Abstract
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.
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Acknowledgements
We gratefully acknowledge support under US National Institutes of Health grant no. CA136590.
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P.J.C. primarily prepared the manuscript. X.C. constructed the current instrument, acquired the data for the manuscript and developed the analysis scripts; S.P. and O.N. constructed the first instrument; P.J.C., S.P., O.N. and others primarily developed the SHG tissue-imaging protocols.
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Chen, X., Nadiarynkh, O., Plotnikov, S. et al. Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure. Nat Protoc 7, 654–669 (2012). https://doi.org/10.1038/nprot.2012.009
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DOI: https://doi.org/10.1038/nprot.2012.009
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