Measuring Off Peak

Because a cytophotometer counts molecules with markedly reduced accuracy at either 1% Tor at 99% T, it is always prudent policy to measure objects

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Wavelength

Fig. 14. Absorption curve of the DNA-Feulgen dye complex in a fat body cell of Drosophila melanogaster. (Rasch, unpublished data; 1984.)

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Wavelength

Fig. 14. Absorption curve of the DNA-Feulgen dye complex in a fat body cell of Drosophila melanogaster. (Rasch, unpublished data; 1984.)

with IOD values in the range 0.2-0.8. Absorbencies above 1.00 and below 0.15 are subject to rapidly rising error functions at either high density (or low transmission) and at high transmission (and low density). The relative error curve in Fig. 5 clearly illustrates the magnitude of this potential photometric pitfall.

As shown initially by Swift (5) in 1950 and subsequently by others (50,51,64,68), if single-point absorbances for nuclei exceed 1.0 at a wavelength of 560 nm, near the peak absorbance of the Feulgen chromophore, it is both possible and feasible to measure such specimens "off peak" (e.g., at 615 nm or at 620 nm), at which wavelengths absorbance of the Feulgen-DNA reaction product is about half of that at the peak (Amax). At the off-peak wavelength, the nuclei will have absorbances in the range 0.40-0.60, instead of 1.0 or above. The wavelength of measuring light must be selected so as to be sufficiently close to Amax to allow accurate measurement of the palest nuclei present in the cell sample (51,64). By measuring on the red side of the Feulgen absorption curve (Fig. 14), the slope of the line plotting concentration vs absorbance is greater at 560 nm and lower at 620 nm. This loss in sensitivity, however, is offset by the enhanced accuracy gained with lower and more reliable IOD values.

This shift in wavelength for measuring large, darkly stained nuclei is a valuable tool to have available when comparing DNA levels in polysomatic tissues from maturing larvae of Drosophila melanogaster (25,31,33,34,36). Obviously, when nuclei of an "unknown" are measured off peak, the internal standard reference cell samples for each slide series also must be measured at the same wavelength with the same optical conditions for magnification, electronic gain settings, scanning spot size and mask size, selection (12).

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