Refractometry of living cells part I. basic principles

Abstract

ABSTRACT The principles underlying a new method of refractometry of living cells are discussed. The method was evolved from the chance observation that the amoebocytes of the blood of the earthworm, examined in their own blood, appeared bright instead of dark by positive phase-contrast microscopy. This was shown to be due to the presence of dissolved haemoglobin which raised the refractive index of the medium above that of the cytoplasm. In order to determine the refractive index of the latter it was only necessary to dilute the blood until the cytoplasm became virtually invisible. Non-pigmented proteins and other high molecular weight substances have now been substituted for haemoglobin. The nature of the initial observations suggested that if the cell could be regarded to a first approximation as being composed entirely of proteins, the cytoplasmic protein concentration could be equated to the protein concentration of the immersion medium which made the cell appear with minimum contrast. This would only be true if equal concentrations of different proteins in solution had the same refractive index. The nature of refractive index and its relationship to density are discussed and it is shown that for nearly all unconjugated proteins so far investigated the specific refraction increments (i.e. the increase in refractive index per 1 per cent, increase in concentration) have almost the same values (00185±2 per cent.). The effects of many factors such as pH, salts, temperature, wavelength, concentration, and nature of the solvent are discussed. Since living cells contain substances other than proteins the specific refraction increments of protein derivatives, lipides, carbohydrates, and salts are considered and it is shown that the presence of moderate amounts of such substances is unlikely to affect the refractive index of cells to any great extent. It is suggested that the mean specific refraction increment of protoplasm should be taken as 0018 and that this value can be used in order to calculate the solid and water content of protoplasm from values of refractive index.