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Introduction:
Optoacoustic (OA) imaging, originally developed for improved structural imaging, can be used to non-invasively map whole hearts in 3D without the need for contrast agents. OA imaging uses light to stimulate molecules which undergo a transient thermoelastic expansion, resulting in a pressure wave that can be detected and located in 3D with an ultrasound transducer. Sound travels further in tissue than light, allowing imaging depths of up to a few centimetres1. In this project, we assess the potential of genetically encoded voltage indicators (GEVI) for OA imaging, thereby exploring 3D functional imaging of electrophysiology with OA. With this novel approach, we hope to non-invasively gain insight into action potential (AP) properties throughout myocardial walls beyond (sub)epicardial tissue layers.
Methods:
We stably expressed several near-infrared (NIR)-fluorescent GEVI in a Human Embryonic Kidney (HEK) cell line that is optically depolariseable due to the expression of a light-sensitive ion channel (CheRiff). A co-expressed potassium channel (Kir2.1) lowers the resting membrane potential to ~ -80 mV. We are hereby able to use blue light to depolarize the cells, which allows us to study changes of the acoustic signal emitted by the voltage reporters under electrical conditions similar to those found in cardiac muscle. Recordings were conducted using both confocal microscopy and OA imaging. Three NIR-GEVI were tested for OA imaging: The ratiometric reporter VSFP6702, the single-opsin GEVI Archon13, and HArcLight, a chemogenetic reporter4.
Results:
Until now, no GEVI have been reported for use with OA imaging. One reason may be the high light absorption of native tissue in broad sections of the visible spectrum, limiting the utility of common fluorescent reporters that work with blue, green or yellow light. However, we found that when using optical reporters that are excited at wavelengths in the NIR part of the spectrum (640 – 950 nm), changes in membrane potential can be recorded as changes in an acoustic signal with OA imaging. So far, we achieved highest signal-to-noise ratios using Archon1, where voltage swings of ~40 mV (from ~ -80 mV to ~ -40 mV) result in an ~10% change in acoustic signal.
Conclusion and outlook:
In the future, we wish to utilise animal models with cardiac expression of the most potent GEVI for OA imaging to overcome limitations of other techniques that record AP on the surface of the heart only, or that destroy tissue integrity by highly invasive approaches. Studying the whole depth of cardiac muscle structurally and electrically at the same time will provide so-far inaccessible information on cardiac electrophysiology, for example to unequivocally distinguish ectopic excitation foci from epicardial breakthrough sites of a steady intramural excitation rotor.
1 Karlas, A. et al. Nat. Rev. Endocrinol. 17, 323–335 (2021)
2 Matlashov, M. E. et al. Nat. Commun. 11, 1–12 (2020)
3 Qian, Y. et al. Nat. Methods 16, (2019)
4 Deo, C. et al. bioRxiv 0–11 (2020)
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