Neural Basis of Breathing, Vocalization, and Diseases of the Larynx

DeRose, James; Davydenko, Olga

May 02, 2023

Neural Basis of Breathing, Vocalization, and Diseases of the Larynx

Visualizing motor neurons in immunostained whole mounts of mouse laryngeal muscle

Raw widefield (left) and Computationally Cleared image (right) of mouse neuromuscular junctions acquired with a THUNDER Imager. Courtesy of A. Yung and M. Krasnow in California, USA. Mouse_neuromuscular_junctions_raw_widefield_and_Computationally_Cleared__teaser.jpg

This study shows the advantages of imaging whole mounts of mouse laryngeal muscle tissue, which may be useful for research on cancer and diseases of the larynx, with a THUNDER Imager. The larynx is important for respiration and vocalization. Research on cancer and diseases is often done with the aid of mouse models. Immunostained neuromuscular junctions and neurofilaments in motor neurons of mouse laryngeal muscle are better distinguished in Computationally Cleared images taken with a THUNDER Imager when compared to conventional widefield microscopy methods.

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 [1]. Scientist doing research on cancer and disease of the larynx and lungs often use mouse models [2]. One thing they may study is the neural basis of breathing and vocalization which depends on the precisely coordinated movement of tiny laryngeal muscles [3].

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.

Figure 1: Left) Raw widefield and Right) THUNDER image, after ICC, of mouse neuromuscular junctions where neurofilaments are indicated with white and alpha-bungarotoxin with red. Image courtesy of Dr. Andrea Yung from the laboratory of Dr. Mark Krasnow in California, USA.

Conclusions

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.