Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

Rasmus Jensen, Niels Ørtenblad, Cristiano Di Benedetto, Klaus Qvortrup, Joachim Nielsen*

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Abstract

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This enables quantifications of ultrastructural aspects such as volume fractions, surface area to volume ratios, morphometry, and physical contact sites of different subcellular structures. In the 1970s, a protocol for enhanced staining of glycogen in cells was developed and paved the way for a string of studies on the subcellular localization of glycogen and glycogen particle size using transmission electron microscopy. While most analyses interpret glycogen as if it is homogeneously distributed within the muscle fibers, providing only a single value (e.g., an average concentration), transmission electron microscopy has revealed that glycogen is stored as discrete glycogen particles located in distinct subcellular compartments. Here, the step-by-step protocol from tissue collection to the quantitative determination of the volume fraction and particle diameter of glycogen in the distinct subcellular compartments of individual skeletal muscle fibers is described. Considerations on how to 1) collect and stain tissue specimens, 2) perform image analyses and data handling, 3) evaluate the precision of estimates, 4) discriminate between muscle fiber types, and 5) methodological pitfalls and limitations are included.

OriginalsprogEngelsk
Artikelnummere63347
TidsskriftJournal of Visualized Experiments
Vol/bind2022
Udgave nummer180
ISSN1940-087X
DOI
StatusUdgivet - feb. 2022

Bibliografisk note

Funding Information:
This work was supported by the Swedish Olympic Committee.

Publisher Copyright:
© 2022 JoVE Journal of Visualized Experiments.

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