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Previous collection of an image at a higher zoom setting has caused slight bleaching in the fluorescent image (arrowed), but the second-harmonic image is unaffected. Note that no SHG signal is visible in the type III collagen (upper right), implying that if it either produces no, SHG signal or a much weaker, SHG signal. reached, for optimal imaging in SHG microscopy the signal has to be able to pass through the entire sample. Thus, in either case, the penetration depth might be 200 µm; however, in SHG, the sample itself cannot be too much thicker than this.
Milthorpe, B. K. (1994) Xenografts for tendon and ligament repair. Biomaterials 15, 745–752. 22. Cox, G. , Sheppard, C. J. , and Ramshaw, J. (2003) Characterization of the second harmonic signal from collagen. Proceedings of SPIE 4963, 32–40. 23. Zipfel, W. , Williams, R. , Nitikin, A. , Hyman, B. , and Webb, W. W. (2003) Live tissue intrinsic emission microscopy using multiphoton excited native fluorescence and second harmonic generation. Proc. Natl. Acad. Sci. USA 100, 7075–7080. 24. , and Fraser, I.
This is more than adequate for effective three-dimensional reconstructions (see Fig. 6). 2. 1. Suitable Samples The SHG signal from type I collagen is very strong—typically stronger than most two-photon excited fluorescence in biological samples—so that low excitation levels can be used. When fluorescence is being detected at the same time, the laser intensity needed will generally be determined by the fluorescent signal. SHG from collagen seems to be unaffected by most preparation techniques. Good images can be obtained from histological paraffin sections, whether unstained (see Fig.