Third-harmonic microscopy is one of the emerging techniques for noninvasive microscopic imaging of biological structures. We use a novel technique for nonlinear optical material characterization and study the effect of different environment and the structural sensitivity of the third harmonic. In particular, a transformation of collagen in solution is observed for the first time using thirdharmonic generation. We also study the ultimate limits of the third harmonic to detect micro- and nanoscopic features inside living cells and find that structures as small as 50 nm can be detected using the current level of technology. (C) 2003 Elsevier Inc. All rights reserved.