2019/02/01 by Jes Dreier, Marco Castello, Giovanna Coceano +4 · 1 voice · 3 citations
Biochemistry, Genetics and Molecular Biology · #Advanced Electron Microscopy Techniques and Applications #Advanced Fluorescence Microscopy Techniques #Cell Image Analysis Techniques
paper · pdf · doi:10.1038/s41467-019-08442-4
openalex publication_date 2019/02/01 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/31
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.