Super-resolved live-cell imaging using Random Illumination Microscopy

Abstract

25Super-resolution fluorescence microscopy has been instrumental to progress in biology. Yet, the photo-26 induced toxicity, the loss of resolution into scattering samples or the complexity of the experimental setups 27 curtail its general use for functional cell imaging. Here, we describe a new technology for tissue imaging 28 reaching a 114nm/8Hz resolution at 30 µm depth. Random Illumination Microscopy (RIM) consists in 29 shining the sample with uncontrolled speckles and extracting a high-fidelity super-resolved image from the 30 variance of the data using a reconstruction scheme accounting for the spatial correlation of the illuminations. 31 Super-resolution unaffected by optical aberrations, undetectable phototoxicity, fast image acquisition rate and 32 ease of use, altogether, make RIM ideally suited for functional live cell imaging in situ. RIM ability to image 33 molecular and cellular processes in three dimensions and at high resolution is demonstrated in a wide range 34 of biological situations such as the motion of Myosin II minifilaments in Drosophila. 35 36 37 38

Random Illumination Microscopy from Variance Images

“…The latter components include both the electronic and the photon counting noise, but also the fluorescence light emitted from out-of-focus planes, real tissues being three-dimensional. AlgoRIM was recently applied to cases of super-resolved functional imaging of living cells and tissues [8], which confirms its practical super-resolution capability, as well as its versatility and robustness. In Section II, we introduce the discretized model for RIM and we express second-order statistical moments of the measurements which are useful in the rest of the paper.…”

“…In Fig. 3, we show results obtained from real data acquired with a two-color fluorescence microscope described in [8]. The sample is a cell membrane with marked podosomes (lifeact-GFP in red and zyxine-mCh in green).…”

“…The maximum super-resolution gain of RIM is the same as SIM [7], i.e., a factor of two in the lateral dimensions. On the other hand, RIM requires only simple modifications of a fluorescent microscope, and it is easy to use [8]. Moreover, it is robust to optical aberrations, which makes it an excellent candidate for functional cell imaging.…”

High resolution dynamic mapping of the C. elegans intestinal brush border

“…Of note, this method allowed to uncover localization differences between in-locus mNG-tagged and overexpressed GFP-tagged proteins (compare Figs 3C and S4A), as already reported for E-cadherin in the same tissue (Cordova-Burgos et al,2021). Individual microvilli were similarly visualized using Random Illumination Microscopy (Mangeat et al, 2021), but not using conventional confocal or Stimulated-emission-depletion (STED) microscopes, probably because of the depth of the intestine inside the nematode body (~15μm) (Fig S4B). The brush border could also be imaged transversally (compare Figs 3D and 1F).…”

“…All images were examined using Fiji software. Random Illumination Microscopy was performed at the LITC Core Facility, Centre de Biologie Integrative, Université de Toulouse, France, using the workflow recently published on ERM-1::GFP expressing strains (Mangeat et al, 2021).…”