Abstract
In spite of a number of examples of the apparently successful application of the two-wavelength method of microspectrophotometry to biological materials, a demonstration of the effective transition from the theoretical to the practical aspects of the method has never been given. The following tests were carried out on model systems in order to confirm the practical validity of the method: (1) Spherical droplets of Sudan blue or Sudan IV (varying in volume by as much as 400 times) were measured with the surrounding blank field included and were found to have amounts of chromophore which conformed to the estimated droplet volume to within 3 to 6 per cent. (2) Repeated determinations on the same isolated nucleus gave results which did not vary in spite of wide variations in the area of the photometric field. (3) The results of repeated determinations on the same droplet of dye were unaffected by gross changes in the focus of the objective. The microspectrophotometer on which these measurements were made has been briefly described; it was designed specifically for the two-wavelength method. Some of the details of instrumentation have been discussed, in particular, the effects of non-uniform illumination and the contrasting demands of the two integral parts of this type of photometry. Although it has certain limitations, the two-wavelength method conforms to its theoretical promise and is a practical method of measuring light absorption in complex objects.
Full Text
The Full Text of this article is available as a PDF (636.3 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- FIRKET H., CHEVREMONT-COMHAIRE S., CHEVREMONT M. Action of ribonuclease on living cells in vitro and synthesis of deoxyribonucleic acid. Nature. 1955 Dec 3;176(4492):1075–1076. doi: 10.1038/1761075a0. [DOI] [PubMed] [Google Scholar]
- MERRIAM R. W., RIS H. Size and DNA content of nuclei in various tissues of male, female, and worker honeybees. Chromosoma. 1954;6(6-7):522–538. doi: 10.1007/BF01259952. [DOI] [PubMed] [Google Scholar]
- MOSES M. J., TAYLOR J. H. Desoxypentose nucleic acid synthesis during microsporogenesis in Tradescantia. Exp Cell Res. 1955 Dec;9(3):474–488. doi: 10.1016/0014-4827(55)90077-x. [DOI] [PubMed] [Google Scholar]
- ORNSTEIN L. The distributional error in microspectrophotometry. Lab Invest. 1952;1(2):250–265. [PubMed] [Google Scholar]
- PATAU K. Absorption microphotometry of irregular-shaped objects. Chromosoma. 1952;5(4):341–362. doi: 10.1007/BF01271492. [DOI] [PubMed] [Google Scholar]
- PATAU K., SWIFT H. The DNA-content (Feulgen) of nuclei during mitosis in a root tip of onion. Chromosoma. 1953;6(2):149–169. doi: 10.1007/BF01259937. [DOI] [PubMed] [Google Scholar]
- SETTERFIELD G., SCHRIEBER R., WOODARD J. Mitotic frequency determinations and microphotometric Feulgen dye measurements in root tips. Stain Technol. 1954 May;29(3):113–120. doi: 10.3109/10520295409115454. [DOI] [PubMed] [Google Scholar]