Skip to main content Accessibility help

Visualization of ATP with Luciferin-Luciferase Reaction in Mouse Skeletal Muscles Using an “In Vivo Cryotechnique”

  • N. Terada (a1) (a2), Y. Saitoh (a1), S. Saitoh (a1), N. Ohno (a1), K. Fujishita (a3), S. Koizumi (a3) and S. Ohno (a1)...


Adenosine triphosphate (ATP) is a well-known energy source for muscle contraction. In this study, to visualize localization of ATP, a luciferin-luciferase reaction (LLR) was performed in mouse skeletal muscle with an “in vivo cryotechnique” (IVCT). First, to confirm if ATP molecules could be trapped and detected after glutaraldehyde (GA) treatment, ATP was directly attached to glass slides with GA, and LLR was performed. The LLR was clearly detected as an intentional design of the ATP attachment. The intensity of the light unit by LLR was correlated with the concentration of the GA-treated ATP in vitro. Next, LLR was evaluated in mouse skeletal muscles with IVCT followed by freeze-substitution fixation (FS) in acetone-containing GA. In such tissue sections the histological structure was well maintained, and the intensity of LLR in areas between muscle fibers and connective tissues was different. Moreover, differences in LLR among muscle fibers were also detected. For the IVCT-FS tissue sections, diaminobenzidine (DAB) reactions were clearly detected in type I muscle fibers and erythrocytes in capillaries, which demonstrated flow shape. Thus, it became possible to perform microscopic evaluation of the numbers of ATP molecules in the mouse skeletal muscles with IVCT, which mostly reflect living states.


Corresponding author

* Corresponding author: E-mail:


Hide All
Allen, D.G., Lannergren, J. & Westerblad, H. (2002). Intracellular ATP measured with luciferin/luciferase in isolated single mouse skeletal muscle fibres. Eur J Physiol 443, 836842.
Cebasek, V., Erzen, I., Vyhnal, A., Janacek, J., Ribaric, S. & Kubinova, L. (2010). The estimation error of skeletal muscle capillary supply is significantly reduced by 3D method. Microvasc Res 79, 4046.
Fouces, V., Torrella, J.R., Palomeque, J. & Viscor, G. (1993). A histochemical ATPase method for the demonstration of the muscle capillary network. J Histochem Cytochem 41, 283289.
Hepple, R.T. & Mathieu-Costello, O. (2001). Estimating the size of the capillary-to-fiber interface in skeletal muscle: A comparison of methods. J Appl Physiol 91, 21502156.
Janacek, J., Cebasek, V., Kubinova, L., Ribaric, S. & Erzen, I. (2009). 3D visualization and measurement of capillaries supplying metabolically different fiber types in the rat extensor digitorum longus muscle during denervation and reinnervation. J Histochem Cytochem 57, 437447.
Khan, H.A. (2003). Bioluminometric assay of ATP in mouse brain: Determinant factors for enhanced test sensitivity. J Biosci 28, 379382.
Lind, A. & Kernell, D. (1991). Myofibrillar ATPase histochemistry of rat skeletal-muscles—A 2-dimensional quantitative approach. J Histochem Cytochem 39, 589597.
Maechler, P., Wang, H. & Wollheim, C.B. (1998). Continuous monitoring of ATP levels in living insulin secreting cells expressing cytosolic firefly luciferase. FEBS Lett 422, 328332.
Manfredi, G., Yang, L., Gajewski, C.D. & Mattiazzi, M. (2002). Measurements of ATP in mammalian cells. Methods 26, 317326.
Mathieu-Costello, O. & Hepple, R.T. (2002). Muscle structural capacity for oxygen flux from capillary to fiber mitochondria. Exercise Sport Sci Rev 30, 8084.
Ohno, S., Terada, N., Ohno, N., Saitoh, S., Saitoh, Y. & Fujii, Y. (2010). Significance of ‘in vivo cryotechnique’ for morphofunctional analyses of living animal organs. J Electron Microsc 59, 395408.
Rizza, T., Vazquez-Memije, M.E., Meschini, M.C., Bianchi, M., Tozzi, G., Nesti, C., Piemonte, F., Bertini, E., Santorelli, F.M. & Carrozzo, R. (2009). Assaying ATP synthesis in cultured cells: A valuable tool for the diagnosis of patients with mitochondrial disorders. Biochemi Biophys Res Comm 383, 5862.
Roels, F. (1974). Letter: Cytochrome c and cytochrome oxidase in diaminobenzidine staining of mitochondria. J Histochem Cytochem 22, 442444.
Saitoh, Y., Terada, N., Saitoh, S., Ohno, N., Fujii, Y. & Ohno, S. (2010). Histochemical approach of cryobiopsy for glycogen distribution in living mouse livers under fasting and local circulation loss conditions. Histochem Cell Biol 133, 229239.
Shi, L., Terada, N., Saitoh, Y., Saitoh, S. & Ohno, S. (2011). Immunohistochemical distribution of serum proteins in living mouse heart with in vivo cryotechnique. Acta Histochem Cytochem 44, 6172.
Terada, N., Ohno, N., Saitoh, S., Saitoh, Y. & Ohno, S. (2009). Immunoreactivity of glutamate in mouse retina inner segment of photoreceptors with in vivo cryotechnique. J Histochem Cytochem 57, 883888.
Terada, N., Saitoh, Y., Ohno, N., Komada, M., Saitoh, S., Peles, E. & Ohno, S. (2012). Essential function of protein 4.1G in targeting of MPP6 into Schmidt-Lanterman incisures in myelinated nerves. Mol Cell Biol 32, 199205.
Terada, N., Saitoh, Y., Saitoh, S., Ohno, N., Jin, T. & Ohno, S. (2010). Visualization of microvascular blood flow in mouse kidney and spleen by quantum dot injection with “in vivo cryotechnique.” Microvasc Res 80, 491498.
Torrella, J.R., Fouces, V., Palomeque, J. & Viscor, G. (1993). A combined myosin ATPase and acetylcholinesterase histochemical method for the demonstration of fibre types and their innervation pattern in skeletal muscle. Histochem 99, 369372.
Vazquez-Memije, M.E., Shanske, S., Santorelli, F.M., Kranz-Eble, P., Davidson, E., DeVivo, D.C. & DiMauro, S. (1996). Comparative biochemical studies in fibroblasts from patients with different forms of Leigh syndrome. J Inherit Metab Dis 19, 4350.


Visualization of ATP with Luciferin-Luciferase Reaction in Mouse Skeletal Muscles Using an “In Vivo Cryotechnique”

  • N. Terada (a1) (a2), Y. Saitoh (a1), S. Saitoh (a1), N. Ohno (a1), K. Fujishita (a3), S. Koizumi (a3) and S. Ohno (a1)...


Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed