Clearing skeletal muscle with CLARITY for light microscopy imaging

Area of Science:

• Biomedical Engineering
• Microscopy
• Histology

Background:

• Deep tissue imaging is crucial for visualizing cellular structures in whole organs.
• Tissue opacity hinders deep imaging, necessitating advanced tissue clearing techniques.
• CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel) is a novel tissue clearing protocol.

Purpose of the Study:

• To evaluate the efficacy of CLARITY for clearing mouse hindlimb skeletal muscles.
• To assess the preservation of muscle and fiber structural integrity after CLARITY processing.
• To determine the feasibility of fluorescent marker application in CLARITY-cleared skeletal muscle.

Main Methods:

• Application of the CLARITY protocol to adult mouse hindlimb skeletal muscle samples.
• Microscopic examination of cleared muscle tissue to assess structural preservation.
• Attempted integration of fluorescent markers for subcellular visualization.

Main Results:

• CLARITY effectively cleared mouse hindlimb skeletal muscles, rendering them transparent.
• The basic structural features of the muscle and its individual fibers were maintained post-clearing.
• Successful integration of fluorescent markers for detailed subcellular imaging was not achieved.

Conclusions:

• CLARITY is a viable method for achieving optical transparency in skeletal muscle tissue.
• While structural integrity is preserved, CLARITY's current limitations impede fluorescent labeling for subcellular analysis in muscle.
• Further optimization is needed to enable detailed subcellular visualization in cleared skeletal muscle using CLARITY.