Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics

Shroff, Hari; Galbraith, Catherine G.; Galbraith, James A.; Betzig, Eric

doi:10.1038/nmeth.1202

Cited by 814 publications

(765 citation statements)

References 15 publications

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“…When the product of this fraction and the density of fluorescent labels reaches approximately one fluorophore per diffraction-limited volume, it becomes difficult to resolve and precisely localize the activated fluorophores. Hence the on-off ratio limits the density of fluorescent labels that can be localized, which in turn affects the effective image resolution based on the Nyquist sampling theorem (10). (iii) The third property is the dimerization tendency.…”

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confidence: 99%

Characterization and development of photoactivatable fluorescent proteins for single-molecule–based superresolution imaging

Wang¹,

Moffitt²,

Dempsey³

et al. 2014

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

332

382

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Photoactivatable fluorescent proteins (PAFPs) have been widely used for superresolution imaging based on the switching and localization of single molecules. Several properties of PAFPs strongly influence the quality of the superresolution images. These properties include (i) the number of photons emitted per switching cycle, which affects the localization precision of individual molecules; (ii) the ratio of the on-and off-switching rate constants, which limits the achievable localization density; (iii) the dimerization tendency, which could cause undesired aggregation of target proteins; and (iv) the signaling efficiency, which determines the fraction of target-PAFP fusion proteins that is detectable in a cell. Here, we evaluated these properties for 12 commonly used PAFPs fused to both bacterial target proteins, H-NS, HU, and Tar, and mammalian target proteins, Zyxin and Vimentin. Notably, none of the existing PAFPs provided optimal performance in all four criteria, particularly in the signaling efficiency and dimerization tendency. The PAFPs with low dimerization tendencies exhibited low signaling efficiencies, whereas mMaple showed the highest signaling efficiency but also a high dimerization tendency. To address this limitation, we engineered two new PAFPs based on mMaple, which we termed mMaple2 and mMaple3. These proteins exhibited substantially reduced or undetectable dimerization tendencies compared with mMaple but maintained the high signaling efficiency of mMaple. In the meantime, these proteins provided photon numbers and on-off switching rate ratios that are comparable to the best achieved values among PAFPs.P hotoactivated localization microscopy, stochastic optical reconstruction microscopy, and related imaging methods take advantage of photoswitching and imaging of single molecules to circumvent the diffraction limit of spatial resolution in light microscopy (1-3). In these methods, only a subset of the fluorescent labels in the sample is switched on at any given time such that the positions of individual fluorophores can be localized from their images with high precision. Iteration of this process allows numerous fluorescent labels to be localized and an image with sub-diffraction-limit resolution to be reconstructed from the fluorophore localizations. Fluorescent proteins that can be activated from dark to fluorescent or converted from one color to another are widely used for such imaging approaches (4, 5). Although photoactivatable fluorescent proteins (PAFPs) are generally dimmer than photoswitchable dyes (6, 7) and hence give lower image resolution, the ease and high specificity of labeling protein targets in living cells with fluorescent proteins makes PAFPs highly appealing probes for imaging the dynamics of cellular structures (4,8).For single-molecule-based superresolution imaging methods, several properties of PAFPs are particularly important for the image quality. Here, we focus on four such key properties. (i) The first property is the photon budget, defined as the average number of ph...

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mentioning

confidence: 99%

Characterization and development of photoactivatable fluorescent proteins for single-molecule–based superresolution imaging

Wang¹,

Moffitt²,

Dempsey³

et al. 2014

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

332

382

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“…It has been further demonstrated that many conventional dyes can be used for super-resolution imaging based on photoswitching/bleaching and localization of single molecules (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Using single-molecule-based super-resolution methods, 3D resolutions down to approximately 10 nm have been demonstrated for fixed samples (21)(22)(23)(24)(25)(26), and live-cell imaging has also been achieved with spatial resolutions of 20-60 nm at time resolutions ranging from 0.5 s to 1 min (27)(28)(29)(30)(31)(32).…”

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confidence: 99%

“…However, membrane proteins are often not distributed uniformly on the membrane, but instead form localized domains nonideal for general membrane imaging. Moreover, since probe density is a key determinant of the image resolution (27,31), protein labels need to be expressed at high levels to achieve high resolutions, which can cause overexpression artifacts. An alternative to protein labels is small-molecule probes that directly bind to membrane structures, which have been widely used for specific labeling of membrane organelles (33).…”

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confidence: 99%

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

Shim

Xia

Zhong

et al. 2012

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

466

455

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Imaging membranes in live cells with nanometer-scale resolution promises to reveal ultrastructural dynamics of organelles that are essential for cellular functions. In this work, we identified photoswitchable membrane probes and obtained super-resolution fluorescence images of cellular membranes. We demonstrated the photoswitching capabilities of eight commonly used membrane probes, each specific to the plasma membrane, mitochondria, the endoplasmic recticulum (ER) or lysosomes. These small-molecule probes readily label live cells with high probe densities. Using these probes, we achieved dynamic imaging of specific membrane structures in living cells with 30-60 nm spatial resolution at temporal resolutions down to 1-2 s. Moreover, by using spectrally distinguishable probes, we obtained two-color super-resolution images of mitochondria and the ER. We observed previously obscured details of morphological dynamics of mitochondrial fusion/fission and ER remodeling, as well as heterogeneous membrane diffusivity on neuronal processes.nanoscopy | diffraction limit | photoswitchable dye | stochastic optical reconstruction microscopy | photoactivation localization microscopy

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“…Finally, the third aspect of imaging that needs to be addressed is resolution, where we find great efforts directed towards super-resolution techniques, including stimulated emission depletion (STED) [63][64][65][66], structured illumination microscopy (SIM) [67,68], photoactivated localization microscopy (PALM) [69,70], and stochastic optical reconstruction microscopy (STORM) [71][72][73][74]. These approaches offer sub-diffraction-limited resolution and have opened new ways of exploring the submicron world within the living cell.…”

Section: Imaging Smaller: Pushing Resolution To the Limitmentioning

confidence: 99%