Enhanced photon collection enables four dimensional fluorescence nanoscopy of living systems

Luciano A. Masullo, Andreas Bodén, Francesca Pennacchietti, Giovanna Coceano, Michael Ratz and Ilaria Testa ()
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Luciano A. Masullo: KTH Royal Institute of Technology
Andreas Bodén: KTH Royal Institute of Technology
Francesca Pennacchietti: KTH Royal Institute of Technology
Giovanna Coceano: KTH Royal Institute of Technology
Michael Ratz: Karolinska Institutet
Ilaria Testa: KTH Royal Institute of Technology

Nature Communications, 2018, vol. 9, issue 1, 1-9

Abstract: Abstract The theoretically unlimited spatial resolution of fluorescence nanoscopy often comes at the expense of time, contrast and increased dose of energy for recording. Here, we developed MoNaLISA, for Molecular Nanoscale Live Imaging with Sectioning Ability, a nanoscope capable of imaging structures at a scale of 45–65 nm within the entire cell volume at low light intensities (W-kW cm−2). Our approach, based on reversibly switchable fluorescent proteins, features three distinctly modulated illumination patterns crafted and combined to gain fluorescence ON–OFF switching cycles and image contrast. By maximizing the detected photon flux, MoNaLISA enables prolonged (40–50 frames) and large (50 × 50 µm2) recordings at 0.3–1.3 Hz with enhanced optical sectioning ability. We demonstrate the general use of our approach by 4D imaging of organelles and fine structures in epithelial human cells, colonies of mouse embryonic stem cells, brain cells, and organotypic tissues.

Date: 2018
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DOI: 10.1038/s41467-018-05799-w

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