The Application of Ultraviolet Absorbence Measurements to Problems in Cell Biology

Ultraviolet (UV) absorption spectra of intracellular structures are an aid in the interpretation of the biological effects of UV radiation and are useful for the identification of the substances localized in the structures. Micro-assays of the total amount of a UV absorbing compound in a structure enable a study of the metabolism of that compound to be made in reference to the function of the structure. Both types of analysis are possible in the spectral range between 230 and 300 mμ where the absorption peaks of the biologically important nucleic acids (260 mμ) and proteins (280 and less than 250 mμ) lie. The components of a microspectrophotometer, source of radiation, monochromator, microscope optics, and detector are discussed with reference to the UV. The main restriction on the design of an instrument is imposed by the necessity to measure the absorption of an extremely small area (of the order of one micron in diameter) in the specimen. High intensity sources, optical components with high transmission values, and sensitive detectors are required. The application of the method to a study of nucleic acid metabolism in chromosomes is outlined. The total amount of UV absorbing material in an intrachromosomal structure was measured by scanning a photomicrograph and integrating the absorbencies over the structure. Nucleic acid was determined by the difference between the total amount of material found before and after extraction with nucleases.