Computational protocol: One, two or three? Probing the stoichiometry of membrane proteins by single-molecule localization microscopy

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Protocol publication

[…] SMLM movies were analyzed using the rapidSTORM software and post-processed using a python-based custom written software. In brief, single mEos2 fluorescent peaks were identified in each frame and fit by a two-dimensional Gaussian function to extract spatial coordinates and the number of photons. Peaks with brightness below 63 photons were discarded. The majority of peaks were localized with a spatial precision <35 nm according to Mortensen et al. PALM images were generated from the coordinate lists of localized peaks using rapidSTORM or ViSP. Spatio-temporal grouping was applied so that peaks appearing in consecutive camera frames within a spatial separation of 90 nm were registered as a single localization with averaged spatial position. The algorithm is based on Kalman filtering and is implemented in rapidSTORM. The distance threshold was chosen so that 99% of peaks with a localization precision of 35 nm were grouped successfully. Peaks that only appeared in one frame were excluded. Note that the temporal cutoff time does not correct for the long-lived fluorescent off times previously described for mEos2 that can last up to several seconds. Individual molecules in a small protein complex (e.g. receptor dimer) cannot be spatially resolved with PALM and will appear as clusters of several localizations with a spatial spread in the range of PALM resolution itself. Before spatio-temporal grouping, even a monomeric protein will give rise to a cluster of localizations due to repeated mEos2 emissions. We made use of the characteristic blinking behavior of mEos2 and defined protein complexes as mEos2 bursts with a maximum spread of 100 nm in the initial PALM image. We rigorously excluded clusters with low brightness, partially overlapping clusters and clusters in close proximity to localizations caused by fluorescent background. mEos2 localizations identified as belonging to the same complex are then used to extract the true number of mEos2 reappearances/blinks (Nblinks) per cluster after spatio-temporal grouping. Distributions of Nblinks were plotted, fitted and statistically evaluated using OriginPro 9.1G. […]

Pipeline specifications

Software tools rapidSTORM, ViSP
Application Super-resolution imaging
Diseases Stomatitis