We have developed a light-sheet microscope which can digitize the whole cochlea. This microscope has air-mounted objectives with a long working distance that can image small mouse cochleas up to a large human temporal bone. It serially sections the cochlea with a laser beam without causing any mechanical destruction.
Our imaging method can be used to assess pathological changes in the cochlea, such as the loss of hair cells and spiral ganglion neurons due to aging. After euthanizing and decapitating a mouse, make a dorsal-ventral incision through the brain to hemisect the skull. Remove the brain and identify the round bulla in the basoventral part of the skull.
Open the bulla with rongeurs. Then, visualize and remove the cochlea. Under a dissection microscope at five times magnification, puncture the oval window and remove the stapes with a sharp pick.
Then, insert a pick into the round window to puncture the membrane. To perform fixation, cover the opened round window with the cut tip of an infusion set which is attached to a one-milliliter syringe filled with two milliliters of formalin. Slowly infuse formalin through the perilymphatic spaces of a cochlea over a two-minute period, ensuring that formalin is exiting the cochlea via the opened oval window.
Trim excess tissue off the cochlea. Immerse it in a bottle containing 10%formalin and place it on a rotator overnight. For decalcification, rinse the cochlea in PBS thrice for five minutes each.
Immerse in a bottle containing 10%solution of EDTA. Incubate it for four days with rotation, changing the solution daily. Then, perfuse the cochlea with PBS thrice and immerse for 15 minutes between changes to remove all EDTA.
After washing, dehydrate the cochlea with ascending concentrations of ethanol for 30 minutes in each concentration. Stain the whole cochlea by immersion in rhodamine B solution overnight with rotation. Remove excess dye from the cochlea with two changes of 100%ethanol with incubation for five minutes for each change.
Transfer the stained cochlea into two changes of Spalteholz solution for 30 minutes in each change. Leave overnight in the clearing solution with rotation. Attach the cochlea to a specimen rod at the oval and round window membrane end.
Ensure that the clearing solution remains within the cochlea and bubbles are not formed. Attach the oval and round window ends of the wet cochlea to the dry specimen rod using UV activating glue. Cure the UV glue for 10 seconds by moving around the cochlea with a UV light.
Suspend the cochlea into the imaging chamber in optically-clear quartz fluorometer cell filled with Spalteholz solution with the specimen rod attached to a rotating holder that is also attached to X/Z translation stage. Then, use a custom designed program to move the specimen through the light-sheet in the X-axis and Z steps and make a stack of 2D images throughout the cochlea. To process the image, transfer the image stack to another computer and load it into a 3D rendering program for 3D reconstruction and quantification.
Direct volume rendering of a mouse cochlea showed the oval and round windows, organ of Corti with hair cells, Rosenthal's canal was segmented and volume rendered, and showed spiral ganglion neurons, or SGNs, along its length. Using TSLIM, the cochlea of a three-month-old young mouse and a 23-month-old mouse were imaged and counted for SGNs. SGN cell counts as a function of Rosenthal's canal distance depicted in a linear plot showed greater SGNs in the younger animal in the middle and apical ends of the cochlea as compared to the older animal.
This apparent loss of SGNs was visualized by using cluster analysis and 3D rendering software. SGN density differences were depicted on a color scale where high-density clusters are yellow and blue represents lower density regions. The cluster analysis clearly depicted the regions of lower cell density along the length of Rosenthal's canal.
Light-sheet microscopy is designed to produce a well-aligned stack of images from which 3D volume renderings of structures can be produced. 3D rendering programs allow for the quantitative assessment of structures and anatomical relationships among different structures. However, since 3D rendering programs offer many different user parameters, there's a significant learning curve to use these programs effectively.
