Optical errors in scanning stage absorbance cytophotometry. II. Application of correction factors for residual distributional error, glare and diffraction error in practical cytophotometry.

In a preceding article theoretical methods were derived for correcting the integrated absorbance values of microscopic objects determined with a scanning stage cytophotometer for the systematical erros due to residual distributional error, diffraction error, and glare error. For an experimental investigation of these results, the local apparant transmission at the center of an opaque particle was determined and this value used as a measure for the substage glare. By sufficient reduction of the size of the illuminated field, this substage glare could be kept below 1% in our scanning stage cytophotometer. The magnitude of the diffraction error was experimentally approached by comparing the values found for the integrated absorbance of the same amount of chromophore, dispersed over two different areas by crushing or centrifugation. After appropriate correction for the residual distributional error, the remaining difference was ascribed to the diffraction error, caused by diffraction at the edges of the object, and to the glare error, present all over the measured area. The local borderline corrections found necessary to obtain the best matching corrected integrated absorbance values were between 3 and 5%, in good agreement with the value derived theoretically. The importance of these corrections for the determination of the integrated absorbance of common biological objects is discussed.