Reversibly photo-switchable proteins are essential for many super-resolution fluorescence microscopic and optoacoustic imaging methods. However, they have yet to be used as sensors that measure the distribution of specific analytes at the nanoscale or in the tissues of live animals. Here we constructed the prototype of a photo-switchable Ca²⁺ sensor based on GCaMP5G that can be switched with 405/488-nm light and describe its molecular mechanisms at the structural level, including the importance of the interaction of the core barrel structure of the fluorescent protein with the Ca²⁺ receptor moiety. We demonstrate super-resolution imaging of Ca²⁺ concentration in cultured cells and optoacoustic Ca²⁺ imaging in implanted tumor cells in mice under controlled Ca²⁺ conditions. Finally, we show the generalizability of the concept by constructing examples of photo-switching maltose and dopamine sensors based on periplasmatic binding protein and G-protein-coupled receptor-based sensors.

Temporally unmixed optoacoustic enables routine in vivo imaging of transgene-labeled cells deep in the mouse body.

Photo-controllable proteins revolutionized life-science imaging due to their contribution to subdiffraction-resolution optical microscopy. They might have yet another lasting impact on Photo- or Optoacoustic imaging (OA). OA combines optical contrast with ultrasound detection enabling high-resolution real-time in vivo imaging well-beyond the typical penetration depth of optical methods. While OA already showed numerous applications relying on endogenous contrast from blood hemoglobin or lipids its application in the life-science was limited by a lack of labels overcoming the strong signal from the aforementioned endogenous absorbers. Here, a number of recent studies showed that photo-controllable proteins provide the means to overcome this barrier eventually enabling OA to image small cell numbers in a complete organism in vivo. In this Feature article, we introduce the key photo-controllable proteins, explain the basic concepts and highlight achievements that have been already made.