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We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to approximately 2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image.(More)
We report a photoactivatable variant of the Aequorea victoria green fluorescent protein (GFP) that, after intense irradiation with 413-nanometer light, increases fluorescence 100 times when excited by 488-nanometer light and remains stable for days under aerobic conditions. These characteristics offer a new tool for exploring intracellular protein dynamics(More)
We combined photoactivated localization microscopy (PALM) with live-cell single-particle tracking to create a new method termed sptPALM. We created spatially resolved maps of single-molecule motions by imaging the membrane proteins Gag and VSVG, and obtained several orders of magnitude more trajectories per cell than traditional single-particle tracking(More)
The reliance of modern microscopy techniques on photoactivatable fluorescent proteins prompted development of mCherry variants that are initially dark but become red fluorescent after violet-light irradiation. Using ensemble and single-molecule characteristics as selection criteria, we developed PAmCherry1 with excitation/emission maxima at 564/595 nm.(More)
Rapidly emerging techniques of super-resolution single-molecule microscopy of living cells rely on the continued development of genetically encoded photoactivatable fluorescent proteins. On the basis of monomeric TagRFP, we have developed a photoactivatable TagRFP protein that is initially dark but becomes red fluorescent after violet light irradiation.(More)
Receptor-regulated cellular signaling often is mediated by formation of transient, heterogeneous protein complexes of undefined structure. We used single and two-color photoactivated localization microscopy to study complexes downstream of the T cell antigen receptor (TCR) in single-molecule detail at the plasma membrane of intact T cells. The kinase ZAP-70(More)
Cell biology is being transformed by the use of fluorescent proteins as fusion tags to track protein behaviour in living cells. Here, we discuss the techniques of photobleaching and photoactivation, which can reveal the location and movement of proteins. Widespread applications of these fluorescent-based methods are revealing new aspects of protein dynamics(More)
The prevailing view of intra-Golgi transport is cisternal progression, which has a key prediction--that newly arrived cargo exhibits a lag or transit time before exiting the Golgi. Instead, we find that cargo molecules exit at an exponential rate proportional to their total Golgi abundance with no lag. Incoming cargo molecules rapidly mix with those already(More)
The ability to visualize, track, and quantify molecules and events in living cells with high spatial and temporal resolution is essential for understanding biological systems. Only recently has it become feasible to carry out these tasks due to the advent of fluorescent protein technology. Here, we trace the development of highly visible and minimally(More)
Superresolution imaging is a rapidly emerging new field of microscopy that dramatically improves the spatial resolution of light microscopy by over an order of magnitude (approximately 10-20-nm resolution), allowing biological processes to be described at the molecular scale. Here, we discuss a form of superresolution microscopy based on the controlled(More)