1. The total mass M of substances other than water (the dry mass) in the living cell can be obtained from the expression M = φA/χ, where φ is the optical path difference (o.p.d.) due to the cell and A its projected area. The method makes use of the fact that the refractive increments α(χ = ioocα) of most substances in cells are approximately the same, and independent of concentration. Values for χ have been tabulated.
Inaccuracies in the measurement of dry mass due to variations in χ (using λ average = 0.18) will be less than ± 10 per cent, in cells containing nucleic acids, proteins, and lipoproteins. When appreciable quantities of other substances are present the inaccuracy may be somewhat greater.
When the total dry mass of living cells is determined in a medium other than water (e.g. isotonic solution), a correction term involving the thickness must be determined; this correction is often small.
3. The total dry masses and, in some cases, the concentrations of dry substance in a variety of biological objects including Amoebae, pollen grains at various stages of development, nuclei of cells in tissue culture, and sperm heads have been determined.
In Tradescantia bracteata, during development from the microspore to the mature pollen grains, the dry mass increases by about tenfold. The dry masses of mature pollen grains were measured before and after successive digestion with ribonuclease, which removed about 4 to 14 per cent, of the dry mass, and with trypsin, after which about 40 per cent, of the original dry mass remained.
In living ram sperm heads the ratio of deoxyribose nucleic acid to total dry mass determined by ultra-violet and interference microscopy respectively is 40 per cent. This is in good agreement with the value 45 per cent, obtained by bulk biochemical methods.
4. The interference microscope has been used to measure the refractive index of cells and, hence, the concentrations of dry substances in them, by immersing them in media of different known refractive indices. The application of this method to fixed cells is discussed theoretically. In experiments on fixed ram sperm heads the expected linear relationship between o.p.d. and refractive index of the immersion medium was obtained. Data on the average concentration of dry substance in ram sperm heads, the localized refractive index, and concentration in the denatured submicroscopic particles in the head, the percentage of the head volume occupied by them, and the geometrical thickness of the head were obtained.
5. Factors affecting the accuracy of the measurements of o.p.d., such as glare in the microscope, light scatter or absorption by the object, &c, are outlined.
Frequency domain fluorescence microspectrometry: Application to cellular uptake and drug distribution