Our experimentally measured intensity variations contain information on the RBC birefringence properties. However, quantification of such birefringence would require use of a general, all-encompassing model. In the case of RBCs, it is not possible to rule out the role played by polarizing-depolarizing interactions, like scattering-induced diattenuation and depolarization. Specifically, a model based on electromagnetic theory, which properly incorporates the effect of the RBC’s scattering and polarization properties, would be required to simulate the variations of transmitted light detected in our experiments. Such simulation would then allow extraction of the birefringence properties of the RBCs in a quantitative fashion. Such a theoretical endeavor is formidable and well beyond the scope of the present studies. However, a simplistic approach is possible to help rationalize the observations we report here. So, we assume that linear birefringence is the dominant effect in RBCs, and we estimate the trend in variation of birefringence of optically trapped, malaria stage-dependent, infected RBCs as well as for RBCs that were not malaria infected but whose shape was perturbed by altering osmolarity. In case of osmolarity-induced shape-distorted RBCs, the resulting form-birefringence induces the cell to rotate within our trap, as indicated in Fig. 4, which also shows rotations due to the intrinsic birefringence of malaria-infected cells. In the former case, we made measurements on cells using osmolarities ranging from 150 to 1200 oSm; the rotational behavior did not depend on osmolarity change, with the peak-to-peak photodiode voltage remaining at 20 mV. The retardance (measured in degrees) measured for 20 mV change in photodiode voltage corresponds to 6 deg, which, in conjunction with the relation31Display Formula
(1)yields a value of for and wavelength . Here, is the refractive index change. In the case of malaria-infected cells, we found that the PD voltage depended on infection stage. Our measurements were made on a synchronous culture of the malarial parasite at fixed time points. In all results that we present in the following, each data point represents an average of measurements made on 25 RBCs. Data obtained after 5 and 11 h of infection (Fig. 4) clearly show the PD voltage increasing as the malarial stage progresses, indicating stage-dependent change in the infected RBC’s intrinsic birefringence. In the early stages of infection (5 h), the peak-to-peak voltage is 20 mV; this almost doubles 11 h postinfection, with a corresponding change of . At these times, RBC shapes are not significantly altered26 and the contributions are mainly from intrinsic changes. Twenty hours postinfection, when shape changes are considerable, . Even though we follow a simplistic approach, we observe that our values of change in birefringence are not inconsistent with the one that we deduced from an earlier report based on tomographic phase microscopy of malaria-infected RBCs16 in which refractive index mapping was carried out.