This research has been supported by grants from the National Institute on Deafness and Other Communication Disorders of the National Institutes of Health, the Kellogg Foundation, and private donations from Bridget Sperl and John McCormick. TSLIM has been developed with the excellent assistance of Matthias Hillenbrand, Kerstin John, Meike Lawin, Michel Layher, Tobias Schroeter, Peter Schacht, Oliver Dannberg, and Julian Wuester from the Technical University of Illmenau, Germany, supervision by their mentors (Stefan Sinzinger and Rene Theska) and James Leger.

All the procedures and the use of live animals have been reviewed and approved (Protocol ID #2010-38573A) by the University of Minnesota Institutional Care and Use Committee (IACUC) and investigators who use these animals have been thoroughly trained and tested by the Research Animal Resources (RAR) Veterinarians before they have access to the animal facilities. Both male and female mice were used in this study.
1. Cochlea removal for fixation and tissue processing for imaging
<ol>
	<li>Euthanize a mouse using CO2 inhalation. Decapitate the mouse with scissors and make a dorsal-ventral incision through the brain to hemisect the skull. Remove the brain, identify the round bulla in the baso-ventral part of the skull, open the bulla with rongeurs, and visualize and remove the cochlea.</li>
	<li>Fixation: Perform this procedure under a fume hood and using a dissection microscope at 5x magnification. Wear gloves and protective clothing. Puncture the oval window and remove the stapes with a sharp pick. Insert a pick into the round window to puncture the membrane.</li>
	<li>Cover the opened round window with the cut tip of an infusion set attached to a 1 mL syringe filled with 2 mL of formalin. Slowly infuse formalin through the perilymphatic spaces of the cochlea over a 2 min period, noting that formalin is exiting the cochlea via the opened oval window. Trim excess tissue off the cochlea and immerse in a bottle containing 10% formalin, and place on a rotator overnight.</li>
	<li>Decalcification: Rinse the cochlea in PBS 3x for 5 min each and immerse in a bottle containing 10% solution of disodium ethylenediaminetetraacetic acid (EDTA) with rotation for 4 days, changing the solution daily.</li>
	<li>Dehydration: Perfuse the cochlea with PBS 3x and immerse for 15 min between changes. Dehydrate the cochlea with ascending concentrations of ethanol 10%, 50%, 70%, 95%, 95%, 100%, 100%; for 30 min in each concentration. 
	&#8203;NOTE: It is important to remove all the EDTA before dehydration as EDTA precipitates in ethanol. Also, cochleae can be left in any concentration of ethanol greater than 70% overnight.</li>
	<li>Staining: Stain the whole cochlea by immersion in a solution of Rhodamine B isothiocynate (5 &#181;g/mL in 100% ethanol) overnight with rotation. Remove excess dye from the cochlea with two changes of 100% ethanol, 5 min each change.</li>
	<li>Clearing: Transfer the stained cochlea into two changes of Spalteholz12 solution (5:3 methyl salicylate:benzyl benzoate), 30 min each change and leave overnight in the clearing solution with rotation. Cochleae can be left in Spalteholz solution indefinitely.</li>
</ol>
2. Imaging of cochleae
<ol>
	<li>Attach the cochleae to a specimen rod at the oval and round window membrane end so that the clearing solution remains within the cochlea and bubbles are not formed (Figure 2). Care must be taken not to let bubbles form within the cochlea as they are difficult to remove and if left in the tissue, they will cause imaging artifacts.</li>
	<li>Use a UV activating glue to attach the wet cochlea to the dry specimen rod (Figure 2). Attach the cochlea at the oval and round window ends. Cure the UV glue for 10 s by moving around the cochlea with the UV light. 
	NOTE: A loose attachment of the cochlea to the specimen rod will result in imaging defects. This rod is specifically manufactured for this protocol (see Santi et al.8 for details) and is specific to our light-sheet microscope.</li>
	<li>Suspend the cochlea into the imaging chamber filled with Spalteholz solution for imaging. The specimen chamber is an optically clear quartz fluorometer cell (Video 1).</li>
	<li>Attach the specimen rod to a rotating holder that is also attached to XZ translation stage. Most stacks are obtained by translating the specimen in the XZ planes, but rotational stacks can also be obtained.</li>
	<li>TSLIM optical sectioning: Use a blue or green laser for excitation depending upon the type of fluorochrome staining. Position the light sheet in the middle of the tissue for focusing and determine the magnification that will be used to illuminate the full width of the cochlea. Then, use a custom-designed program to move the specimen through the light-sheet across the specimen in the X-axis (stitching the image) and in Z-steps to make a stack of 2D images throughout the cochlea.</li>
	<li>For the first image, the beam waist of the light-sheet is positioned at the edge of the specimen and the program scans the full width of the specimen collecting columns of data (see image stitching; Santi et al.8) that are the width of the confocal parameter (Figure 3) to produce a composite 2D image of maximum resolution across the width of the specimen. The program automates image stitching for each Z-step until the specimen is completely imaged.</li>
	<li>Image processing: Transfer the image stack to another computer and load it into a 3D rendering program for 3D reconstruction and quantification.</li>
</ol>

<ol>
	<li>Deafness and hearing loss. World Health Organization Available from: <a target="_blank" href="https://www.who.int/news-room/fact-sheets/deafness-and-hearing-loss">https://www.who.int/news-room/fact-sheets/deafness-and-hearing-loss</a> (2021)</li><li>Vater, M., K&#246;ssl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+aspects+of+cochlear+functional+organization+in+mammals.">Comparative aspects of cochlear functional organization in mammals.</a> Hearing Research. 273 (1-2), 89-99 (2011).</li><li>Santi, P. A., Blair, A., Bohne, B. A., Lukkes, J., Nietfeld, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+digital+cytocochleogram.">The digital cytocochleogram.</a> Hearing Research. 192 (1-2), 75-82 (2004).</li><li>Voie, A. H., Burns, D. H., Spelman, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthogonal-plane+fluorescence+optical+sectioning:+three-dimensional+imaging+of+macroscopic+biological+specimens.">Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens.</a> Journal of Microscopy. 170, 229-236 (1993).</li><li>Voie, A. H., Spelman, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+of+the+cochlea+from+two-dimensional+images+of+optical+sections.">Three-dimensional reconstruction of the cochlea from two-dimensional images of optical sections.</a> Computerized Medical Imaging and Graphics. 19 (5), 377-384 (1995).</li><li>Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J., Stelzer, E. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+sectioning+deep+inside+live+embryos+by+selective+plane+illumination+microscopy.">Optical sectioning deep inside live embryos by selective plane illumination microscopy.</a> Science. 305 (5686), 1007-1009 (2004).</li><li>Santi, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+sheet+fluorescence+microscopy:+a+review.">Light sheet fluorescence microscopy: a review.</a> The Journal of Histochemistry and Cytochemistry. 59 (2), 129-138 (2011).</li><li>Santi, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thin-sheet+laser+imaging+microscopy+for+optical+sectioning+of+thick+tissues.">Thin-sheet laser imaging microscopy for optical sectioning of thick tissues.</a> BioTechniques. 46 (4), 287-294 (2009).</li><li>Schr&#246;ter, T. J., Johnson, S. B., John, K., Santi, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+thin-sheet+laser+imaging+microscopy+(sTSLIM)+with+structured+illumination+and+HiLo+background+rejection.">Scanning thin-sheet laser imaging microscopy (sTSLIM) with structured illumination and HiLo background rejection.</a> Biomedical Optics Express. 3 (1), 170-177 (2012).</li><li>Keller, P. J., Schmidt, A. D., Wittbrodt, J., Stelzer, E. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+zebrafish+early+embryonic+development+by+scanned+light+sheet+microscopy.">Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy.</a> Science. 322 (5904), 1065-1069 (2008).</li><li>Buytaert, J. A. N., Dirckx, J. J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+quantitative+resolution+measurements+of+an+optical+virtual+sectioning+three-dimensional+imaging+technique+for+biomedical+specimens,+featuring+two-micrometer+slicing+resolution.">Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution.</a> Journal of Biomedical Optics. 12 (1), 014039 (2007).</li><li>Spalteholz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> On making human and animal preparations transparent. , (1914).</li><li>Johnson, S., Schmitz, H., Santi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TSLIM+imaging+and+a+morphometric+analysis+of+the+mouse+spiral+ganglion.">TSLIM imaging and a morphometric analysis of the mouse spiral ganglion.</a> Hearing Research. 278 (1-2), 34-42 (2011).</li><li>Santi, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+of+Corti+surface+preparations+for+computer-assisted+morphometry.">Organ of Corti surface preparations for computer-assisted morphometry.</a> Hearing Research. 24 (3), 179-187 (1986).</li><li>Brown, D., Pastras, C., Curthoys, I., Southwell, C., Van Roon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endolymph+movement+visualized+with+light+sheet+fluorescence+microscopy+in+an+acute+hydrops+model.">Endolymph movement visualized with light sheet fluorescence microscopy in an acute hydrops model.</a> Hearing Research. 339, 112-124 (2016).</li><li>White, J. A., Burgess, B. J., Hall, R. D., Nadol, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+of+degeneration+of+the+spiral+ganglion+cell+and+its+processes+in+the+C57BL/6J+mouse.">Pattern of degeneration of the spiral ganglion cell and its processes in the C57BL/6J mouse.</a> Hearing Research. 141 (1-2), 12-18 (2000).</li><li>Grierson, K. E., Hickman, T. T., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopaminergic+and+cholinergic+innervation+in+the+mouse+cochlea+after+noise-induced+or+age-related+synaptopathy.">Dopaminergic and cholinergic innervation in the mouse cochlea after noise-induced or age-related synaptopathy.</a> Hearing Research. 422, 108533 (2022).</li></ol>

The authors have nothing to disclose.

Optical sectioning by light sheet microscopy for examination of cochlear structures is not mechanically destructive like other more traditional histological methods, and it provides a complete digital view of cochlear structures relative to one another. Previous methods such as surface preparations of the organ of Corti14 provided a map of hair cell loss along the length of the basilar membrane, but SGN loss could not be assessed since the tissue had been dissected away to reveal the organ of Cort...

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&#39;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&#39;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 Schr&#246;ter 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&#39;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&#39;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&#39;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&#39;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.

<table><tbody><tr><td>Amira 3D Rendering Software</td><td>ThermoFisher Scientific</td><td></td><td>Address: 501 90th Ave NW, Coon Rapids, MN 55433</td></tr><tr><td>benzyl benzoate (W213810)</td><td>Sigma-Aldrich, Inc.&amp;nbsp;</td><td></td><td>Address: PO Box, 14508, St. Louis, MO 68178</td></tr><tr><td>Bondic&amp;nbsp;</td><td>Bondic&amp;nbsp;</td><td></td><td>Address: 235 Industrial Parkway S., Unit 18 Aurora, ON L4G 3V5 Canada</td></tr><tr><td>Ethanol 95% and 100%&amp;nbsp;</td><td>University of Minnesota</td><td></td><td>Address: General Storehouse, Minneapolis, MN 55455</td></tr><tr><td>Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)&amp;nbsp; (E5134)</td><td>Sigma-Aldrich, Inc.&amp;nbsp;</td><td></td><td>Address: PO Box, 14508, St. Louis, MO 68178</td></tr><tr><td>LabVIEW graphical program and Vision</td><td>National Instruments</td><td></td><td>Address: 11500 N Mopac Expwy Austin, TX 78759-3504</td></tr><tr><td>methyl salicylate (M6742)</td><td>Sigma-Aldrich, Inc.&amp;nbsp;</td><td></td><td>Address: PO Box, 14508, St. Louis, MO 68178</td></tr><tr><td>Olympus MVX10 dissection microscope</td><td>Olympus Corp</td><td></td><td>Address: 3500 Corporate Parkway, Center Valley, PA 18034</td></tr><tr><td>Rhodamine B isothiocynate, (283924)&amp;nbsp;</td><td>Sigma-Aldrich, Inc.&amp;nbsp;</td><td></td><td>Address: PO Box, 14508, St. Louis, MO 68178</td></tr><tr><td>Starna Flurometer Cell (3-G-20)</td><td>Starna Cells</td><td></td><td>Address: PO Box 1919, Atascadero, CA 82423</td></tr></tbody></table>

imaging the aging cochlea with light-sheet fluorescence microscopy

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&#39;s canal along its 3D volume. These approaches demonstrate TSLIM microscopy&#39;s ability to quantify structure-function relationships within and between cochleae.

Since the theme of this special issue is imaging the effects of aging in the cochlea, a young (3-month-old, HS2479, CBA strain mouse) and aged (23-month-old, HS2521, C57 strain mouse) cochleae will be used as examples. It should be noted that TSLIM is capable of imaging a variety of specimens, including cochleae from humans, mammals, other rodents, and fish, as well as other organs such as the brain.
Johnson et al. 13 published an article on SGNs in young (3-week-old) C...

Watch this Scientific Journal Video about Imaging the Aging Cochlea with Light-Sheet Fluorescence Microscopy at JoVE.com

Imaging the Aging Cochlea with Light-Sheet Fluorescence Microscopy

A light-sheet microscope was developed to image and digitize whole cochlea.

Deafness is the most common sensory impairment, affecting approximately 5% or 430 million people worldwide as per the World Health Organization1. Aging or ...

imaging-the-aging-cochlea-with-light-sheet-fluorescence-microscopy

Research

JoVE Journal

Neuroscience

2.1K Views.  University of Minnesota. We hebben een lichtplaatmicroscoop ontwikkeld die het hele slakkenhuis kan digitaliseren. Deze microscoop heeft op lucht gemonteerde objectieven met een lange werkafstand die kleine muizenslakkenhuizen tot een groot menselijk temporale bot kunnen afbeelden. Het snijdt het slakkenhuis serieel met een laserstraal zonder mechanische vernietiging te veroorzaken.Onze beeldvormingsmethode kan worden gebruikt om pathologische veranderingen in het slakkenhuis te beoordelen, zoals het verlies v...

Video: Imaging the Aging Cochlea with Light-Sheet Fluorescence Microscopy

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These amyloid fibrils can sequester endogenous metastable proteins as well as components of the proteostasis network (PN) and thereby exacerbate protein misfolding in the cell. There are a limited number of tools available to assess the aggregation process of amyloid proteins within an animal. We present a protocol for fluorescence lifetime microscopy (FLIM) that allows monitoring as well as quantification of the amyloid fibrilization in specific cells, such as neurons, in a noninvasive manner and with the progression of aging and upon perturbation of the PN. FLIM is independent of the expression levels of the fluorophore and enables an analysis of the aggregation process without any further staining or bleaching. Fluorophores are quenched when they are in close vicinity of amyloid structures, which results in a decrease of the fluorescence lifetime. The quenching directly correlates with the aggregation of the amyloid protein. FLIM is a versatile technique that can be applied to compare the fibrilization process of different amyloid proteins, environmental stimuli, or genetic backgrounds in vivo in a non-invasive manner.

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Fluorescence lifetime imaging monitors, quantifies and distinguishes the aggregation tendencies of proteins in living, aging, and stressed C. elegans disease models.

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These ...

Biochemistry

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Dextran Labeling and Uptake in Live and Functional Murine Cochlear Hair Cells

The hair cell mechanotransduction (MET) channel plays an important role in hearing. However, the molecular identity and structural information of MET remain unknown. Electrophysiological studies of hair cells revealed that the MET channel has a large conductance and is permeable to relatively large fluorescent cationic molecules, including some styryl dyes and Texas Red-labeled aminoglycoside antibiotics. In this protocol, we describe a method to visualize and evaluate the uptake of fluorescent dextrans in hair cells of the organ of Corti explants that can be used to assay for functional MET channels. We found that 3 kDa Texas Red-labeled dextran specifically labels functional auditory hair cells after 1&#8211;2 h incubation. In particular, 3 kDa dextran labels the two shorter stereocilia rows and accumulates in the cell body in a diffuse pattern when functional MET channels are present. An additional vesicle-like pattern of labeling was observed in the cell body of hair cells and surrounding supporting cells. Our data suggest that 3 kDa Texas-Red dextran can be used to visualize and study two pathways for cellular dye uptake; a hair cell-specific entry route through functional MET channels and endocytosis, a pattern also available to larger dextran.

Here, we present a method for visualizing the uptake of 3 kDa Texas Red-labeled dextran in auditory hair cells with functional mechanotransduction channels. In addition, dextrans of 3&#8211;10 kDa can be used to study endocytosis in hair and supporting cells of the organ of Corti.

The hair cell mechanotransduction (MET) channel plays an important role in hearing. However, the molecular identity and structural information of MET ...

Bioengineering

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

Here, we describe a protocol for visualizing motor neuron projection and axon arborization in transgenic Hb9::GFP mouse embryos. After immunostaining for motor neurons, we used light sheet fluorescence microscopy to image embryos for subsequent quantitative analysis. This protocol is applicable to other neuron navigation processes in the central nervous system.

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Stereocilia Bundle Imaging with Nanoscale Resolution in Live Mammalian Auditory Hair Cells

Inner ear hair cells detect sound-induced displacements and transduce these stimuli into electrical signals in a hair bundle that consists of stereocilia that are arranged in rows of increasing height. When stereocilia are deflected, they tug on tiny (~5 nm in diameter) extracellular tip links interconnecting stereocilia, which convey forces to the mechanosensitive transduction channels. Although mechanotransduction has been studied in live hair cells for decades, the functionally important ultrastructural details of the mechanotransduction machinery at the tips of stereocilia (such as tip link dynamics or transduction-dependent stereocilia remodeling) can still be studied only in dead cells with electron microscopy. Theoretically, scanning probe techniques, such as atomic force microscopy, have enough resolution to visualize the surface of stereocilia. However, independent of imaging mode, even the slightest contact of the atomic force microscopy probe with the stereocilia bundle usually damages the bundle. Here we present a detailed protocol for the hopping probe ion conductance microscopy (HPICM) imaging of live rodent auditory hair cells. This non-contact scanning probe technique allows time lapse imaging of the surface of live cells with a complex topography, like hair cells, with single nanometers resolution and without making physical contact with the sample. The HPICM uses an electrical current passing through the glass nanopipette to detect the cell surface in close vicinity to the pipette, while a 3D-positioning piezoelectric system scans the surface and generates its image. With HPICM, we were able to image stereocilia bundles and the links interconnecting stereocilia in live auditory hair cells for several hours without noticeable damage. We anticipate that the use of HPICM will allow direct exploration of ultrastructural changes in the stereocilia of live hair cells for better understanding of their function.

Here we present a protocol for the Hopping Probe Ion Conductance Microscopy (HPICM), a non-contact scanning probe technique that allows nanoscale imaging of stereocilia bundles in live auditory hair cells.

Inner ear hair cells detect sound-induced displacements and transduce these stimuli into electrical signals in a hair bundle that consists of stereocilia ...

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining endocochlear potential, ion transport, and fluid balance. Different forms of hearing disorders are reported to involve abnormal microcirculation in the cochlea. Investigation of how cochlear blood flow (CoBF) pathology affects hearing function is challenging due to the lack of feasible interrogation methods and the difficulty in accessing the inner ear. An open vessel-window in the lateral cochlear wall, combined with fluorescence intravital microscopy, has been used for studying CoBF changes in vivo, but mostly in guinea pig and only recently in the mouse. This paper and the associated video describe the open vessel-window method for visualizing blood flow in the mouse cochlea. Details include 1) preparation of the fluorescent-labeled blood cell suspension from mice; 2) construction of an open vessel-window for intravital microscopy in an anesthetized mouse, and 3) measurement of blood flow velocity and volume using an offline recording of the imaging. The method is presented in video format to show how to use the open window approach in mouse to investigate structural and functional changes in the cochlear microcirculation under normal and pathological conditions.

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining ...

Medicine

Immunolabeling and Counting Ribbon Synapses in Young Adult and Aged Gerbil Cochleae

The loss of ribbon synapses connecting inner hair cells and afferent auditory nerve fibers is assumed to be one cause of age-related hearing loss. The most common method for detecting the loss of ribbon synapses is immunolabeling because it allows for quantitative sampling from several tonotopic locations in an individual cochlea. However, the structures of interest are buried deep inside the bony cochlea. Gerbils are used as an animal model for age-related hearing loss. Here, routine protocols for fixation, immunolabeling gerbil cochlear whole mounts, confocal imaging, and quantifying ribbon synapse numbers and volumes are described. Furthermore, the particular challenges associated with obtaining good material from valuable aging individuals are highlighted. 
Gerbils are euthanized and either perfused cardiovascularly, or their tympanic bullae are carefully dissected out of the skull. The cochleae are opened at the apex and base and directly transferred to the fixative. Irrespective of the initial method, the cochleae are postfixed and subsequently decalcified. The tissue is then labeled with primary antibodies against pre- and postsynaptic structures and hair cells. Next, the cochleae are incubated with secondary fluorescence-tagged antibodies that are specific against their respective primary ones. The cochleae of aged gerbils are then treated with an autofluorescence quencher to reduce the typically substantial background fluorescence of older animals' tissues. 
Finally, cochleae are dissected into 6-11 segments. The entire cochlear length is reconstructed such that specific cochlear locations can be reliably determined between individuals. Confocal image stacks, acquired sequentially, help visualize hair cells and synapses at the chosen locations. The confocal stacks are deconvolved, and the synapses are either counted manually using ImageJ, or more extensive quantification of synaptic structures is carried out with image analysis procedures custom-written in Matlab.

A protocol for processing young adult and aged gerbil cochleae by immunolabeling the afferent synaptic structures and hair cells, quenching autofluorescence in aged tissue, dissecting and estimating the length of the cochleae, and quantifying the synapses in image stacks obtained with confocal imaging is presented.

The loss of ribbon synapses connecting inner hair cells and afferent auditory nerve fibers is assumed to be one cause of age-related hearing loss. The ...

Imaging the Aging Cochlea with Light-Sheet Fluorescence Microscopy

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