Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes

Shim, Sang‐Hee; Xia, Chenglong; Zhong, Guisheng; Babcock, Hazen P.; Vaughan, Joshua C.; Huang, Bo; Wang, Xun; Xu, Chengpei; Bi, Guo-Qiang; Zhuang, Xiaowei

doi:10.1073/pnas.1201882109

Cited by 462 publications

(497 citation statements)

References 47 publications

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“…However, spatial resolution is limited by the wave nature of light, which makes it difficult to resolve details in cells smaller than about half the illumination wavelength. In the past 20 years, the diffraction barrier has been overcome by several advanced techniques in far-field fluorescence microscopy such as stimulated emission depletion microscopy [1,2], photoactivated localization microscopy [3,4], stochastic optical reconstruction microscopy [5][6][7], saturated structured illumination microscopy [8][9][10][11] and so on. Recently, superresolution fluorescence imaging of thick specimens was also achieved [12,13].…”

Section: Introductionmentioning

confidence: 99%

Saturated excitation of fluorescent proteins for subdiffraction-limited imaging of living cells in three dimensions

et al. 2013

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We report, for the first time, the saturated excitation (SAX) of fluorescent proteins for subdiffraction-limited imaging of living cells in three-dimensions. To achieve saturation, a bright yellow and green fluorescent protein (Venus and EGFP) that exhibits a strong nonlinear fluorescence response to the high excitation intensity at the laser focus is used. Harmonic demodulation of the fluorescence signal produced by a modulated excitation light extracts the nonlinear fluorescence signals. After constructing the image from the nonlinear components, we obtain fluorescence images of living cells with spatial resolution beyond the diffraction limit. We also applied linear deconvolution to SAX microscopy and found it effective in further enhancing the contrast of small intracellular structures in the SAX image, confirming the expansion of the optical transfer function in SAX microscopy.

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Section: Introductionmentioning

confidence: 99%

Saturated excitation of fluorescent proteins for subdiffraction-limited imaging of living cells in three dimensions

et al. 2013

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“…The microtubule network has been imaged using these methods in 3D with ∼20-nm lateral and ∼55-nm axial resolution inside fixed cells (14) and in 2D with ∼70-nm lateral resolution inside living cells (15). However, the imaging speed of live-cell superresolution microscopy has been limited because of the frame rates of modern cameras and the performance of photoswitchable fluorescent probes (15)(16)(17)(18). Therefore, spatial and temporal resolution must often be balanced against each other in live-cell superresolution microscopy, making it challenging to observe fast cargo dynamics on the microtubule network with the needed spatiotemporal resolution.…”

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confidence: 99%

Correlative live-cell and superresolution microscopy reveals cargo transport dynamics at microtubule intersections

Bálint

Vilanova

Álvarez

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

198

230

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Intracellular transport plays an essential role in maintaining the organization of polarized cells. Motor proteins tether and move cargos along microtubules during long-range transport to deliver them to their proper location of function. To reach their destination, cargo-bound motors must overcome barriers to their forward motion such as intersection points between microtubules. The ability to visualize how motors navigate these barriers can give important information about the mechanisms that lead to efficient transport. Here, we first develop an all-optical correlative imaging method based on single-particle tracking and superresolution microscopy to map the transport trajectories of cargos to individual microtubules with high spatiotemporal resolution. We then use this method to study the behavior of lysosomes at microtubulemicrotubule intersections. Our results show that the intersection poses a significant hindrance that leads to long pauses in transport only when the separation distance of the intersecting microtubules is smaller than ∼100 nm. However, the obstructions are typically overcome by the motors with high fidelity by either switching to the intersecting microtubule or eventually passing through the intersection. Interestingly, there is a large tendency to maintain the polarity of motion (anterograde or retrograde) after the intersection, suggesting a high degree of regulation of motor activity to maintain transport in a given direction. These results give insights into the effect of the cytoskeletal geometry on cargo transport and have important implications for the mechanisms that cargo-bound motors use to maneuver through the obstructions set up by the complex cytoskeletal network.C ells rely on a two-way transport system to deliver important proteins and organelles to their location of function. Kinesin and dynein motors are responsible for long-range transport along microtubules (1). Although dynein walks toward the (−) end of the microtubule (retrograde), carrying cargo toward the cell nucleus, most kinesins walk toward the (+) end (anterograde), carrying cargo toward the cell periphery (1). Microtubules organize into a complex, 3D network inside cells, and the intersections between microtubule filaments or between microtubules and other cytoskeletal filaments (actin, intermediate filaments) likely have important consequences on the efficiency and accuracy of cargo transport (2). For example, microtubule-microtubule intersections can serve as switching points or barriers that disrupt continuous transport in a given direction.The effect of microtubule-microtubule intersections on the movement of individual motors and motor-decorated beads has been studied using in vitro reconstituted microtubules deposited on top of each other (3, 4). These studies showed that although single motors can have varied behavior (passing, dissociation, switching), at high motor densities dynein-decorated beads stop and tether at the intersection (3). On the basis of these results, it was suggested that microtubul...

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“…Thus, each localized singlemolecule emitter contains spatial and temporal information that provides quantitative data that surpass those obtained from other super-resolution techniques. We took advantage of recent developments in instrumentation and data analysis methods (26)(27)(28)(29)(30)(31)(32) to perform FPALM super-resolution microscopy at ∼35-nm resolution (localization precision, σ loc , ∼14 nm). These methods enabled us to observe that nodes are uniform units with stoichiometric ratios and distinct distributions of constituent proteins, and provided the quantitative data necessary to build a molecular model of the node.…”

mentioning

confidence: 99%

Molecular organization of cytokinesis nodes and contractile rings by super-resolution fluorescence microscopy of live fission yeast

Laplante

Huang

Tebbs

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

127

287

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Cytokinesis in animals, fungi, and amoebas depends on the constriction of a contractile ring built from a common set of conserved proteins. Many fundamental questions remain about how these proteins organize to generate the necessary tension for cytokinesis. Using quantitative high-speed fluorescence photoactivation localization microscopy (FPALM), we probed this question in live fission yeast cells at unprecedented resolution. We show that nodes, protein assembly precursors to the contractile ring, are discrete structural units with stoichiometric ratios and distinct distributions of constituent proteins. Anillin Mid1p, Fes/CIP4 homology-Bin/amphiphysin/ Rvs (F-BAR) Cdc15p, IQ motif containing GTPase-activating protein (IQGAP) Rng2p, and formin Cdc12p form the base of the node that anchors the ends of myosin II tails to the plasma membrane, with myosin II heads extending into the cytoplasm. This general node organization persists in the contractile ring where nodes move bidirectionally during constriction. We observed the dynamics of the actin network during cytokinesis, starting with the extension of short actin strands from nodes, which sometimes connected neighboring nodes. Later in cytokinesis, a broad network of thick bundles coalesced into a tight ring around the equator of the cell. The actin ring was ∼125 nm wide and ∼125 nm thick. These observations establish the organization of the proteins in the functional units of a cytokinetic contractile ring.T he mechanism of cell division by a contractile ring of actin and myosin II appeared in the common ancestor of amoebas, fungi, and animals (1). Although much is known about the protein composition of contractile rings, relatively little is known about the 3D organization of these proteins. This information is required to formulate computer models and simulations to test ideas regarding the mechanisms of contractile ring function.Electron microscopy has shown actin filaments in contractile rings of animal cells (2, 3) and yeast cells (4,5). Myosin II concentrates in cleavage furrow (6) and is the main motor for constricting contractile rings (7,8). In animal cells, electron microscopy has revealed rods the size of myosin II minifilaments in contractile rings (2, 9), and structured-illumination fluorescence microscopy has shown that this myosin II is organized in bipolar assemblies (10). Contractile rings contain other structural and regulatory proteins, including anillin (11), IQ motif containing GTPase-activating proteins (IQGAP) (12), formins (13), alphaactinin (14), and Fes/CIP4 homology-Bin/amphiphysin/Rvs (F-BAR) proteins (15), but how these proteins are anchored to the plasma membrane or organized into functional complexes in animal cells is unknown.Our understanding of the cytokinetic apparatus is most advanced in fission yeast (16). Only in fission yeast do we know the concentrations of the major cytokinesis proteins (17) and the time course of events leading to cellular division (18,19). Molecularly explicit computer models can account for both the ...

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Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes

Cited by 462 publications

References 47 publications

Saturated excitation of fluorescent proteins for subdiffraction-limited imaging of living cells in three dimensions

Saturated excitation of fluorescent proteins for subdiffraction-limited imaging of living cells in three dimensions

Correlative live-cell and superresolution microscopy reveals cargo transport dynamics at microtubule intersections

Molecular organization of cytokinesis nodes and contractile rings by super-resolution fluorescence microscopy of live fission yeast

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