Center components appropriately to eliminate uneven color fringes.

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