PT  - JOURNAL ARTICLE
AU  - Luciano A. Masullo
AU  - Andreas Bodén
AU  - Francesca Pennacchietti
AU  - Giovanna Coceano
AU  - Michael Ratz
AU  - Ilaria Testa
TI  - Enhanced photon collection enables four dimensional fluorescence nanoscopy of living systems
AID  - 10.1101/248880
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 248880
4099  - http://biorxiv.org/content/early/2018/01/17/248880.short
4100  - http://biorxiv.org/content/early/2018/01/17/248880.full
AB  - 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/cm2). Our approach, based on reversibly switchable fluorescent proteins, features three distinctly modulated illumination patterns crafted and combined in a novel way 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 x 50 μm2) recordings at 0.3–1.3 Hz with enhanced optical sectioning ability. We demonstrated 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.