1. Each compensator rotation was digitised and subtracted from the respective background image. The mean intensity for each compensator step was registered and the compensator angle values were plotted against the respective mean grey intensity. Finally, constants a and b of equation (2) were determined by an iterative process using a Kaleida Graph software (Synergy Software, PA, USA) to determine the standard calibration curve required to convert the observed grey intensity of the specimen into ot values. A set of measurements was carried out using manual compensation with the Senarmont A/4 compensator plate in the samples. The theoretically derived constants were applicable to this real dataset with very close agreement (r2 = 0 986), which indicated that virtually no light loss occurred because of unstained samples. Consequently, the optical path difference of the birefringent material (F) was calculated using the Senarmont method,27 as follows: F= (otX) / 1800 (3)
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