Imaging elastic and collagen fibers with sulforhodamine B and second-harmonic generation

摘要

Since the early nineties, multiphoton microscopy has become a powerful tool to investigate morphological and physiological parameters in vivo or on thick ex vivo sections. To stain structures of interest many dyes have been developed and two-photon properties (cross section, excitation and emission spectra) of existing ones have been characterized. Recently, our team has shown a new property of sulforhodamine B (SRB). This dye has the ability to bind specifically elastic fibers. The observation of elastin using its endofluorescence properties was already widely described but required long exposition delays up to 10s and the imaging depth was limited to approximately 50 μm. With a multiphoton microscope and SRB, it is possible to observe elastic fibers directly in the living animal or on thick tissue sections with a micrometric spatial resolution in less than one second per image with an imaging depth of ～ 200 μm. Moreover, with an appropriate set of filters, we can acquire simultaneously the SRB and the second harmonic generation (SHG) signals of collagen fibers. Here, we report various applications of this new staining method on different arterial rings. The layers of the arterial wall, as well as, the elastic lamellae are observed and are numbered. With the addition of a nuclear stain such as the Hoechst 33342, a more accurate morphological study of the arterial walls can be accomplished. Finally, an intravital observation of the saphenous artery morphology is presented.