Optimisation of specimen handling & mitochondrial analysis in patient skeletal muscle biopsies

Skeletal muscles harbour an unparalleled structural complexity, with a distinctive architecture that sets them apart from other tissues. Skeletal muscle biopsies are invaluable tools for investigation of numerous physiological and pathological muscle conditions. When diagnosing conditions based on muscle biopsies, appropriate handling of skeletal muscles is imperative when it comes to subsequent histological and immunohistochemical analysis to ensure differentiation between actual biology and artifact. Our study aimed to optimise specimen handling of muscle biopsies by utilising samples from healthy human volunteers and rat skeletal muscles.
We conducted six different cryopreservation techniques on rat samples (direct immersion in freezing medium, dipping in optimal cutting temperature compound prior to immersion, and placement of sample in histocassettes prior to immersion – these were done for cryopreservation with liquid nitrogen and precooled isopentane as freezing media) to assess which one is most ideal for further experiments. This assessment was done based on histological staining techniques, and cellular structure was further investigated using fluorescence microscopy for myofibril and mitochondrial architecture. Human biopsy samples were then tested.
Our results demonstrated that placement of sample in histocassettes prior to immersion in precooled isopentane preserves skeletal muscle samples free from any ice crystal artifacts. Following, specimen handling optimisation, we also developed a workflow for mitochondrial analysis using existing Fiji functions and plugins, to accurately obtain various quantitative parameters from skeletal muscles tissue sections stained with mitochondrial dye. The methods developed so far will be employed for a future study on patient muscle biopsy samples.

Challenges faced:
Developing an experimental setup for freezing tissues
Working with Fiji to analyse mitochondria in confocal images with high tissue background
Learning to cryosection skeletal muscle tissues without embedding medium and ultra-thin slices