Accuracy of optical density measurement of cells 1: Low resolution

In this paper we analyze factors influencing the accuracy and precision of optical density measurement of light microscopical objects. The study is applied to the DNA content of Feulgen-stained cells using a lowresolution, TV-equipped microscope connected to a digital image processing system. Factors influencing the accuracy of density measurement include staining (not considered here), image formation (glare, focus, diffraction), image sampling (distribution, sampling density, noise), and computational accuracy. These factors are reviewed with respect to potential remedies. Evaluation of the measurement error contribution is performed on the level of the individual cell and on the specimen level. It is concluded that a coefficient of variation of 5% (in contrast to 3.5% using flow cytometry of the same specimen) in the measured values of normal cells of one specimen is attainable using adequate shading correction. With relatively simple computational methods, image cytometry may be well suited for pathology practice.