Photoactivatable Fluorophore for Stimulated Emission Depletion (STED) Microscopy and Bioconjugation Technique for Hydrophobic Labels

Weber, Michael; Khan, Taukeer A.; Patalag, Lukas J.; Bossi, Mariano L.; Leutenegger, Marcel; Belov, Vladimir N.; Hell, Stefan W.

doi:10.1002/chem.202004645

Cited by 33 publications

(52 citation statements)

References 37 publications

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“…Additionally, a high planarity and C 2v symmetry tend to trigger aggregation processes, either of the single fluorophore itself [4,7] or of labeled (bio-)molecules. [8] This aggregation can impose severe limitations on conjugation processes, [9] the image quality in modern (super-resolution) microscopy techniques, [10] and general applications in aqueous media (sensors, photosensitizers etc.). [11] Carbohydrates, on the other hand, encompass a diverse set of modularly assembled biomolecules of the highest structural and chiral complexity.…”

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confidence: 99%

GlycoBODIPYs: Sugars Serving as a Natural Stock for Water‐soluble Fluorescent Probes of Complex Chiral Morphology

et al. 2021

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A range of unprocessed, reducing sugar substrates (mono-, di-, and trisaccharides) is shown to take part in a straightforward four-step synthetic route to water-soluble, uncharged BODIPY derivatives with unimpaired chiral integrity and high fluorescence efficiency. A wide compatibility with several postfunctionalizations is demonstrated, thus suggesting a universal utility of the multifunctional glycoconjugates, which we call GlycoBODIPYs. Knoevenagel condensations are able to promote a red-shift in the spectra, thereby furnishing strongly fluorescent red and far-red glycoconjugates of high hydrophilicity. The synthetic outcome was studied by Xray crystallography and by comprehensive photophysical investigations in several solvent systems. Furthermore, cell experiments illustrate efficient cell uptake and demonstrate differential cell targeting as a function of the integrated chiral information.

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confidence: 99%

GlycoBODIPYs: Sugars Serving as a Natural Stock for Water‐soluble Fluorescent Probes of Complex Chiral Morphology

et al. 2021

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“…Antibody conjugation. The labelling of the antibody using glycan modification and strain-promoted click chemistry, together with the synthesis of the dye used was as described previously 13 . In short, the rabbit monoclonal antibody (ab245764, Abcam) was modified with azide groups using a commercial enzyme system (GlyClick, Genovis).…”

Section: Methodsmentioning

confidence: 99%

“…The confocal and STED images were recorded using a commercial Abberior Instruments Expert Line microscope equipped with a 775 nm 40 MHz STED laser and a 640 nm excitation laser after activation with a spectrally broad 405 nm light-emitting diode as described in ref. 13 . For MINSTED, the labelled cells were incubated with freshly diluted silica shelled silver nanoplates in PBS for 20 min and then washed with PBS.…”

Section: Methodsmentioning

confidence: 99%

“…The separation of emitters in MINSTED nanoscopy requires fluorophores that can be transferred from a lasting state that is non-responsive to excitation light into a semi-stable state leading to fluorescence upon excitation. Silicon rhodamine (SiR) fluorophores with two unsubstituted photoactivatable ortho-nitrobenzyl (ONB) caging groups proved suitable for MINSTED because photoactivation at the 355 nm wavelength activated the SiR fluorophores enabling STED at a wavelength of 775 nm with no concurrent two-photon activation 13 .…”

Section: Minsted Fluorescence Nanoscopy In Cellsmentioning

confidence: 99%

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MINSTED fluorescence localization and nanoscopy

et al. 2021

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We introduce MINSTED, a fluorophore localization and super-resolution microscopy concept based on stimulated emission depletion (STED) that provides spatial precision and resolution down to the molecular scale. In MINSTED, the intensity minimum of the STED doughnut, and hence the point of minimal STED, serves as a movable reference coordinate for fluorophore localization. As the STED rate, the background and the required number of fluorescence detections are low compared with most other STED microscopy and localization methods, MINSTED entails substantially less fluorophore bleaching. In our implementation, 200–1,000 detections per fluorophore provide a localization precision of 1–3 nm in standard deviation, which in conjunction with independent single fluorophore switching translates to a ~100-fold improvement in far-field microscopy resolution over the diffraction limit. The performance of MINSTED nanoscopy is demonstrated by imaging the distribution of Mic60 proteins in the mitochondrial inner membrane of human cells.

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“…Separation of emitters in MINSTED nanoscopy requires fluorophores that can be transferred from a lasting state that is non-responsive to excitation light into a semi-stable state leading to fluorescence upon excitation. Silicon rhodamine (SiR) fluorophores with two unsubstituted photoactivatable ortho -nitrobenzyl (ONB) caging groups proved suitable for MINSTED because photoactivation at 355 nm wavelength activated the SiR fluorophores enabling STED at 775 nm wavelength with no concurrent two-photon activation 13 .…”

Section: Resultsmentioning

confidence: 99%

MINSTED fluorescence localization and nanoscopy

Weber

Leutenegger

Stoldt

et al. 2020

Preprint

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We introduce MINSTED, a stimulated-emission-depletion (STED) based fluorescence localization and super-resolution microscopy concept providing spatial precision and resolution down to the molecular scale. In MINSTED, the intensity minimum of the STED donut, and hence the point of minimal STED, serves as a movable reference coordinate for fluorophore localization. As the STED rate, the background, and the required number of fluorescence detections are low compared to most other STED microscopy and localization methods, MINSTED entails substantially less fluorophore bleaching. In our implementation, 200-1000 detections per fluorophore provide a localization precision of 1-3 nm in standard deviation, which in conjunction with independent single fluorophore switching translates to a ~100-fold improvement of far-field microscopy resolution over the diffraction limit. The performance of MINSTED nanoscopy is demonstrated by imaging the distribution of Mic60 proteins in the mitochondrial inner membrane of human cells.

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Photoactivatable Fluorophore for Stimulated Emission Depletion (STED) Microscopy and Bioconjugation Technique for Hydrophobic Labels

Cited by 33 publications

References 37 publications

GlycoBODIPYs: Sugars Serving as a Natural Stock for Water‐soluble Fluorescent Probes of Complex Chiral Morphology

GlycoBODIPYs: Sugars Serving as a Natural Stock for Water‐soluble Fluorescent Probes of Complex Chiral Morphology

MINSTED fluorescence localization and nanoscopy

MINSTED fluorescence localization and nanoscopy

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