Challenging a Preconception: Optoacoustic Spectrum Differs from the Optical Absorption Spectrum of Proteins and Dyes for Molecular Imaging

Abstract

Optoacoustic (photoacoustic) imaging has seen marked technological advances in detection and data analysis, but there is less progress in understanding the photophysics of optoacoustic signal generation of commonly used contrast agents, such as dyes and chromoproteins. This gap blocks the precise development of novel agents and the accurate analysis and interpretation of Multispectral Optoacoustic Tomography (MSOT) images. To close it, we developed a multimodal laser spectrometer (MLS) to enable the simultaneous measurement of optoacoustic, absorbance, and fluorescence spectra. MLS provides reproducible, high-quality optoacoustic (non-radiative) 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 optical absorption spectra do not correlate with the optoacoustic 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 optoacoustic signal generation efficiency than the monomer transition. Our analyses for proteins point to a favored vibrational relaxation and optoacoustic signal generation that stems from the neutral or zwitterionic chromophores. We were able to crystalize HcRed in its optoacoustic state, confirming the change isomerization respect to its fluorescence state. Such data is highly relevant for the engineering of tailored contrast agents for optoacoustic imaging. Furthermore, discrepancies between absorption and optoacoustic spectra underline the importance of correct spectral information as a prerequisite for the spectral-unmixing schemes that are often required for in vivo imaging. Finally, optoacoustic spectra of some of the most commonly used proteins and dyes in optical imaging, recorded on our MLS, reveal previously unknown photophysical characteristics, such as unobserved photo-switching behavior.

VEGF-Targeted Multispectral Optoacoustic Tomography and Fluorescence Molecular Imaging in Human Carotid Atherosclerotic Plaques

“…Both samples showed optoacoustic peaks at ~700 and ~780 nm, with bevacizumab-800CW showing slightly higher signal at ~700 nm, similar to previous studies with IRDye800CW conjugated to a monoclonal antibody ( Figure 2 ). The second of IRDye800CW-peak at ~700 nm has previously been identified, and is possibly caused by stronger optoacoustic generation efficiency attributable to H-aggregates [ 21 ].…”

Brilliant cresyl blue enhanced optoacoustic imaging enables non-destructive imaging of mammalian ovarian follicles for artificial reproduction

“…As described previously [25], we diluted a 13 mM stock solutions of 1 M BCB in Dulbecco's phosphate-buffered saline (DPBS) to six working concentrations (25 × to 250 × dilutions). We used 10 wavelengths to derive the reference optoacoustic spectrum for BCB at the designated concentrations [26]. An automatic segmentation and tracking algorithm developed in-house [19] was used to automatically annotate the region of interest (ROI) (approximately 25 ± 2 pixels) in real time and 50 frame averaging was done to achieve high signal-to-noise ratio.…”

Facile Synthesis of a Croconaine‐Based Nanoformulation for Optoacoustic Imaging and Photothermal Therapy

“…Optoacoustic spectra of samples were carried out using an MSOT inVision 256‐TF (iThera Medical, Munich, Germany) and done the correction with India ink and the normalization with Brilliant Black BN (BBN). [ 26 ] Briefly, the Indian ink was used to correct the known pulse‐to‐pulse fluctuations from the OPO laser. The BBN was selected as reference because its photostability and lack of any fluorescence generation allowed to assign an OGE value of 1.0, assuming no decay channels taking place.…”

“…The OGE of samples is equal to the slope of the line where corrected optoacoustic intensities are plotted against absorbance. [ 26 ] The photostability of samples was detected by an MSOT inVision 256‐TF with pulsed laser irradiation for 30 min (fluence 10 mJ cm −2 ).…”