Characterization of connective tissues using near-infrared spectroscopy and imaging


Near-infrared (NIR) spectroscopy is a powerful analytical method for rapid, non-destructive and label-free assessment of biological materials. Compared to mid-infrared spectroscopy, NIR spectroscopy excels in penetration depth, allowing intact biological tissue assessment, albeit at the cost of reduced molecular specificity. Furthermore, it is relatively safe compared to Raman spectroscopy, with no risk of laser-induced photothermal damage. A typical NIR spectroscopy workflow for biological tissue characterization involves sample preparation, spectral acquisition, pre-processing and analysis. The resulting spectrum embeds intrinsic information on the tissue’s biomolecular, structural and functional properties. Here we demonstrate the analytical power of NIR spectroscopy for exploratory and diagnostic applications by providing instructions for acquiring NIR spectra, maps and images in biological tissues. By adapting and extending this protocol from the demonstrated application in connective tissues to other biological tissues, we expect that a typical NIR spectroscopic study can be performed by a non-specialist user to characterize biological tissues in basic research or clinical settings. We also describe how to use this protocol for exploratory study on connective tissues, including differentiating among ligament types, non-destructively monitoring changes in matrix formation during engineered cartilage development, mapping articular cartilage proteoglycan content across bovine patella and spectral imaging across the depth-wise zones of articular cartilage and subchondral bone. Depending on acquisition mode and experiment objectives, a typical exploratory study can be completed within 6 h, including sample preparation and data analysis.

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Fig. 1
Fig. 2: Schematic description of NIR spectroscopy workflow.
Fig. 3
Fig. 4: Near-infrared fiber optic probe.
Fig. 5: Diagrammatic illustration of the steps required for NIR spectroscopic mapping of specific target features on the desired tissue area.
Fig. 6: Effect of different pre-processing options and their combinations on NIR spectral data.
Fig. 7: NIR spectroscopy of tissue-engineered cartilage.
Fig. 8: Mapping the proteoglycan content across bovine patella articular surface.
Fig. 9: NIR spectral imaging of bone and cartilage.

Data availability

The source data underlying Figs. 79 and Supplementary Fig. 1 are provided as Source Data files. Raw imaging files (Fig. 9 and Supplementary Fig. 1) can be opened with MATLAB via the MIA toolbox add-on. Source data for Fig. 6 can be obtained from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

nippy, the Python package used for spectral pre-processing in Fig. 6, is open source and available at under the MIT license.


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This work was financially supported by the Academy of Finland (315820), the European Union (Horizon 2020 Research and Innovation Programme, 780598), the SCITECO Doctoral Programme of the University of Eastern Finland, State Research Funding of Kuopio University Hospital (5203111) and the U.S. National Institutes of Health (R01 AR056145).

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I.O.A., R.S., W.Q., N.P. and J. Töyräs contributed to conceptualization, overall framing of the protocol and funding acquisition. I.O.A., E.N., W.Q., J. Torniainen, J.K.S., S.K. and N.P. contributed to data analysis, interpretation and figure preparation. I.O.A., R.S., E.N., W.Q., J. Torniainen, J.K.S. and S.K. wrote sections of the manuscript, and I.O.A. led the organization of the original manuscript draft. All authors contributed to and edited/revised the original draft and approved the final version.

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Correspondence to Isaac O. Afara.

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The authors declare no competing interests as defined by Nature Research or other interests that might be perceived to influence the interpretation of the article.

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Peer review information Nature Protocols thanks Daniel Cozzolino, Ilias Tachtsidis and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol:

Afara, I. O. et al. Sci. Rep. 7, 11463 (2017):

Ala-Myllymäki, J. et al. Ann. Biomed. Eng. 2020,

Rajapakse, C. S. et al. Bone 103, 116–124 (2017):

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2.

Supplementary Data

Hyperspectral imaging file containing data for Supplementary Fig. 1.

Source data

Source Data Fig. 7

NIR-mapped proteoglycan content of bovine patella (zipped file contains a .mat file).

Source Data Fig. 8

Raw data file for spectra of tissue-engineered cartilage.

Source Data Fig. 9

Hyperspectral NIR imaging file containing data for Fig. 9 (zipped file contains an .fsm file).

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Afara, I.O., Shaikh, R., Nippolainen, E. et al. Characterization of connective tissues using near-infrared spectroscopy and imaging. Nat Protoc 16, 1297–1329 (2021).

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