Mancebo, A. | Search bioRxiv

bioRxiv2020

Quantitative super-resolution microscopy reveals distinct ULK1 oligomeric states and nanoscopic morphologies during autophagy initiation

Autophagy induction involves extensive molecular and membrane reorganization. Despite significant progress, the mechanism underlying autophagy initiation remains poorly understood. Here, we used quantitative Photoactivated Localization Microscopy with single-molecule sensitivity to analyze the nanoscopic distribution of endogenous ULK1, the kinase that triggers autophagy. Under amino acid starvation, ULK1 formed large clusters containing up to 161 molecules at the endoplasmic reticulum. Cross-correlation analysis revealed that ULK1 clusters engaging in autophagosome formation require 30 or more molecules. The ULK1 structures with more than the threshold number contained varying levels of Atg13, Atg14, Atg16, LC3B, GEC1 and WIPI2. We found that ULK1 activity is dispensable for the initial clustering of ULK1, but necessary for the subsequent expansion of the clusters, which involves interaction with Atg14, Atg16, and LC3B and relies on Vps34 activity. This quantitative analysis at the single molecule level has provided unprecedented insights into the behavior of ULK1 during autophagy initiation.

biophysics↗

bioRxiv2019

Calibrated feedback illumination for precise conventional fluorescence and PALM imaging applications

Spatial light modulation using cost efficient digital mirror arrays (DMA) is finding broad applications in fluorescence microscopy due to the reduction of phototoxicity and bleaching and the ability to manipulate proteins in optogenetic experiments. However, the precise calibration of DMAs and their application to single-molecule localization microscopy (SMLM) remained a challenge because of non-linear distortions between the DMA and camera coordinate system caused by optical components. Here we develop a fast and easy to implement calibration procedure that determines these distortions by means of an optical feedback and matches the DMA and camera coordinate system with ~50 nm precision. As a result, a region from a fluorescence image can be selected with a higher precision for illumination compared to manual alignment of the DMA. We first demonstrate the application of our precisely calibrated light modulation by performing a proof-of concept fluorescence recovery after photobleaching experiment with the endoplasmic reticulum-localized protein IRE1 fused to GFP. Next, we develop a spatial feedback photoactivation approach for SMLM in which only regions of the cell are selected for photoactivation that contain photoactivatable fluorescent proteins. The reduced exposure of the cells to 405 nm light increases the possible imaging time by 44% until phototoxic effects cause a dominant fluorescence background and a change in the cells morphology. As a result, the mean number of reliable single molecule localizations is also significantly increased by 28%. Since the localization precision and the ability for single molecule tracking is not altered compared to traditional photoactivation of the entire field of view, spatial feedback photoactivation significantly improves the quality of SMLM images and the precision of single molecule tracking. Our calibration method therefore lays the foundation for improved SMLM with active feedback photoactivation far beyond the applications in this work.\n\nStatement of significanceActively patterned illumination in fluorescence microscopy can reduce bleaching and phototoxicity as well as actively manipulate proteins in optogenetic applications. Matching the coordinate system of the camera and the light patterning device such as digital mirror arrays (DMA) remains a challenge. We developed a fast and easy calibration procedure that determines and corrects for the transformation between the camera and DMA coordinate system with ~50 nm precision. Using this approach, we develop spatial feedback photoactivation for Single Molecule Localization Microscopy (SMLM) to photoswitch only intracellular regions containing photoswitchable fluorophores. Our results show a 44% improvement in the possible data acquisition time before phototoxic effects become detectable and a 28% increase in detected localizations. Spatial feedback photoactivation thus significantly improves SMLM experiments.

biophysics↗