Optical Redox Imaging of Fixed Unstained Muscle Slides Reveals Useful Biological Information



Optical redox imaging (ORI) technique images cellular autofluorescence of nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp containing FAD, i.e., flavin adenine dinucleotide). ORI has found wide applications in the study of cellular energetics and metabolism and may potentially assist in disease diagnosis and prognosis. Fixed tissues have been reported to exhibit autofluorescence with similar spectral characteristics to those of NADH and Fp. However, few studies report on quantitative ORI of formalin-fixed paraffin-embedded (FFPE) unstained tissue slides for disease biomarkers. We investigate whether ORI of FFPE unstained skeletal muscle slides may provide relevant quantitative biological information.


Living mouse muscle fibers and frozen and FFPE mouse muscle slides were subjected to ORI. Living mouse muscle fibers were imaged ex vivo before and after paraformaldehyde fixation. FFPE muscle slides of three mouse groups (young, mid-age, and muscle-specific overexpression of nicotinamide phosphoribosyltransferase (Nampt) transgenic mid-age) were imaged and compared to detect age-related redox differences.


We observed that living muscle fiber and frozen and FFPE slides all had strong autofluorescence signals in the NADH and Fp channels. Paraformaldehyde fixation resulted in a significant increase in the redox ratio Fp/(NADH + Fp) of muscle fibers. Quantitative image analysis on FFPE unstained slides showed that mid-age gastrocnemius muscles had stronger NADH and Fp signals than young muscles. Gastrocnemius muscles from mid-age Nampt mice had lower NADH compared to age-matched controls, but had higher Fp than young controls. Soleus muscles had the same trend of change and appeared to be more oxidative than gastrocnemius muscles. Differential NADH and Fp signals were found between gastrocnemius and soleus muscles within both mid-aged control and Nampt groups.


Aging effect on redox status quantified by ORI of FFPE unstained muscle slides was reported for the first time. Quantitative information from ORI of FFPE unstained slides may be useful for biomedical applications.

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We thank Dr. William Quinn for providing the living muscle samples, Annemarie Jacob for her assistance during imaging the fixation effects, and Dr. Gordon Ruthel for his assistance in two-photon imaging. We also thank the Cell and Developmental Biology (CDB) Microscopy Core, Perelman School of Medicine and Penn Vet Imaging Core, School of Veterinary Medicine, University of Pennsylvania.


This work was supported by the NIH Grants R01CA191207 (L.Z. Li) and R01 DK098656 (J. A. Baur) and a pilot grant (L.Z. Li and J. A. Baur) from the University of Pennsylvania Institute on Aging.

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Xu, H.N., Zhao, H., Chellappa, K. et al. Optical Redox Imaging of Fixed Unstained Muscle Slides Reveals Useful Biological Information. Mol Imaging Biol 21, 417–425 (2019). https://doi.org/10.1007/s11307-019-01348-z

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Key words

  • NADH and flavoproteins containing FAD
  • Redox ratio
  • Autofluorescence
  • Formalin-fixed unstained tissue slide
  • Muscle aging