Impact of photobleaching on quantitative, spatio-temporal, super-resolution imaging of mitochondria in live C. elegans larvae

Super-resolution (SR) 3D rendering allows superior quantitative analysis of intracellular structures but has largely been limited to fixed or ex vivo samples. Here we developed a method to perform SR live imaging of mitochondria during post-embryonic development of <i>C</i>. <i>elegans</i> larvae. Our workflow includes the drug-free mechanical immobilisation of animals using polystyrene nanobeads, which has previously not been used for in vivo SR imaging. Based on the alignment of moving objects and global threshold-based image segmentation, our method enables an efficient 3D reconstruction of individual mitochondria. We demonstrate for the first time that the frequency distribution of fluorescence intensities is not affected by photobleaching, and that global thresholding alone enables the quantitative comparison of mitochondria along timeseries. Our composite approach significantly improves the study of biological structures and processes in SR during <i>C. elegans</i> post-embryonic development. Furthermore, the discovery that image segmentation does not require any prior correction against photobleaching, a fundamental problem in fluorescence microscopy, will impact experimental strategies aimed at quantitatively studying the dynamics of organelles and other intracellular compartments in any biological system.