Several techniques have been developed over the past few decades to assess the mechanical properties of biological samples, which has fueled a rapid growth in the fields of biophysics, bioengineering, and mechanobiology. In this context, Brillouin optical spectroscopy has long been known as an intriguing modality for noncontact material characterization. However, limited by speed and sample damage, it had not translated into a viable imaging modality for biomedically relevant materials. Recently, based on a novel spectroscopy strategy that substantially improves the speed of Brillouin measurement, confocal Brillouin microscopy has emerged as a unique complementary tool to traditional methods as it allows noncontact, nonperturbative, label-free measurements of material mechanical properties. The feasibility and potential of this innovative technique at both the cell and tissue level have been extensively demonstrated over the past decade. As Brillouin technology is rapidly recognized, a standard approach for building and operating Brillouin microscopes is required to facilitate the widespread adoption of this technology. In this protocol, we aim to establish a robust approach for instrumentation, and data acquisition and analysis. By carefully following this protocol, we expect that a Brillouin instrument can be built in 5–9 days by a person with basic optics knowledge and alignment experience; the data acquisition as well as postprocessing can be accomplished within 2–8 h.
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The authors declare that all data supporting the findings of this study are available within the paper and its Supplementary Information files. The raw Brillouin spectra of Figs. 8 and 9 are available via Figshare (https://figshare.com/articles/dataset/raw_data_to_Fig_8_9/13135760). Source data are provided with this paper.
The MATLAB code to analyze images as well as representative raw data are provided as Supplementary Data 1.
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The authors thank M. Nikolic and A. Fiore for helpful discussions, and H. Zhang and E. Frank for helping with the LabVIEW program. This work was supported in part by the National Institutes of Health (K25HD097288, R33CA204582, U01CA202177, R01EY028666 and R01HD095520) and the National Science Foundation (CMMI 1929412 and DBI 1942003).
G.S holds patents related to Brillouin technology (US7898656B2, US8115919B2 and US20200278250A1) and is a consultant for Intelon Optics. The other authors declare no competing interests.
Peer review information Nature Protocols thanks Robert Prevedel, Vladislav Yakovlev, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Key references using this protocol:
Scarcelli, G. et al. Nat. Methods 12, 1132-1134 (2015): https://doi.org/10.1038/nmeth.3616
Wisniewski, E. O. et al. Sci. Adv. 6, eaba6505 (2020): https://doi.org/10.1126/sciadv.aba6505
Zhang, J. et al. Small 16, 1907688 (2020): https://doi.org/10.1002/smll.201907688
Supplementary Figs. 1–4 and Data 2–4.
Supplementary Data 1
MATLAB codes and example data.
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Source Data Fig. 9
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Zhang, J., Scarcelli, G. Mapping mechanical properties of biological materials via an add-on Brillouin module to confocal microscopes. Nat Protoc 16, 1251–1275 (2021). https://doi.org/10.1038/s41596-020-00457-2
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