ELECTRON-PROBE MICROANALYSIS OF THE OTOLITHIC MEMBRANE - A METHODOLOGICAL AND QUANTITATIVE STUDY

The effect of tissue preparation on calcium an potassium weight percents in the otoconial layer in the utricle and saccule was studied in four groups of OF1 mice with electron probe X-ray microanalysis. Glutaraldehyde and freeze-drying, glutaraldehyde and air-drying, air-drying, and cryo-fixation and freeze-drying were compared. Ca and K changed significantly in the utricle depending on the method used (P < 0.001), and K changed significantly in the saccule (P < 0.001 ). We chose cryo-fixation with freeze-drying for the quantitative analysis of the otolithic membrane because this method provided the highest values of Ca and K with minimum loss of Ca and K. Microcrystalline salt standards mounted on scanning electron microscopy holders were used for the quantification of Ca and K by the peak-to-local-background (P/B) ratio method. The P/B ratio in standards with reproducible results, when plotted against weight percent, gave a straight line for Ca (r = 0.99, P < 0.001) and K (r = 0.98, P < 0.001). The Ca and K weight percents in otoconia showed similar frequency distributions in the utricle and saccule.