Resolution in optical nanoscopy depends on the localization uncertainty of single fluorescent labels, the density of labels covering the sample, and the sample’s spatial structure. Currently there is no integral, practical resolution measure that takes all factors into account. Here we introduce such a measure that can be computed directly from the image. We demonstrate its validity and benefits on 2D and 3D localization microscopy images of tubulin and actin filaments. Our approach makes it possible to compare achieved resolutions in images taken with different nanoscopy methods, optimize and rank different emitter localization and labeling strategies, define a stopping criterion for data acquisition, describe image anisotropy and heterogeneity, and, surprisingly, estimate the average number of localizations per emitter. Our findings challenge the current focus on obtaining the best localization precision, but instead show how the best image resolution can be achieved as fast as possible.

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The extent to which ligand occupancy and dimerization contribute to erbB1 signaling is controversial. To examine this, we utilized two-color Quantum Dot tracking for visualization of erbB1 homodimerization and quantification of the dimer off rate (koff) on living cells. Kinetic parameters were extracted using a 3-state Hidden Markov Model to identify transition rates between free, co-confined, and dimerized states. We report that dimers composed of 2 ligand-bound receptors are long-lived and their koff is independent of kinase activity. By comparison, unliganded dimers have >4-fold faster koff. Transient co-confinement of receptors promotes repeated encounters and enhances dimer formation. Mobility decreases >6-fold when ligand-bound receptors dimerize. Blockade of erbB1 kinase activity or disruption of actin networks results in faster diffusion of receptor dimers. These results implicate both signal propagation and the cortical cytoskeleton in reduced mobility of signaling-competent erbB1 dimers.

The actin cytoskeleton has been implicated in restricting diffusion of plasma membrane components. Here, simultaneous observations of quantum dot-labelled FcεRI motion and GFPtagged actin dynamics provide direct evidence that actin filament bundles define micron-sized domains that confine mobile receptors. Dynamic reorganisation of actin structures occurs over seconds, making the location and dimensions of actin-defined domains time dependent. Multiple FcεRI often maintain extended close proximity without detectable correlated motion, suggesting that they are co-confined within membrane domains. FcεRI signalling is activated by cross-linking with multivalent antigen. We show that receptors become immobilised within seconds of crosslinking. Disruption of the actin cytoskeleton results in delayed immobilisation kinetics and increased diffusion of cross-linked clusters. These results implicate actin in membrane partitioning that not only restricts diffusion of membrane proteins, but also dynamically influences their longrange mobility, sequestration, and response to ligand binding.Signal transduction from the external environment to the cell interior is typically mediated by ligand-bound transmembrane receptors embedded in a lipid bilayer. In many systems, receptor activation is associated with changes in receptor dynamics and membrane topography1 -3 . Among these are the multi-chain immune recognition receptor family members that include the B-cell receptor (BCR) of B-cells, the T-cell receptor (TCR) of Tcells, and the high affinity IgE receptor (FcεRI) of mast cells and basophils, which are crucial to the execution of key events in the immune response. Cross-linking of these transmembrane receptors induces receptor oligomerisation, protein and lipid kinase activation and Ca 2+ mobilisation, leading in turn to cytoskeletal reorganisation, receptor trafficking and cell-specific responses including altered gene expression [4][5][6] . These signalling events have been well studied by biochemical techniques, but the precise mechanism by which oligomerisation initiates these events has remained elusive. Full understanding of these complex signalling cascades will require a more complete description of receptor movements in the membrane, including restrictions that might limit receptor diffusion and accessibility.Correspondence should be addressed to D.S.L. (dlidke@salud.unm.edu). 4 These authors contributed equally to this work. NIH Public Access Author ManuscriptNat Cell Biol. Author manuscript; available in PMC 2011 January 18. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptA rich literature on single particle tracking (SPT) methods to follow the lateral diffusion of transmembrane and membrane-associated proteins7 -10 has revealed nanometer-scale "confinement zones" that restrict lateral diffusion and supports the general notion that plasma membrane organisation is more structured than originally postulated by the fluid mosaic model11. A membrane-skeleton fence (picket fence) model ...

Autophagy is a process delivering cytoplasmic components to lysosomes for degradation. Autophagy may, however, play a role in unconventional secretion of leaderless cytosolic proteins. How secretory autophagy diverges from degradative autophagy remains unclear. Here we show that in response to lysosomal damage, the prototypical cytosolic secretory autophagy cargo IL-1b is recognized by specialized secretory autophagy cargo receptor TRIM16 and that this receptor interacts with the R-SNARE Sec22b to recruit cargo to the LC3-II + sequestration membranes. Cargo secretion is unaffected by downregulation of syntaxin 17, a SNARE promoting autophagosome-lysosome fusion and cargo degradation. Instead, Sec22b in combination with plasma membrane syntaxin 3 and syntaxin 4 as well as SNAP-23 and SNAP-29 completes cargo secretion. Thus, secretory autophagy utilizes a specialized cytosolic cargo receptor and a dedicated SNARE system. Other unconventionally secreted cargo, such as ferritin, is secreted via the same pathway.

Single molecule localization based super-resolution imaging techniques require repeated localization of many single emitters. We describe a method that uses the maximum likelihood estimator to localize multiple emitters simultaneously within a single, two-dimensional fitting sub-region, yielding an order of magnitude improvement in the tolerance of the analysis routine with regards to the single-frame active emitter density. Multiple-emitter fitting enables the overall performance of single-molecule super-resolution to be improved in one or more of several metrics that result in higher single-frame density of localized active emitters. For speed, the algorithm is implemented on Graphics Processing Unit (GPU) architecture, resulting in analysis times on the order of minutes. We show the performance of multiple emitter fitting as a function of the single-frame active emitter density. We describe the details of the algorithm that allow robust fitting, the details of the GPU implementation, and the other imaging processing steps required for the analysis of data sets.

In microscopy, single fluorescence point sources can be localized with a precision several times greater than the resolution limit of the microscope. We show that the intermittent fluorescence or 'blinking' of quantum dots can analyzed by an Independent Component Analysis so as to identify the light emitted by each individual nanoparticle, localize it precisely, and thereby resolve groups of closely spaced (< lambda / 30) quantum dots. Both simulated and experimental data demonstrate that this technique is superior to localization based on Maximum Likelihood Estimation of the sum image under the assumption of point emitters. This technique has general application to any emitter with non-Gaussian temporal intensity distribution, including triplet state blinking. When applied to the labeling of structures, a high resolution "image" consisting of individually localized points may be reconstructed leading to the term "Pointillism".

Highlights d A system for membrane repair, removal, and replacement is coordinated by galectins d Galectin-3 recruits ESCRT components to damaged lysosomes to repair them d Galectins induce autophagy to remove damaged lysosomes and activate their biogenesis d The galectin-directed systems protect against M. tuberculosis and neurotoxic tau