Combined two photon excited fluorescence and second harmonic generation imaging microscopy of collagen structures

Collagen is known to be a very efficient producer of both second harmonic generation (SHG) and two-photon excited fluorescence and the combined use of those nonlinear signals is emerging as a new imaging probe to be used as a diagnostic tool. By recording structural information of collagen between different samples, the technique shows 9 promising for the study of the distribution of collagen in tissue and for identifying pathologic conditions. Unique information about the molecular organization of collagen can be extracted from SHG and TPEF imaging data in several ways and we have initiated a systematic Study of these issues. The main objectives of this work are to combine TPEF and SHG methodologies, in order to elucidate and quantify cross-linking and to describe a model of fibrils orientation within different samples. In this early approach we discuss fundamental principles governing SHG and TPEF and present the first results of applying these rules to collagen type I images analysis. By comparing signals between lyophilized and soluble collagen we validate that the SHG signal arises from dipolar interactions that are enhanced by the quaternary structure of collagen fibrils, while TPEF arises from fluorophores which are suggested to be products Of cross-linking. Using a homogenization protocol of acid treated collagen gels we manage to produce SHG and TPEF active thin films, which characterized by means of their contrast capability. A home-built scanning microscope employing SHG and TPEF was used for the high-resolution imaging of endogenous SHG and TPEF signals, without exogenous dyes.