Objectives: The investigation of cochlear hair cells and lateral wall is a time-consuming and labor-intensive process. However, it is a mandatory experiment in audiology research. Here we suggest a novel method for investigating the inner ear microstructures from intact cochleae using two-photon laser scanning microscopy (TPLSM). This technique guarantees fewer artifacts and technical simplicity.
Methods: Using TPLSM, we investigated the whole mount cochleae, decalcified cochleae, and cleared cochleae of wild type C57BL/6 mice. CX3CR1+/GFP mice were used to investigate the feasibility of visualizing cellular structures in the cochlear spiral ligament. All samples were investigated without staining.
Results: Endogenous fluorescence emission from the outer hair cells was strong enough to be distinguished from the other structures in all samples. From the single apical view, 50 and 90% of the whole hair cells of the decalcified cochleae and cleared cochleae, respectively, could be visualized without staining using TPLSM. Capillary structure of stria vascularis and spiral ligament could be visualized by endogenous fluorescence without staining.
Conclusion: We successfully investigated the hair cells and lateral wall of mouse cochleae using TPLSM without using staining or any destructive procedures. This method is easier, faster, and more reliable than conventional methods.
Keywords: auditory hair cells; cochlea; multiphoton fluorescence microscopy; organ of Corti; two-photon fluorescence microscopy.
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