Breaking the Resolution Limit in Two-Photon Microscopy Using Molecular Photoswitches

Research Project, 2021 –

Multiphoton microscopy is a benchmark tool used for e.g. fluorescence imaging in cellular environments. Two-photon microscopy is typically used in this context. Theory foresees that the spatial resolution can be dramatically improved by drawing on higher-order processes such as four-photon absorption.
However, its practical implementation imposes major technical challenges, such as extreme laser intensities in the deep NIR. For this reason, four-photon microscopy has so far attracted academic interest only.The present proposal addresses this issue and provides an innovative answer to the question: Can we develop a technique that offers spatial resolution of four-photon microscopy, but relies on two-photon absorption? This would combine the upsides of two-photon microscopy with the superior spatial resolution of four-photon microscopy. The result would be nothing less than a true paradigm shift in multiphoton microscopy. Purposefully designed switchable fluorophores, that unify several photophysical assets, will be developed (year 1) and their performance will be critically validated in a multi-angle work-flow, including spectroscopic characterization (year 1-2), proof-of-principle microscopy experiments in model systems (year 2-3) and the demonstration in application-relevant biological environments (year 3-4).