Deafness is the most common sensory impairment, affecting approximately 5% or 430 million people worldwide as per the World Health Organization1. Aging or presbycusis is a primary cause of sensorineural hearing loss and is characterized by damage to hair cells, spiral ganglion neurons (SGNs), and the stria vascularis. These structures reside within the cochlea, which has a complex, spiral-shaped anatomy of membranous tissues suspended in fluid and surrounded by bone. These properties make it technically difficult to investigate and quantify histopathological changes. To address this need, we developed a light-sheet microscope (TSLIM) that can image and digitize the whole cochlea to facilitate the study of structure-function relationships in the inner ear. Well-aligned serial sections of the whole cochlea result in a stack of images for three-dimensional (3D) volume rendering and segmentation of individual structures for 3D visualization and quantitative analysis (i.e., length, width, surface, volume, and number). Cochleae require minimal processing steps (fixation, decalcification, dehydration, staining, and optical clearing), all of which are compatible with subsequent high-resolution imaging by scanning and transmission electron microscopy. Since all the tissues are present in the stacks, each structure can be assessed individually or relative to other structures. In addition, since imaging uses fluorescent probes, immunohistochemistry and ligand binding can be used to identify specific structures and their 3D volume or distribution within the cochlea. Here we used TSLIM to examine cochleae from aged mice to quantify the loss of hair cells and spiral ganglion neurons. In addition, advanced analyses (e.g., cluster analysis) were used to visualize local reductions of spiral ganglion neurons in Rosenthal's canal along its 3D volume. These approaches demonstrate TSLIM microscopy's ability to quantify structure-function relationships within and between cochleae.