Instant Live-Cell Super-Resolution Imaging of Cellular Structures by Nanoinjection of Fluorescent Probes

Hennig, Simon; Linde, Sebastian van de; Lummer, Martina; Simonis, Matthias; Huser, Thomas; Sauer, Markus

doi:10.1021/nl504660t

Cited by 56 publications

(47 citation statements)

References 36 publications

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“…To explain this observation, we hypothesize that some of the larger probes do get washed out, but not fast enough before the membrane starts to heal, and only smaller probes are given enough time to be diffuse out completely. This background-free staining makes permeabilization by SLO advantageous over other methods like microinjection or microporation, where free probes cannot be washed away (Hennig et al, 2015). In addition, the required concentration of fluorescent chloroalkane with SLO-treated cells was a factor of ten lower than that recommended by the manufacturer.…”

Section: Resultsmentioning

confidence: 99%

Labeling proteins inside living cells using external fluorophores for microscopy

Teng

Ishitsuka

Ren

et al. 2016

eLife

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Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial toxin which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG’s to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20–30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes.DOI: http://dx.doi.org/10.7554/eLife.20378.001

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Section: Resultsmentioning

confidence: 99%

Labeling proteins inside living cells using external fluorophores for microscopy

Teng

Ishitsuka

Ren

et al. 2016

eLife

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“…[60,61] Both cell-permeable and impermeable fluorescent molecules canb ei njected into as inglec ell.M oreover,s everal typeso fm oleculesc an be injected simultaneouslyb y applying differentp otentials. [62] External cellular delivery known as cellular extraction is important in understanding fundamental cellular processes. [63] Theapplication of anano-Angewandte Chemie Minireviews 3710 www.angewandte.org pipettea sa ne lectrochemical attosyringew as achieved by filling it with organic solvent and immersingitinan aqueous environment.…”

Section: Single-cell Analysismentioning

confidence: 99%

Confined Nanopipette Sensing: From Single Molecules, Single Nanoparticles, to Single Cells

et al. 2018

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Nanopipette provides a promising confined space, which exhibits advances in electrochemical, optical and mass spectrometric measurements at the nanoscale. It has been employed to reveal the hidden population properties and dynamics of single molecules and single particles. Moreover, new detection mechanisms developed on nanopipette lead to the discovery of detailed single cell information at high spatial and high temporal resolution. Herein, we focus on the fabrication and characterization of nanopipettes, summarize their wide applications for single entity analysis, and conclude with an outlook for advanced practical sensing.

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“…Although temporal resolutions in the order of seconds are possible using PALM8, the SMLM method most often reported to achieve such a temporal resolution is (direct) stochastic optical reconstruction microscopy ((d)STORM)910. However, delivery of (d)STORM dyes to intracellular targets remains difficult11. PALM is well suited for live-cell imaging of focal adhesions since it uses genetically expressed fluorescent proteins known for being well tolerated in living cells.…”

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confidence: 99%

Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions

et al. 2016

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Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min−1. The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics.

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Instant Live-Cell Super-Resolution Imaging of Cellular Structures by Nanoinjection of Fluorescent Probes

Cited by 56 publications

References 36 publications

Labeling proteins inside living cells using external fluorophores for microscopy

Labeling proteins inside living cells using external fluorophores for microscopy

Confined Nanopipette Sensing: From Single Molecules, Single Nanoparticles, to Single Cells

Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions

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