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Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure

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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Figure 1: Montage of SHG imaging of collagen tissues.
Figure 2: Schematic of the optical layout of the SHG microscope, showing the optical components before the scan head and the detection pathways.
Figure 3: Comparison of SHG images of self-assembled collagen gels using linear and circular excitation polarization.
Figure 4
Figure 5
Figure 6
Figure 7: Flowchart of the day-to-day operational procedures for typical SHG imaging of tissues with approximate times and corresponding step numbers.
Figure 8: Two-photon excited fluorescence images of giant vesicles labeled with the membrane staining dye Di-8-ANEPPS with the rotation of the λ/2 plate.
Figure 9: SHG intensity dependence on the laser polarization.
Figure 10: Representative SHG polarization anisotropy images for mouse tendon are shown with the GLP oriented parallel and perpendicular to the laser polarization.
Figure 11: Using ImageJ to measure collagen fiber lengths.
Figure 12: Forward-backward analysis of SHG image from a fibrillar collagen gel using MATLAB.
Figure 13: 3D renderings of SHG images of a fibrillar collagen gel.
Figure 14: Forward SHG anisotropy images from a fibrillar collagen gel.

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Acknowledgements

We gratefully acknowledge support under US National Institutes of Health grant no. CA136590.

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Contributions

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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Correspondence to Paul J Campagnola.

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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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