Smart scanning for low-illumination and fast RESOLFT nanoscopy in vivo

Jes Dreier, Marco Castello, Giovanna Coceano, Rodrigo Cáceres, Julie Plastino, Giuseppe Vicidomini () and Ilaria Testa ()
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Jes Dreier: KTH Royal Institute of Technology
Marco Castello: Istituto Italiano di Tecnologia
Giovanna Coceano: KTH Royal Institute of Technology
Rodrigo Cáceres: PSL Research University, CNRS
Julie Plastino: PSL Research University, CNRS
Giuseppe Vicidomini: Istituto Italiano di Tecnologia
Ilaria Testa: KTH Royal Institute of Technology

Nature Communications, 2019, vol. 10, issue 1, 1-11

Abstract: Abstract RESOLFT fluorescence nanoscopy can nowadays image details far beyond the diffraction limit. However, signal to noise ratio (SNR) and temporal resolution are still a concern, especially deep inside living cells and organisms. In this work, we developed a non-deterministic scanning approach based on a real-time feedback system which speeds up the acquisition up to 6-fold and decreases the light dose by 70–90% for in vivo imaging. Also, we extended the information content of the images by acquiring the complete temporal evolution of the fluorescence generated by reversible switchable fluorescent proteins. This generates a series of images with different spatial resolution and SNR, from conventional to RESOLFT images, which combined through a multi-image deconvolution algorithm further enhances the effective resolution. We reported nanoscale imaging of organelles up to 35 Hz and actin dynamics during an invasion process at a depth of 20–30 µm inside a living Caenorhabditis elegans worm.

Date: 2019
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DOI: 10.1038/s41467-019-08442-4

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