Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure
Open accessAbstract
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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Second harmonic generation microscopy is a label-free imaging modality capable of visualizing collagen-rich structures within intact spheroids.
“second harmonic generation (SHG) microscopy has emerged as a label-free imaging modality capable of visualizing collagen-rich structures within intact spheroids [259,260].”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [259]
The Introduction’s comparison with other methods says SHG directly visualizes protein assemblies without exogenous labels, and the experimental-design section describes imaging through thick tissue. Neither passage establishes its use for visualizing collagen-rich structures within intact spheroids; the spheroid-specific part is unsupported by our copy. Page numbers are unavailable. (LLM, conf=0.86)
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- Authors
- Chen, X., Nadiarynkh, O., Plotnikov, S., & Campagnola, P. J.
- Affiliations
- University of Wisconsin–Madison (US)
- National Institutes of Health (US)
- Corresponding author
- Campagnola, Paul J
- Venue
- Nature Protocols
- Published
- 2012-03-08
- Volume
- 7
- Issue
- 4
- Pages
- 654-669
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- PMC
- PMC4337962
- OpenAlex
- W1996841581
- Cited by
- 985
- References
- 69
- Citations by year
- 2026: 67 · 2025: 81 · 2024: 91 · 2023: 81 · 2022: 80 · 2021: 93 · 2020: 101 · 2019: 73 · 2018: 82 · 2017: 71 · 2016: 51 · 2015: 44 · 2014: 38 · 2013: 23 · 2012: 7
- FWCI
- 16.39
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Cancer Institute (R01CA136590, CA136590)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Spectroscopy Techniques in Biomedical and Chemical Research
- Physical Sciences › Materials Science › Biomaterials › Collagen: Extraction and Characterization
- Keywords
- second harmonic generation microscopy, collagen fibrils, collagen fibril structure, collagen self-assembly, instrument calibration, connective tissue diseases, fibrosis, cancer, nonlinear optical imaging
- MeSH terms
- Calibration, Collagen, Microscopy, Optics and Photonics
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