Optical errors in scanning stage absorbance cytophotometry. I. Procedures for correcting apparent integrated absorbance values for distributional, glare, and diffraction errors.

The main optical errors in the cytophotometric determination of the integrated absorbance of an ideal amplitude object are distributional error, glare error and diffraction error. Scanning reduces the magnitude of the distributional error considerably, but the remaining (residual) distributional error still may be important, depending on the shape and absorbance of the object and on the size of the smallest step of the scanning raster in relation to the object size. A theoretical relationship is derived for model objects which allows an estimation of the magnitude of this residual distributional error at various conditions of measurement. Glare is caused by optical imperfections, especially in the substage optics. Its effect is a uniform redistribution over the image plane of that fraction of the incident light that passes outside the projection of the