PROTOCOL Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure Xiyi Chen1, Oleg Nadiarynkh1,2, Sergey Plotnikov1,2 & Paul J Campagnola1 1Department of Biomedica l Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA. 2Present addresses: Department of Physics, University of Utrecht, Utrecht, The Netherlands (O.N.); US National Institutes of Health, Heart, Lung and Blood Institute, Bethesda, Maryland, USA (S.P.). Correspondence should be addressed to P.J.C. (
[email protected]). Published online 8 March 2012; doi:10.1038/nprot.2012.009 Second-harmonic generation (SHG) microscopy has emerged as a powerful modality for imaging fibrillar collagen in a diverse range of tissues. Because of its underlying physical origin, it is highly sensitive to the collagen fibril/fiber structure, and, importantly, to changes that occur in diseases such as cancer, fibrosis and connective tissue disorders. We discuss how SHG can be used to obtain more structural information on the assembly of collagen in tissues than is possible by other microscopy techniques. We first provide an overview of the state of the art and the physical background of SHG microscopy, and then describe the optical modifications that need to be made to a laser-scanning microscope to enable the measurements. Crucial aspects for biomedical applications are the capabilities and limitations of the different experimental configurations. We estimate that the setup and calibration of the SHG instrument from its component parts will require 2–4 weeks, depending on the level of the user′s experience. INTRODUCTION Over the past decade, the nonlinear optical method of SHG micros on the harmonophores and their assembly that can be imaged, as copy has emerged as a powerful tool for visualizing the supra the environment must be noncentrosymmetric on the size scale molecular assembly of collagen in tissues at an unprecedented level of of λSHG; otherwise, the signal will vanish.