Theranostics 2021; 11(6):2876-2891. doi:10.7150/thno.51558

Research Paper

An advanced optical clearing protocol allows label-free detection of tissue necrosis via multiphoton microscopy in injured whole muscle

Dominik Schneidereit1,2, Anita Bröllochs1, Paul Ritter1,2, Lucas Kreiß1,2, Zeinab Mokhtari3, Andreas Beilhack3, Gerhard Krönke4, Jochen A Ackermann4, Maria Faas4, Anika Grüneboom4, Sebastian Schürmann1,2,5, Oliver Friedrich1,2,5,6,✉

1. Institute of Medical Biotechnology, Friedrich-Alexander University Erlangen-Nürnberg, Paul-Gordan-Str. 3, 91052 Erlangen, Germany
2. Graduate School in Advanced Optical Technologies (SAOT), Friedrich-Alexander University Erlangen-Nürnberg, Paul-Gordan-Str. 7, 91052 Erlangen, Germany
3. Interdisciplinary Center for Clinical Research Laboratory for Experimental Stem Cell Transplantation, Department of Medicine II, Würzburg University Medical School, Zinklesweg 10, 97078 Würzburg, Germany
4. Clinics of Internal Medicine III (Rheumatology and Immunology), University Hospitals Erlangen, Friedrich-Alexander University Erlangen-Nürnberg, Ulmenweg 18, 91054 Erlangen, Germany
5. Muscle Research Centre Erlangen (MURCE), Henkestr. 91, 91052 Erlangen, Germany
6. School of Medical Sciences (SOMS), University of New South (UNSW), Kensington Campus, High St, 2052 NSW, Sydney, Australia

This is an open access article distributed under the terms of the Creative Commons Attribution License ( See for full terms and conditions.
Schneidereit D, Bröllochs A, Ritter P, Kreiß L, Mokhtari Z, Beilhack A, Krönke G, Ackermann JA, Faas M, Grüneboom A, Schürmann S, Friedrich O. An advanced optical clearing protocol allows label-free detection of tissue necrosis via multiphoton microscopy in injured whole muscle. Theranostics 2021; 11(6):2876-2891. doi:10.7150/thno.51558. Available from

File import instruction


Rationale: Structural remodeling or damage as a result of disease or injury is often not evenly distributed throughout a tissue but strongly depends on localization and extent of damaging stimuli. Skeletal muscle as a mechanically active organ can express signs of local or even systemic myopathic damage, necrosis, or repair. Conventionally, muscle biopsies (patients) or whole muscles (animal models) are mechanically sliced and stained to assess structural alterations histologically. Three-dimensional tissue information can be obtained by applying deep imaging modalities, e.g. multiphoton or light-sheet microscopy. Chemical clearing approaches reduce scattering, e.g. through matching refractive tissue indices, to overcome optical penetration depth limits in thick tissues.

Methods: Here, we optimized a range of different clearing protocols. We find aqueous solution-based protocols employing (20-80%) 2,2'-thiodiethanol (TDE) to be advantageous over organic solvents (dibenzyl ether, cinnamate) regarding the preservation of muscle morphology, ease-of-use, hazard level, and costs.

Results: Applying TDE clearing to a mouse model of local cardiotoxin (CTX)-induced muscle necrosis, a complete loss of myosin-II signals was observed in necrotic areas with little change in fibrous collagen or autofluorescence (AF) signals. The 3D aspect of myofiber integrity could be assessed, and muscle necrosis in whole muscle was quantified locally via the ratios of detected AF, forward- and backward-scattered Second Harmonic Generation (fSHG, bSHG) signals.

Conclusion: TDE optical clearing is a versatile tool to study muscle architecture in conjunction with label-free multiphoton imaging in 3D in injury/myopathy models and might also be useful in studying larger biofabricated constructs in regenerative medicine.

Keywords: skeletal muscle, optical clearing, muscle injury, multiphoton imaging, light-sheet microscopy