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Fuenzalida Werner, J.P. ; Huang, Y. ; Mishra, K. ; Janowski, R. ; Vetschera, P. ; Heichler, C.* ; Chmyrov, A. ; Neufert, C.* ; Niessing, D. ; Ntziachristos, V. ; Stiel, A.-C.

Challenging a preconception: Optoacoustic spectrum differs from the absorption spectrum of proteins and dyes for molecular imaging.

Anal. Chem. 92, 10717-10724 (2020)
Postprint DOI PMC
Open Access Green
Optoacoustic (photoacoustic) imaging has seen marked advances in detection and data analysis, but there is less progress in understanding the photophysics of common optoacoustic contrast agents. This gap blocks the development of novel agents and the accurate analysis and interpretation of multispectral optoacoustic images. To close it, we developed a multimodal laser spectrometer (MLS) to enable the simultaneous measurement of optoacoustic, absorbance, and fluorescence spectra. Herein, we employ MLS to analyze contrast agents (methylene blue, rhodamine 800, Alexa Fluor 750, IRDye 800CW, and indocyanine green) and proteins (sfGFP, mCherry, mKate, HcRed, iRFP720, and smURFP). We found that the optical absorption spectrum does not correlate with the optoacoustic spectrum for the majority of the analytes. We determined that for dyes, the transition underlying an aggregation state has more optoacoustic signal generation efficiency than the monomer transition. For proteins we found a favored optoacoustic relaxation that stems from the neutral or zwitterionic chromophores and unreported photoswitching behavior of tdTomato and HcRed. We then crystalized HcRed in its photoswitch optoacoustic state, confirming structurally the change in isomerization with respect to HcReds' fluorescence state. Finally, on the example of the widely used label tdTomato and the dye indocyanine green, we show the importance of correct photophysical (e.g., spectral and kinetic) information as a prerequisite for spectral-unmixing for in vivo imaging.
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Publication type Article: Journal article
Document type Scientific Article
Corresponding Author
Keywords Fluorescent Proteins; Indocyanine Green; Red; Aggregation; Tomography; Mesoscopy; Models; Probes; Limits
ISSN (print) / ISBN 0003-2700
e-ISSN 1520-6882
Journal Analytical Chemistry
Quellenangaben Volume: 92, Issue: 15, Pages: 10717-10724 Article Number: , Supplement: ,
Publisher American Chemical Society (ACS)
Publishing Place 1155 16th St, Nw, Washington, Dc 20036 Usa
Non-patent literature Publications
Reviewing status Peer reviewed
Institute(s) Institute of Biological and Medical Imaging (IBMI)
Institute of Structural Biology (STB)