Actin, Spectrin, and Associated Proteins Form a Periodic Cytoskeletal Structure in Axons

Xu, Ke; Zhong, Guisheng; Zhuang, Xiaowei

doi:10.1126/science.1232251

Cited by 1,093 publications

(1,403 citation statements)

References 41 publications

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“…These probes can be activated in sparse numbers over time such that their images are optically resolvable and can be precisely localized 4 . These techniques have been exploited to image sub-cellular structures [5][6][7] , visualize protein (co)-organization [8][9][10] and quantify protein stoichiometry [11][12][13][14][15] .…”

Section: Introductionmentioning

confidence: 99%

Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

et al. 2014

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Photoswitchable fluorescent probes are central to localization-based super-resolution microscopy. Among these probes, fluorescent proteins are appealing because they are genetically encoded. Moreover, the ability to achieve a one to one labeling ratio between the fluorescent protein and the protein of interest makes them attractive for quantitative single molecule counting. The percentage of fluorescent protein that is photoactivated into a fluorescently detectable form (i.e. photoactivation efficiency) plays a critical role in properly interpreting the quantitative information. It is important to characterize the photoactivation efficiency at the single molecule level to replicate the conditions used in super-resolution imaging. Here, we used the human Glycine receptor expressed in Xenopus oocytes and stepwise photobleaching or single molecule counting-PALM to determine the percentage of photoactivated fluorescent protein for mEos2, mEos3.1, mEos3.2, Dendra2, mClavGR2, mMaple, PA-GFP and PA-mCherry. This analysis provides important information that must be considered when using these fluorescent proteins in quantitative super-resolution microscopy.

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Section: Introductionmentioning

confidence: 99%

Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

et al. 2014

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“…We then tested the utility of the phalloidin conjugate 17 to image cellular F‐actin on fixed COS‐7 cells, comparing it to the commercially available Alexa Fluor 647‐phalloidin conjugate (AF647‐phalloidin), which has been used extensively in localization microscopy experiments 13. Under optimal d STORM buffer conditions using a highly inclined and laminated optical sheet (HILO) setup,14 the AF647‐phalloidin gave high photon counts (median=5547) and a localization precision of 5.7 nm (Figure S2).…”

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confidence: 99%

Synthesis of a Far‐Red Photoactivatable Silicon‐Containing Rhodamine for Super‐Resolution Microscopy

et al. 2015

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The rhodamine system is a flexible framework for building small‐molecule fluorescent probes. Changing N‐substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si‐containing analogue of Q‐rhodamine. This probe is the first example of a “caged” Si‐rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red‐shifted to allow multicolor imaging. The dye is a useful label for super‐resolution imaging and constitutes a new scaffold for far‐red fluorogenic molecules.

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“…At room temperature, the surprising single-molecule blinking and photoswitching for single GFP molecules provided a pathway to the active control that was needed for PALM super-resolution microscopy and its relatives. Today, super-resolution microscopy is a powerful application of single molecules that has broad impact across many fields of science (Figure 23), and new and amazing discoveries continue, such as the observation of actin bands in axons (183). All of this has occurred due not only to my efforts, but also due in major part to the clever and insightful research performed by many researchers around the world too numerous to mention 19 here.…”

Section: Concluding Remarks and Acknowledgementsmentioning

confidence: 99%

Nobel Lecture: Single-molecule spectroscopy, imaging, and photocontrol: Foundations for super-resolution microscopy

Moerner

2015

Rev. Mod. Phys.

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The initial steps toward optical detection and spectroscopy of single molecules in condensed matter arose out of the study of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral signatures relating to the fluctuations of the number of molecules in resonance led to the attainment of the single-molecule limit in 1989 using frequency-modulation laser spectroscopy. In the early 90's, many fascinating physical effects were observed for individual molecules, and the imaging of single molecules as well as observations of spectral diffusion, optical switching and the ability to select different single molecules in the same focal volume simply by tuning the pumping laser frequency provided important forerunners of the later superresolution microscopy with single molecules. In the room temperature regime, imaging of single copies of the green fluorescent protein also uncovered surprises, especially the blinking and photoinduced recovery of emitters, which stimulated further development of photoswitchable fluorescent protein labels. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic observation and localization of individual fluorophores is a key ingredient to imaging beyond the optical diffraction limit. Combining this with active control of the number of emitting molecules in the pumped volume led to the super-resolution imaging of Eric Betzig and others, a new frontier for optical microscopy beyond the diffraction limit. The background leading up to these observations is described and current developments are summarized. The early days Introduction and early inspirationsI want to thank the Nobel Committee for Chemistry, the Royal Swedish Academy of Sciences, and the Nobel Foundation for selecting me for this prize recognizing the development of super-resolved fluorescence microscopy. I am truly honored to share the prize with my two esteemed colleagues, Stefan Hell and Eric Betzig. My primary contributions center on the first optical detection and spectroscopy of single molecules in the condensed phase(1), and on the observations of imaging, blinking and photocontrol not only for single molecules at low temperatures in solids, but also for useful variants of the green fluorescent protein at room temperature (2). This lecture describes the context of the events leading up to these advances as well as a portion of the subsequent developments both around the world and in my laboratory.In the mid-1980's, I derived much early inspiration from amazing advances that were occurring around the world where single nanoscale quantum systems were detected and explored for both scientific and technological reasons. Some of these were (i) the spectroscopy of single electrons or ions confined in vacuum electromagnetic traps (3-5), (ii) scanning tunneling microscopy (STM) (6) and atomic force microscopy (AFM) (7), and (iii) the study of ion currents in single membrane-embedded ion channels (8). But why not optical detection and ...

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Actin, Spectrin, and Associated Proteins Form a Periodic Cytoskeletal Structure in Axons

Cited by 1,093 publications

References 41 publications

Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

Synthesis of a Far‐Red Photoactivatable Silicon‐Containing Rhodamine for Super‐Resolution Microscopy

Nobel Lecture: Single-molecule spectroscopy, imaging, and photocontrol: Foundations for super-resolution microscopy

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