3D super-resolution microscopy of living cells using reversibly switchable fluorophores
Time: Fri 2021-10-01 13.30
Subject area: Biological Physics
Doctoral student: Andreas Bodén , Biofysik, Science for Life Laboratory, SciLifeLab, KTH Royal Institute of Technology, Testa
Opponent: PhD Francisco Balzarotti, IMP Vienna
Supervisor: Associate professor Ilaria Testa, Biofysik, Science for Life Laboratory, SciLifeLab
Traditional optical microscopy techniques are limited in spatial resolution due to the wave nature of light. This means that neighboring objects separated by a distance smaller than about 200 nm cannot be distinguished. Super‑resolution microscopy techniques overcome this limitation by utilizing specific light-matter interactions of fluorescent labels to encode finer spatial detail into the recorded data. Regrettably, current super‑resolution approaches often increase the complexity of sample preparation as well as the energy, time, and invasiveness of the imaging scheme compared to conventional imaging techniques. This makes many of these techniques ill‑suited for imaging the dynamics of living cells. Since many biological studies rely on highly spatially resolved data containing three‑dimensional and temporally dynamic information, developing super‑resolution techniques toward the goal of acquiring such data is vital. With this work, we take several important steps in this direction by utilizing reversibly switchable fluorescence proteins (RSFPs) together with new illumination patterns that allow for a parallelized data acquisition scheme. Even low intensity illumination patterns can induce photo‑switching of the RSFPs and generate specific patterns of fluorescent emission that carry high‑resolution spatial information in all three dimensions. By using RSFPs in a parallelized acquisition scheme, temporally extended recordings can be acquired with low illumination intensities and at high speed. In addition to the imaging schemes, we present a theoretical framework for modelling the impact that RSFP properties on image formation and show how different imaging parameters affect the final image quality. We predict and explore the effect of labelling density and photobleaching on single and timelapse recordings, taking into consideration the stochasticity of labelling and fluorophore fatigue. We also present a new family of red‑shifted RSFPs that can be imaged without the need for near‑UV illumination, allowing even less invasive live‑cell imaging. This work aims to not only provide new tools for imaging, but also to contribute to a better understanding of the underlying concepts and to facilitate future developments of super-resolution microscopy for bio-imaging applications.