Imaging the Aging Cochlea with Light-Sheet Fluorescence Microscopy

The cochlea is the peripheral sensory organ for hearing in mammals. It has a complex spiral anatomy of repeating sensory and supporting cells that are anatomically specialized to detect sound vibrations and transmit them to the brain for the perception of hearing. The main sensory elements are the inner and outer hair cells and their innervating nerve fibers whose cell bodies compose the spiral ganglion, which resides within Rosenthal's canal (Figure 1). These sensory and neural structures are tonotopically arranged such that high-frequency sounds are transduced in the cochlear base and low-frequency sounds are transduced in the cochlea apex². An anatomical map of this sensory cell distribution along the spiral length of the supporting basilar membrane is called a cytocochleogram³ and can be compared with hearing loss as a function of frequency as depicted in an audiogram.

The membranous labyrinth of the cochlea, which is surrounded by dense bone, makes it technically difficult to examine more than one cochlear structure at a time. Therefore, the rationale for developing a light-sheet microscope is to produce well-aligned serial sections of the complete cochlea so that all cochlear structures can be examined relative to one another in 3D reconstructions. Voie et al.⁴ and Voie and Spelman⁵ designed the first light-sheet microscope, called orthogonal plane fluorescence optical sectioning (OPFOS) microscope, to optically section the whole cochlea. However, this microscope was never commercially developed; so, our aim was to construct a light sheet microscope called a thin sheet laser imaging microscope (TSLIM; Figure 2). The design and construction details for TSLIM have previously been published⁸. TSLIM made several improvements over the OPFOS, including using a low-light digital camera versus a CCD camera for image collection, optically encoded micropositioners for accurate and reproducible movement of the specimen through the light-sheet, use of a commercially available, optically clear specimen chamber, and Rhodamine staining in ethanol rather than in the clearing solution to prevent stain precipitation within the tissue. Commercial development of light-sheet microscopes such as SPIM⁶ have focused on high-resolution imaging of live, small transparent specimens but are unsuitable for whole cochlear imaging as they lack adequate working distance. A review of the development of other light-sheet microscopes was published by Santi⁷. TSLIM's primary advantage over other histological methods to examine the cochlea is to optically section tissues for 3D reconstruction while preserving the integrity of the specimen so that it can be used by other histological methods. Another advantage of TSLIM imaging is that only a thin light-sheet produced by a laser is exposed to the tissue, compared with whole tissue thickness exposure to the laser as in confocal microscopy. Tissue clearing to minimize light scatter and the fact that only a small portion of the tissue is exposed to the laser results in minimal fluorochrome fading (photobleaching) with light-sheet laser imaging. However, the process of fixation, dehydration, and clearing does alter the morphology of cochlear structures and results in tissue shrinkage compared with living tissue. The actual amount of tissue shrinkage that occurs was not determined.

TSLIM was developed by Shane Johnson and eight German optical engineering students (see Acknowledgements). TSLIM construction details were provided by Santi et al.⁸ and a scanning version (sTSLIM) by Schröter et al.⁹. TSLIM functions as a nondestructive microtome to optically section specimens and as a microscope to collect 2D serial sections through the full width and thickness of the cochlea. TSLIM can image both small (mm) and large (cm), thick specimens. Lenses are air mounted to allow for long working distances with collection objectives of 1x and 2x on a dissection microscope. The dissection microscope also has zoom optics that allow TSLIM to resolve subcellular and synaptic structures on cells. TSLIM is equipped with both a blue (473 nm) and green (532 nm) laser for illumination that allows for a variety of fluorescent probes to be used for imaging. The goal of TSLIM is to produce well-aligned 2D optical sections through a whole cochlea for a complete digital reconstruction of cochlear tissues. Since it is a fluorescent method, ligands and immunohistochemistry can also be used to identify specific cochlear structures.

Initially, a cylindrical lens was used to produce two opposed Gaussian light sheets, but it produced absorption imaging artifacts. Due to the work of Keller et al.¹⁰, the fixed cylindrical lens was replaced by a scanning galvanometer mirror to produce the light sheet⁹. In addition, since the center of the light sheet is the thinnest at the beam waist, sTSLIM 2D images are produced by collecting a composite of X-axis columns of data across the specimen's width (Figure 3). This method was first described by Buytaert and Dircks¹¹. TSLIM custom software to drive and collect images was developed using a graphical program for instrument control. The light sheet travels through the specimen and illuminates a fluorescent plane within the tissue. This fluorescent plane is projected orthogonally through the transparent specimen and is collected by a dissection microscope. Optically encoded micropositioners allow scanning through the beam waist in the X-axis to collect a single composite 2D image and, subsequently, the Z-axis micropositioner moves the specimen to a deeper plane within the tissue to obtain a stack of serial, sectioned 2D images (Video 1, Figure 4). A stack of translational images is collected through the entire width, thickness, and length of the cochlea, and stitching of images is not required (Video 2). The image stack is transferred to another computer and loaded into a 3D rendering program for 3D reconstruction and quantification. The image stacks contain all the digital information about the morphology of a cochlea at the resolution of the microscope. However, if a higher resolution is required, the intact cochlea can be further processed by destructive histological methods such as microtome sectioning, scanning, and transmission electron microscopy.

The 3D rendering program is used to segment different cochlear structures for 3D rendering and quantitative analysis. For segmentation, each structure in every 2D image of the stack is traced using a different color by a graphics tablet and pen (Figure 5). To date, 20 different cochlear structures have been segmented (Figure 6). After segmentation, a variety of 3D analyses can be performed. For example, 3D rendering software can virtually resection the cochlea in any plane along the structure's centroid. Video 3 shows sectioning tangential to the organ of Corti, which reveals the hair cells along the length of the basilar membrane. This process first requires manual segmentation of the structure of interest. Next, the structure's centroid is calculated based on the least squares fit of spline points placed along the center of the structure from its base to its apex, thus allowing an approximation of the structure's length (Video 4). A similar process called skeletonization can be used to visualize the radial width of the structure along its length using a color map (Video 4). The total volume of each structure is calculated by the program after segmentation, but relative distances can also be quantified and visualized with color maps in a 3D rendering software (Figure 7). Segmented structures can also be exported to produce enlarged, solid-plastic model renderings (Figure 8). In addition, semi-automated cell counting can also be performed using 3D rendering software (Figure 9). Immunohistochemistry and ligand binding can be used to stain specific cochlear structures and these structures can be isolated from other cochlear structures for morphometrical assessment such as producing a cytocochleogram (Figure 10). Length, width, surface, volume, and number of all cochlear structures can be determined from the 3D models, making this approach ideal for mapping cochlear damage to functional impairments. Specifically, cochlear damage due to aging, noise-induced trauma, or other insults can be shown and quantified in 3D cochlear reconstructions from 2D optical sections. Once a cochlea has been digitized there are numerous imaging algorithms that can be used to assess cochlear damage of any tissue within the cochlea in the anatomical registry to other cochlear tissues.