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High-resolution scanning-densitometry of photographic negatives of human metaphase chromosomes

II. Feulgen-DNA measurements

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Summary

In photomicrographic negatives of cytochemically stained human metaphase preparations, images of individual chromosomes were scanned interactively with a Zeiss SMP interfaced to a PDP-12 computer.

By means of the CHROSCAN computer program spot-scanning of selected chromosomes was performed in a direction perpendicular to the length axis, each measured value being corrected for background absorbance taken on both sides of the chromosome image. Plotting of the integrated absorbance values of each transversal scanline results in a graphic representation of the absorbance distribution over the chromsome length. Following this procedure, longitudinal curves were obtained which showed the characteristic patterns obtained after Q or G banding, and, in the case of Feulgen-staining, represented quantitative variations of DNA mass along the individual chromosomes. For Feulgen-stained chromosomes, the total integrated absorbance value and the ratio of integrated absorbance in the long arm over the total integrated absorbance, correspond with the DNA-absorbance and-arm ratio values per chromosome respectively.

The results of investigations concerning the reproducibility and accuracy of cytochemical Feulgen staining and of the photographic procedures are presented, together with total integrated Feulgen-DNA absorbance and arm ratio values for a number of human chromosomes.

For several chromosomes, Feulgen absorbance arm ratio measurements were found to result in values more constant over different metaphases when the long arm was considered to start at the lowest dip in the longitudinal absorbance curve, than when the microscopically observable primary constriction was taken to represent the centromere. The results indicate that the present method allows accurate photometry of naturally absorbing, or of stained or fluorescent objects, with measuring intervals of 0.16 μ. In addition it is shown that the arm ratio values and total DNA content can serve as very constant parameters for karyotype analysis.

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Authors and Affiliations

  1. Department of Histochemistry and Cytochemistry, University of Leiden, Leiden, The Netherlands

    M. van der Ploeg, P. van Duijn & J. S. Ploem

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  1. M. van der Ploeg
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  2. P. van Duijn
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  3. J. S. Ploem
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Supported by grant nr. 28-16915 of the Praeventiefonds, 's Gravenhage.

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van der Ploeg, M., van Duijn, P. & Ploem, J.S. High-resolution scanning-densitometry of photographic negatives of human metaphase chromosomes. Histochemistry 42, 31–46 (1974). https://doi.org/10.1007/BF00498477

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  • DOI: https://doi.org/10.1007/BF00498477

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