Development of genetically encoded biosensors for super-resolution optical fluctuation imaging

Life is dynamic, defined by the ability to maintain homeostasis in ever-changing environments. On a cellular level, this requires an intricate regulatory network to manage activities and reactivities in response to external stimuli. Fluorescence microscopy provides us a window into this dynamic realm of subcellular signalling. The complex spatiotemporal regulation of this network can be investigated with fluorescent indicators, remarkable molecules whose fluorescence emission depends on the presence of a specific stimulus. However, nanoscale compartmentalization is thought to play a major role in shaping these dynamic architectures, and currently quantitative observation of dynamic events at these smallest scales remains challenging. 

In this thesis, we describe novel methodologies that take advantage of these fluorescent indicators to observe and manipulate dynamic activities at the nanoscale. We first discuss our perspectives on the potential and pitfalls of genetically-encoded biosensors, including potential challenges for high-resolution biosensing. The specific challenges for ratiometric Förster resonance energy transfer biosensing are extended in the next chapter, where we use simulations to highlight spurious structuring that arises due to resolution mismatch in multicolor imaging. We then describe a correction strategy based on the optical transfer functions corresponding to both color channels to render multicolor images quantitatively comparable. This strategy is then applied to in vivo biosensing of mitochondrial protein kinase A activity in a proof-of-concept experiment.
In the final chapter, we demonstrate that the conjunction of fluorescent indicators and biological nanopores can be used to create nanoscale compartmentalization in vitro. Using a nanopore as an electrically gated valve, we are able to establish attoscale reaction volumes with sub-millisecond actuation times. We confirm that the reaction volume can be accurately controlled and provide evidence for sub-millisecond switching of the nanopore, thereby generating the smallest rapidly controllable reaction volume presented to date.