Selection of operating wavelength-pair for two-wavelength microspectrophotometry

The two-wave length method is the microspectrophotometric method of choice for thick specimens. Its accuracy is dependent on the accuracy of selection of the operating wavelenght-pair. The "slope zero test", offered as a check upon the accuracy of this selection (Garcia and Iorio, '66) is shown to be invalid by analysis of experimental data, by computer model and by mathematical derivation. A means of checking the accuracy of selection of the operating wavelength-pair via scanning measurements is suggested. A new formulation of the two-wavelength method, permitting use of different operating wavelength-pairs, is also given.