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Published February 3, 2014 | Published + Supplemental Material
Journal Article Open

Multicontrast photoacoustic in vivo imaging using near-infrared fluorescent proteins


Non-invasive imaging of biological processes in vivo is invaluable in advancing biology. Photoacoustic tomography is a scalable imaging technique that provides higher resolution at greater depths in tissue than achievable by purely optical methods. Here we report the application of two spectrally distinct near-infrared fluorescent proteins, iRFP670 and iRFP720, engineered from bacterial phytochromes, as photoacoustic contrast agents. iRFPs provide tissue-specific contrast without the need for delivery of any additional substances. Compared to conventional GFP-like red-shifted fluorescent proteins, iRFP670 and iRFP720 demonstrate stronger photoacoustic signals at longer wavelengths, and can be spectrally resolved from each other and hemoglobin. We simultaneously visualized two differently labeled tumors, one with iRFP670 and the other with iRFP720, as well as blood vessels. We acquired images of a mouse as 2D sections of a whole animal, and as localized 3D volumetric images with high contrast and sub-millimeter resolution at depths up to 8 mm. Our results suggest iRFPs are genetically-encoded probes of choice for simultaneous photoacoustic imaging of several tissues or processes in vivo.

Additional Information

© 2016 Macmillan Publishers Limited. This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/ Received 16 September 2013; Accepted 15 January 2014; Published 3 February 2014. We thank M. Lin (Stanford University), B. Glick (University of Chicago) and K. Lukyanov (Institute of Bioorganic Chemistry, Russia) for the plasmids encoding the mNeptune, E2-Crimson and eqFP670 proteins, respectively. We thank L. Luecking for assistance with maintaining cell cultures and J. Ballard for help with editing the manuscript. This work was supported by the grants GM073913 and CA164468 (to V.V.V.) and EB016963 and Director's Pioneer Award EB016986 (to L.V.W.) from the National Institutes of Health. Arie Krumholz & Daria M. Shcherbakova: These authors contributed equally to this work. Author Contributions: D.M.S. prepared iRFP670 and iRFP720 expressing cells, characterized iRFP670 and iRFP720 expression by means of fluorescent imaging. A.K., J.X. and L.V.W. designed and conducted photoacoustic experiments to image iRFP670 and iRFP720. V.V.V. designed and coordinated the project, and together with D.M.S. and A.K. wrote the manuscript. Competing interests: L.V.W. has a financial interest in Microphotoacoustics, Inc. and Endra, Inc., which, however, did not support this work.

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Supplemental Material - srep03939-s1.pdf


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