Sense and switch across scales – Prototyping genetically encoded, reversibly switchable indicators for sub-diffraction microscopy and whole animal optoacoustic Ca2+ imaging (Resubmission)

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The indispensable role of calcium ions (Ca2+) as signaling molecules in virtually all cell types, and in particular in the nervous system, makes the study of their dynamic distribution a topic of singular relevancy. Thus, imaging of Ca2+ was always a topic of high interest, however, only the last decade saw the rise of genetically encodable Ca2+ indicators (GECI). One of the key advantages of the genetic approach is the uninterrupted de novo production of the indicator by the cellular machinery. This enables in vivo longitudinal animal studies of neuronal dynamics. While the indicators provide good temporal accuracy, their spatial resolution is limited by the boundaries of the optical resolution of conventional fluorescence microcopy. Likewise, the field-of-view of microscopy is frequently very limited precluding measurements of Ca2+ dynamics at the tissue or organ level. Thus, so to speak, on both end of the scale there is the need for suitable imaging methods and tailored indicators. On the instrumentation side, two novel developments recently overcame those limitations: first, at the microscopic frontier, the resolution limit has been made obsolete by the advance of super-resolution (SR) fluorescence imaging techniques; secondly, the depth limitation of optical methods was overcome by the development of optoacoustics (OA) enabling high-resolution real-time in vivo imaging to depth of centimeters, readily allowing whole animal imaging. Genetically encoded photo-switchable labels, are pivotal to both techniques: for SR the required transitions of the label between two states can be accomplished by photo-switching between a fluorescent and non-fluorescent state. For OA imaging, the same photo-switching allows modulation of the labels’ signal for subsequent locked-in separation from background; thus, providing a crucial boost in contrast-to-noise (CNR) and enabling the detection of low cell numbers in living animals. Accordingly, we propose to exploit the revolutionary benefits photo-switchable proteins have for both modalities in the construction of transgene Ca2+ indicators. Such GECIs based on photo-switching (rsGECIs) will allow to study Ca2+ distributions at unprecedented spatial resolution and increase the CNR in OA to a level that makes Ca2+ imaging in whole animals, in vivo feasible. Focusing on green fluorescent proteins as templates for SR tailored rsGECIs and near-infrared proteins for OA tailored rsGECIs the goal of the proposal is to construct rsGECIs that will prototype the use of photo-switching in designing transgene indicators and provide a scaffold for further developments of this concept. RsGECIs will facilitate research of a wide range of processes regulated by Ca2+ gradients like cell signaling, metabolic regulation or developmental processes. Further, this work will provide a blueprint for the design of switchable indicators for other ions or small molecules enabling functional studies from the nanoscale to whole animals