Tissue Clearing and Confocal Microscopic Imaging for Skeletal Muscle

Smrithi Karthikeyan, Yoko Asakura, Mayank Verma, Atsushi Asakura

Research output: Chapter in Book/Report/Conference proceedingChapter


Skeletal muscle is a highly ordered tissue composed of a complex network of a diverse variety of cells. The dynamic spatial and temporal interaction between these cells during homeostasis and during times of injury gives the skeletal muscle its regenerative capacity. To properly understand the process of regeneration, a three-dimensional (3-D) imaging process must be conducted. With the advancement of imaging and computing technology, it has become powerful to analyze spatial data from confocal microscope images. In order to prepare whole tissue skeletal muscle samples for confocal imaging, the muscle must be subjected to tissue clearing. With the use of an ideal optical clearing protocol – one that minimizes light scattering via refractive index mismatching – a more accurate 3-D image of the muscle can be produced as it does not involve the physical sectioning of the muscle. While there have been several protocols relating to the study of 3-D biology in whole tissue, these protocols have primarily been focused on the nervous system. In this chapter, we present a new method for skeletal muscle tissue clearing. In addition, this protocol aims to outline the specific parameters required for taking 3-D images of immunofluorescence-stained skeletal muscle samples using a confocal microscope.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages10
StatePublished - 2023

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Bibliographical note

Funding Information:
We thank the Minnesota Supercomputing Institute, the University of Minnesota Imaging Center. We also thank Dr. Masatsugu Ema (Siga University of Medical Science) for providing Flk1+/GFP mice. This work was supported by NIHR01AR062142, NIH-R21AR070319, and Regenerative Medicine Minnesota (RMM) Grant to AA.

Publisher Copyright:
© 2023, Springer Science+Business Media, LLC, part of Springer Nature.


  • 3D imaging
  • Angiogenesis
  • Endothelial cell
  • Muscle regeneration
  • Muscle stem cell
  • Muscular dystrophy
  • Myogenesis
  • Satellite cell
  • Skeletal muscle
  • Tissue clearing
  • Imaging, Three-Dimensional/methods
  • Microscopy, Confocal
  • Fluorescent Antibody Technique
  • Muscle, Skeletal

PubMed: MeSH publication types

  • Research Support, Non-U.S. Gov't
  • Journal Article
  • Research Support, N.I.H., Extramural


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