Selection of optimal transmittances with the cytophotometric two-wavelength method.

Abstract

With the help of a computer program using Newton's iterative method, it is possible to obtain a practically instantaneous solution for the two-wavelenght method equation even for values different from 2 for the ratio k of the extinction coefficients. The influence of the extinction ratio and of the mean transmittance on the correction of the distributional error and on the propagation of stochastic and systematic errors into the computed chromophore amount has been studied. After due precautions are taken to minimize chromatic and non-specific light loss errors, the selection of the optimal wavelenght couple will basically depend only on the compromise between correction of the distributional error and the least propagation of stochastic errors into the computed chromophore amount. In many instances, the use of interference filters will be convenient, since their use will eliminate the uncertainties of monochromator readjustment and will allow evenly illuminated field to be obtained more easily, particularly in the case of large measuring fields.