Engineering genetically encoded photoswitching small molecule / ion sensors for in vivo imaging

In vivo visualization of the spatio-temporal distribution of small molecules and ions is essential to decipher pathophysiologies and fundamental biological mechanisms. For example, the heterogeneity of metabolites in the tumor microenvironment is thought to be crucial for both determining the disease progression and the success of therapeutic interventions. Whole animal imaging techniques for metabolites are often limited to ex vivo approaches (mass spectrometry imaging) or methods with lower resolution (positron emission tomography or hyperpolarized magnetic resonance imaging). Genetically encoded small molecule and ion sensors, which are known from fluorescence microscopy, are currently only rarely used. Similarly, current genetically encoded fluorescence biosensors are not suitable for subcellular resolution imaging (i.e. super-resolution fluorescence microscopy).

Our work aims to approach both topics by engineering photoswitchable sensors for small molecules/ions. Due to their photoswitching capability, such sensors will allow to resolve the distribution of small molecules in vivo with high resolution using advanced whole-animal optical imaging methods such as photo-/optoacoustic. In addition, the concept will enable the development of sensors for high-resolution fluorescence microscopy to visualize small molecule or ion microdomains in the cell that are thought to determine regulatory mechanisms and functions in the cell. The general feasibility of the photoswitching sensor approach was recently demonstrated by us in a proof-of-concept study (Nature Biotechnology, 2022).

We employ protein engineering methods, using rational protein structure-guided, computational and directed evolution approaches to tailor genetically encoded sensors for advanced in vivo imaging from whole animals down to subcellular details.