摘要：

The investigation of molecular processes in vivo requires dedicated imaging methods allowing visualization of protein-protein interactions, enzymatic activities and intracellular Ca++ fluxes. Fluorescence resonance energy transfer (FRET) detection methods are ideally suited, but have not been adapted so far to 3D intravital imaging. We have combined here multiphoton microscopy (MPM), which is method of choice for non-destructive living tissue inspection, and FRET imaging to monitor calpain proteolytic activity in living mouse muscle. We engineered a FRET-based indicator of calpain activity containing eCFP and eYFP fused to the ubiquitous calpains cleavage site of α-fodrin (Calpain-sensor) and expressed this transgene in mouse muscle 7 days prior to MPM. To extract accurate FRET efficiency images and convert data into knowledge, we performed corrections for depth attenuation and used acceptor photobleaching experiments to calibrate the final results. Our experiments highlights: (i) the feasibility and accuracy of three dimensional multiphoton quantitative FRET imaging in vivo (ii) the ability of this method to detect FRET at cellular and subcellular levels up to 200 μm in living organs. This report demonstrates that numerous applications based on FRET biosensors developed in cell cultures can be applied in living animals. References [1]. Vanderklish, P. W., Krushel, L.A., Holst, B.H., Gally, J.A., Crossin, K.L., Edelman, G.M. (2000). Marking synaptic activity in dendritic spines with a calpain substrate exhibiting fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. U S A. 2000 97, 2253-8. [2]. Miyawaki, A., Llopis, J., Heim, R, McCaffery, J.M., Adams, J.A., Ikura, M., Tsien, R.Y. (1997). Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882-7. [3]. Zhang, J., Campbell, R. E., Ting, A. Y. & Tsien, R. Y. (2002). Creating new fluorescent probes for cell biology. Nat Rev Mol Cell Biol 3, 906-18. [4]. Gordon, G. W., Berry, G., Liang, X. H., Levine, B. & Herman, B. (1998). Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys J. 74, 2702-13. [5]. Stockholm, D., Bartoli, M., Sillon, G., Bourg, N., Davoust, J. & Richard, I. (2005). Imaging Calpain Protease Activity by Multiphoton FRET in Living Mice. J. Mol. Biol. 346, 215-222

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