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Synthetic and genetically encoded chromo- and fluorophores have become indispensable tools for biomedical research enabling a myriad of applications in imaging modalities based on biomedical optics. The versatility offered by the optoacoustic (photoacoustic) contrast mechanism enables to detect signals from any substance absorbing light, and hence these probes can be used as optoacoustic contrast agents. While contrast versatility generally represents an advantage of optoacoustics, the strong background signal generated by light absorption in endogeneous chromophores hampers the optoacoustic capacity to detect a photo-absorbing agent of interest. Increasing the optoacoustic sensitivity is then determined by the capability to differentiate specific features of such agent. For example, multispectral optoacoustic tomography (MSOT) exploits illuminating the tissue at multiple optical wavelengths to spectrally resolve (unmix) the contribution of different chromophores. Herein, we present an alternative approach to enhance the sensitivity and specificity in the detection of optoacoustic contrast agents. This is achieved with photoswitchable probes that change optical absorption upon illumination with specific optical wavelengths. Thereby, temporally unmixed MSOT (tuMSOT) is based on photoswitching the compounds according to defined schedules to elicit specific time-varying optoacoustic signals, and then use temporal unmixing algorithms to locate the contrast agent based on their particular temporal profile. The photoswitching kinetics is further affected by light intensity, so that tuMSOT can be employed to estimate the light fluence distribution in a biological sample. The performance of the method is demonstrated herein with the reversibly switchable fluorescent protein Dronpa and its fast-switching fatigue resistant variant Dronpa-M159T.
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