The effect of swelling on the interference microscopy of fixed biological material

Abstract

SYNOPSISMeasurements with the interference microscope show the optical‐path difference relative to the medium of many biological objects after histological processing to be less in water than would be expected on the basis of measurements made in other media. This anomalous optical‐path difference is found also with sections of gelatin (autoradiographic stripping film), but not if the gelatin is mounted on the slide in such a way that swelling is constrained to a direction parallel to the optical axis of the microscope. Swelling in water has been demonstrated in a variety of formalin‐fixed and de‐fatted tissues.It is suggested that the anomalous optical‐path differences in water are due to swelling causing a change in the lateral dimensions of the object, and hence loss of material from the optical path. The fact that the anomaly has also been found with scanning and integrating interference microscopes is discussed, and it is suggested that the validity of such methods needs re‐examination.