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

Despite the tremendous technological advances in detection and data analysis in optoacoustic (OA) imaging, there is no detailed knowledge and understanding of the photophysics of OA signal generation of commonly used contrast agents, such as dyes and chromoproteins. This gap blocks the further development of dedicated labels for optoacoustics. To close it, we developed a multi-modal laser spectrometer (MLS) to enable the simultaneous measurement of OA, absorbance, and fluorescence spectra. MLS provides reproducible, high-quality OA spectra by using correction and referencing workflow. Herein, we employ MLS to analyze several common dyes (Methylene Blue, Rhodamine 800, Alexa Fluor 750, IRDye 800CW and Indocyanine green) and proteins (sfGFP, mCherry, mKate, HcRed, iRFP720 and smURFP) and shed light on their internal conversion properties. Our data shows that the absorption spectra do not correlate with the OA spectra for the majority of the analytes. We determine that for dyes, the transition underlying the high energy shoulder, which mostly correlates with an aggregation state of the dyes, has significantly more OA generation efficiency than the monomer transition. Our analyses for proteins point to a favored vibrational relaxation and OA signal generation that stems from the neutral or zwitterionic chromophores. Such data is highly relevant for the engineering of tailored contrast agents for OA imaging. Furthermore, discrepancies between absorption and OA spectra underline the importance of correct spectral information as a prerequisite for the spectral-unmixing schemes that are often required for in vivo OA imaging. Finally, OA-spectra recorded on our MLS of some of the most commonly used proteins and dyes in optical imaging reveal previously unknown photophysical characteristics, such as unobserved photoswitching behavior.