Interest of multimodal imaging in tissue engineering.

One of the major limitations when imaging biological tissues results in their thickness and opacity. To overcome these limitations and to visualize some structures of interest and to precise the relationship between the structure and the biological function [6], new imaging methods have been developed. Confocal Laser Scanning Microscopy (CLSM) is the reference technique for biological imaging. But CLSM induces some problems of photo toxicity for tissue which are observed in hard conditions. To overcome this gap, linear optical microscopy also called multiphoton microscopy continues to prove its useful for imaging live tissues in particular with reduced toxicity [9,10]. Multiphoton imaging is noninvasive, by keeping the cells alive and enables to be close to physiological conditions and allows a deeper penetration into samples [3,11] than more usual UV-visible techniques [7]. MP-excitation needs a quasi simultaneous absorption of two (or 3) photons, that means a spatio-temporal confinement of photons. A way to satisfy it and to bring a molecule into its excited state consists in using short impulsions at very high frequency. To come back to its fundamental state, many desexcitation ways among which fluorescence emission are possible. The emitted signal is specific to a molecule and its physicochemical environment and can be resolved as well spatially as temporally or spectrally. Some new tools have been developed to obtain complementary information. FLIM (Fluorescence Lifetime Imaging Microscopy) provides a discrimination of molecules on their different lifetimes (contrast image) whereas their emission spectra overlap. Importantly, the lifetime value measured for by FLIM is a property of individual fluorescent molecules and is therefore largely independent of fluorophore concentration and photobleaching [5]. In addition, the emission of specific fluorophores can be contrasted against a strong auto-fluorescence background as encountered in many biological tissues.