An Integration Method for the Interference Microscope

Abstract

ABSTRACT A method is described of measuring the integrated phase retardation of an object with a Baker interference microscope. With certain limitations, the total dry mass of the object can be derived from this figure. This method of ‘weighing’ objects is of particular value in measuring the growth of single cells, since it is very sensitive and can be used on a living cell without damaging it. The method consists of restricting the field round the object with an aperture of known area and then measuring the total retardation within this field due to the presence of the object. The compensator is adjusted until there is an equal light intensity in the field with and without the presence of a quarter-wave plate. A similar adjustment is made with the object moved out of the field. The difference between the two compensator settings gives the integrated area retardation due to the object. The quarter-wave plate is motor-driven, and the light intensity is measured by a photomultiplier and presented on a cathode ray tube. The instrument is sensitive to a change of about 5 × 10_ 14 g in the dry weight of a biological object. For an object of known specific refractive increment and mounted in water, the total dry mass can be derived from the retardation without the dimensions of the object being known. There is, however, a severe limitation with this method of integration— that the maximum retardation of the object must not exceed about 45 º or λ/8. This can be shown by theory, and has also been confirmed by model experiments with small celloidin spheres. Most biological objects have larger retardations in water, so it is necessary to mount them in media of higher refractive index. In this case, the volume of the object must also be known for the dry mass calculation. Although this limits the use of the method, it is important in measuring the growth of a cell to know the volume as well as the dry mass. An example is shown of the application of this method in measuring the growth of a fission yeast, Schizosaccharomyces pombe. Curves are given showing the changes in dry mass, volume, and concentration of a single growing and dividing cell of this yeast.