Quantitative Real-Time Analysis of Living Materials by Stimulated Raman Scattering Microscopy

Creators: Qian, Chenxi; Liu, Hanwei; Chittur, Priya K.; Chadha, Rahuljeet S.; Yao, Yuxing; Kornfield, Julia A.¹; Tirrell, David A.¹; Wei, Lu¹

1. California Institute of Technology

Abstract

Composite materials built in part from living organisms have the potential to exhibit useful autonomous, adaptive, and self-healing behavior. The physicochemical, biological, and mechanical properties of such materials can be engineered through the genetic manipulation of their living components. Successful development of living materials will require not only new methods for design and preparation but also new analytical tools that are capable of real-time noninvasive mapping of chemical compositions. Here, we establish a strategy based on stimulated Raman scattering microscopy to monitor phosphatase-catalyzed mineralization of engineered bacterial films in situ. Real-time label-free imaging elucidates the mineralization process, quantifies both the organic and inorganic components of the material as functions of time, and reveals spatial heterogeneity at multiple scales. In addition, we correlate the mechanical performance of films with the extent of mineralization. This work introduces a promising strategy for quantitatively analyzing living materials, which should contribute to the accelerated development of such materials in the future.

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Acknowledgement

This work was supported by the DARPA Engineered Living Materials program (Award#HR0011-17-2-0037) and the Army Research Office through the Institute for Collaborative Biotechnologies (Award#W911NF-19-2-0026). Chenxi Qian acknowledges the support of the Natural Sciences and Engineering Research Council of Canada (NSERC Postdoctoral Fellowship). Lu Wei acknowledges support from the Heritage Medical Research Institute at the California Institute of Technology. The authors thank Dr. Andres Collazo for assistance with some of the preliminary data by confocal microscopy collected in the Biological Imaging Facility of the Caltech Beckman Institute, which is supported by the Arnold and Mabel Beckman Foundation.

Contributions

C.Q and H.L contributed equally to this work as cofirst authors. C.Q. and H.L. conceived and designed the experiments under the supervision of D.A.T. and L.W. C.Q. was responsible for parts of the biofilm preparation and performed all SRS imaging and analysis. H.L. was responsible for all the cell experiments and most of the sample preparation. P.K.C. performed all experiments related to biofilm mechanical property analysis under the supervision of J.A.K. R.S.C., and Y.Y. performed parts of the SRS and EM imaging, respectively. The manuscript was written by C.Q., H.L., P.K.C., J.A.K., D.A.T., and L.W. with contributions from all coauthors.

Data Availability

For all imaging experiments yielding the micrographs reported herein, at least three independent experiments were repeated with similar results.
Additional methods and materials including bacterial strain, plasmid construct, protein amino acid sequence, viability change over the time, high-quality SRS and calcein stain images, mechanical analysis methods, and all stress versus strain curves (PDF)

Conflict of Interest

The authors declare no competing financial interest.

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