Laser interference microscopy in erythrocyte study

Abstract

With the laser interference microscopy (LIM) technique, one can measure phase height of cells—a variable proportional to the cell thickness and the difference in the refractive indices of the cell and the surrounding medium. This makes functional optical cell imaging possible, and estimation of shape, thickness, and area of erythrocytes feasible. In this paper, we studied changes in erythrocyte shape and volume with osmolarity and pH. Obtained from the LIM technique, erythrocyte phase heights and area values, as well as the hematocrit-measured erythrocyte volume, were used to estimate changes in the refractive index with osmolarity and pH. A comparison between the estimated refractive index with the refractive index, calculated in the assumption that it can only depend on the hemoglobin concentration in the cell, indicates that these two estimates are identical in the range of osmolarity (250–1000 mOsm) and pH (4.5–10.0) values. Thus, refractive index changes result exclusively from the changes in hemoglobin concentration with the changes in erythrocyte volume. Under these conditions, it is possible to estimate the amount of hemoglobin in an erythrocyte from its phase height and area, obtained from LIM.