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

Nat Commun. 2019 Feb 1;10(1):556. doi: 10.1038/s41467-019-08442-4.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Caenorhabditis elegans
  • Cell Line
  • Cell Survival
  • Decision Making
  • Hippocampus / cytology
  • Humans
  • Image Processing, Computer-Assisted
  • Lighting*
  • Microscopy, Fluorescence*
  • Nanotechnology / methods*
  • Neurons / cytology
  • Rats, Sprague-Dawley
  • Time Factors
  • Time-Lapse Imaging