Neural basis of breathing and vocalization and diseases of the larynx
The larynx is an organ located at the beginning of the trachea in the front part of the neck or throat of humans that plays an important role in both vocalization and breathing . Scientist doing research on cancer and disease of the larynx and lungs often use mouse models . One thing they may study is the neural basis of breathing and vocalization which depends on the precisely coordinated movement of tiny laryngeal muscles .
Challenges imaging whole-mount tissue
A significant challenge that often arises when widefield imaging thick whole-mount specimens of tissue is the out-of-focus blur or haze produced by light scattering [4,5]. Such haze can it make difficult to resolve structures of interest inside the whole-mount specimen.
Methods to investigate whole mounts of mouse laryngeal muscles
To determine the identities of motor neurons in the laryngeal muscle, whole-mount specimens of mouse laryngeal muscle tissue were prepared with immunostaining of neurofilaments (white) and neuromuscular junctions (alpha-bungarotoxin, red). A THUNDER Imager Tissue with a 40x planapo objective having a 0.95 numerical aperture (NA) was used to acquire images of neuromuscular junctions. To capture the entire thickness of the 40-μm whole-mount tissue specimen, a maximum intensity projection of a 50-position z-stack was acquired at each tile-scanned position. The original tile-scanned image with z-stacking was acquired in 5 minutes with a total of 2200 frames. Instant Computational Clearing (ICC) was applied on the fly and processing happened during acquisition.
Results concerning motor neurons
The image of the whole-mount specimen of mouse laryngeal muscle tissue seen in figure 1, showing the neuromuscular junctions and neurofilaments of the motor neurons, is the result of a 22-position tilescan.
The images show how THUNDER imaging of whole-mount mouse laryngeal muscle tissue distinguishes better the immunostained neuromuscular junctions and neurofilaments in motor neurons compared to conventional widefield microscopy. THUNDER Imagers may help improve research on cancer and diseases of the larynx.
- K. Shiba, Functions of larynx in breathing, vocalization, and airway protective reflexes, Ch. 9.1 in Handbook of Behavioral Neuroscience, Ed. S.M. Brudzynski (Elsevier, 2010) vol. 19, pp. 373-381, DOI: 10.1016/B978-0-12-374593-4.00034-6.
- Veerakumar, A., Yung, A.R., Liu, Y., Krasnow, M.A., Molecularly defined circuits for cardiovascular and cardiopulmonary control, Nature (2022) vol. 606, pp. 739-746, DOI: 10.1038/s41586-022-04760-8.
- T.J. Glass, C.A. Kelm-Nelson, J. A. Russell, J.C. Szot, J.M. Lake, N.P. Connor, M.R. Ciucci, Laryngeal muscle biology in the Pink1 −/− rat model of Parkinson disease, J. Appl. Physiol. (2019) vol. 126, pp. 1326-1334, DOI: 10.1152/japplphysiol.00557.2018.
- J. Schumacher, L. Bertrand, Real Time Images of 3D Specimens with Sharp Contrast Free of Haze: Technology Note THUNDER Imagers: How do they really work? Science Lab (2019) Leica Microsystems.
- L. Felts, V. Kohli, J.M. Marr, J. Schumacher, O. Schlicker, An Introduction to Computational Clearing: A New Method to Remove Out-of-Focus Blur, Science Lab (2020) Leica Microsystems.
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