We demonstrate live-cell super-resolution imaging using photoactivated localization microscopy (PALM). The use of photon-tolerant cell lines in combination with the high resolution and molecular ...sensitivity of PALM permitted us to investigate the nanoscale dynamics within individual adhesion complexes (ACs) in living cells under physiological conditions for as long as 25 min, with half of the time spent collecting the PALM images at spatial resolutions down to approximately 60 nm and frame rates as short as 25 s. We visualized the formation of ACs and measured the fractional gain and loss of individual paxillin molecules as each AC evolved. By allowing observation of a wide variety of nanoscale dynamics, live-cell PALM provides insights into molecular assembly during the initiation, maturation and dissolution of cellular processes.
A key challenge when imaging living cells is how to noninvasively extract the most spatiotemporal information possible. Unlike popular wide-field and confocal methods, plane-illumination microscopy ...limits excitation to the information-rich vicinity of the focal plane, providing effective optical sectioning and high speed while minimizing out-of-focus background and premature photobleaching. Here we used scanned Bessel beams in conjunction with structured illumination and/or two-photon excitation to create thinner light sheets (<0.5 μm) better suited to three-dimensional (3D) subcellular imaging. As demonstrated by imaging the dynamics of mitochondria, filopodia, membrane ruffles, intracellular vesicles and mitotic chromosomes in live cells, the microscope currently offers 3D isotropic resolution down to ∼0.3 μm, speeds up to nearly 200 image planes per second and the ability to noninvasively acquire hundreds of 3D data volumes from single living cells encompassing tens of thousands of image frames.
Super-resolution microscopy at a glance Galbraith, Catherine G; Galbraith, James A
Journal of cell science,
05/2011, Letnik:
124, Številka:
Pt 10
Journal Article
Statistical analysis is critical in the interpretation of experimental data across the life sciences, including neuroscience. The nature of the data collected has a critical role in determining the ...best statistical approach to take. One particularly prevalent type of data is referred to as "clustered data." Clustered data are characterized as data that can be classified into a number of distinct groups or "clusters" within a particular study. Clustered data arise most commonly in neuroscience when data are compiled across multiple experiments, for example in electrophysiological or optical recordings taken from synaptic terminals, with each experiment providing a distinct cluster of data. However, there are many other types of experimental design that can yield clustered data. Here, we provide a statistical model for intracluster correlation and systematically investigate a range of methods for analyzing clustered data. Our analysis reveals that it is critical to take data clustering into account and suggests appropriate statistical approaches that can be used to account for data clustering.
Despite more than 100 years of study, it is unclear if the movement of proteins inside the cell is best described as a mosh pit or an exquisitely choreographed dance. Recent studies suggest the ...latter. Local interactions induce molecular condensates such as liquid-liquid phase separations (LLPSs) or non-liquid, functionally significant molecular aggregates, including synaptic densities, nucleoli, and Amyloid fibrils. Molecular condensates trigger intracellular signaling and drive processes ranging from gene expression to cell division. However, the descriptions of condensates tend to be qualitative and correlative. Here, we indicate how single-molecule imaging and analyses can be applied to quantify condensates. We discuss the pros and cons of different techniques for measuring differences between transient molecular behaviors inside and outside condensates. Finally, we offer suggestions for how imaging and analyses from different time and space regimes can be combined to identify molecular behaviors indicative of condensates within the dynamic high-density intracellular environment.
Migrating cells extend protrusions, probing the surrounding matrix in search of permissive sites to form adhesions. We found that actin fibers polymerizing along the leading edge directed local ...protrusions and drove synchronous sideways movement of β₁ integrin adhesion receptors. These movements lead to the clustering and positioning of conformationally activated, but unligated, β₁ integrins along the leading edge of fibroblast lamellae and growth cone filopodia. Thus, rapid actin-based movement of primed integrins along the leading edge suggests a "sticky fingers" mechanism to probe for new adhesion sites and to direct migration.
Accurate determination of the relative positions of proteins within localized regions of the cell is essential for understanding their biological function. Although fluorescent fusion proteins are ...targeted with molecular precision, the position of these genetically expressed reporters is usually known only to the resolution of conventional optics (almost equal to200 nm). Here, we report the use of two-color photoactivated localization microscopy (PALM) to determine the ultrastructural relationship between different proteins fused to spectrally distinct photoactivatable fluorescent proteins (PA-FPs). The nonperturbative incorporation of these endogenous tags facilitates an imaging resolution in whole, fixed cells of almost equal to20-30 nm at acquisition times of 5-30 min. We apply the technique to image different pairs of proteins assembled in adhesion complexes, the central attachment points between the cytoskeleton and the substrate in migrating cells. For several pairs, we find that proteins that seem colocalized when viewed by conventional optics are resolved as distinct interlocking nano-aggregates when imaged via PALM. The simplicity, minimal invasiveness, resolution, and speed of the technique all suggest its potential to directly visualize molecular interactions within cellular structures at the nanometer scale.
Cardiac rehabilitation (CR) is an efficacious yet underused treatment for patients with coronary artery disease. The objective of this study was to determine the association between CR completion and ...mortality and resource use.
We conducted a prospective cohort study of 5886 subjects (20.8% female; mean age, 60.6 years) who had undergone angiography and were referred for CR in Calgary, AB, Canada, between 1996 and 2009. Outcomes of interest included freedom from emergency room visits, hospitalization, and survival in CR completers versus noncompleters, adjusted for clinical covariates, treatment strategy, and coronary anatomy. Hazard ratios for events for CR completers versus noncompleters were also constructed. A propensity model was used to match completers to noncompleters on baseline characteristics, and each outcome was compared between propensity-matched groups. Of the subjects referred for CR, 2900 (49.3%) completed the program, and an additional 554 subjects started but did not complete CR. CR completion was associated with a lower risk of death, with an adjusted hazard ratio of 0.59 (95% confidence interval, 0.49-0.70). CR completion was also associated with a decreased risk of all-cause hospitalization (adjusted hazard ratio, 0.77; 95% confidence interval, 0.71-0.84) and cardiac hospitalization (adjusted hazard ratio, 0.68; 95% confidence interval, 0.55-0.83) but not with emergency room visits. Propensity-matched analysis demonstrated a persistent association between CR completion and reduced mortality.
Among those coronary artery disease patients referred, CR completion is associated with improved survival and decreased hospitalization. There is a need to explore reasons for nonattendance and to test interventions to improve attendance after referral.
Much is known about the composition and function of the postsynaptic density (PSD), but less is known about its molecular organization. We use EM tomography to delineate the organization of PSDs at ...glutamatergic synapses in rat hippocampal cultures. The core of the PSD is dominated by vertically oriented filaments, and ImmunoGold labeling shows that PSD-95 is a component of these filaments. Vertical filaments contact two types of transmembrane structures whose sizes and positions match those of glutamate receptors and intermesh with two types of horizontally oriented filaments lying 10-20 nm from the postsynaptic membrane. The longer horizontal filaments link adjacent NMDAR-type structures, whereas the smaller filaments link both NMDA- and AMPAR-type structures. The orthogonal, interlinked scaffold of filaments at the core of the PSD provides a structural basis for understanding dynamic aspects of postsynaptic function.
Recent advances in light microscopy permit visualization of the behavior of individual molecules within dense macromolecular ensembles in live cells. It is now conceptually possible to relate the ...dynamic organization of molecular machinery to cellular function. However, inherent heterogeneities, as well as disparities between spatial and temporal scales, pose substantial challenges in deriving such a relationship. New approaches are required to link discrete single-molecule behavior with continuous cellular-level processes. Here we combined intercalated molecular and cellular imaging with a computational framework to detect reproducible transient changes in the behavior of individual molecules that are linked to cellular behaviors. Applying our approach to integrin transmembrane receptors revealed a spatial density gradient underlying characteristic molecular density increases and mobility decreases, indicating the subsequent onset of local protrusive activity. Integrin mutants further revealed that these density and mobility transients are separable and depend on different binding domains within the integrin cytoplasmic tail. Our approach provides a generalizable paradigm for dissecting dynamic spatiotemporal molecular behaviors linked to local cellular events.
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