Institute of Biological and Medical Imaging (IBMI), Helmholtz Zentrum München, D-85764 Neuherberg, Germany.
Chair of Biological Imaging, Technische Universitat München, D-81675 Munich, Germany.
Anal Chem. 2020 Aug 4;92(15):10717-10724. doi: 10.1021/acs.analchem.0c01902. Epub 2020 Jul 20.
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.
光声（光声）成像是一种具有明显进展的检测和数据分析方法，但对于常见光声对比剂的光物理特性的理解进展较少。这一差距阻碍了新型对比剂的开发以及多光谱光声图像的准确分析和解释。为了弥补这一差距，我们开发了一种多模态激光光谱仪（MLS），以实现光声、吸光度和荧光光谱的同时测量。在此，我们使用 MLS 来分析对比剂（亚甲蓝、罗丹明 800、Alexa Fluor 750、IRDye 800CW 和吲哚菁绿）和蛋白质（sfGFP、mCherry、mKate、HcRed、iRFP720 和 smURFP）。我们发现，对于大多数分析物，光学吸收光谱与光声光谱不相关。我们确定，对于染料，处于聚集态的跃迁比单体跃迁具有更高的光声信号产生效率。对于蛋白质，我们发现了一种有利的光声弛豫，它源于中性或两性离子色团，以及 tdTomato 和 HcRed 的未报道的光致开关行为。然后，我们将 HcRed 晶体化到其光声开关状态，从结构上证实了与 HcReds 荧光状态相比，其异构化的变化。最后，以广泛使用的标签 tdTomato 和染料吲哚菁绿为例，我们展示了正确的光物理（例如光谱和动力学）信息作为体内成像光谱解混的前提的重要性。
