15 results on '"Alexey N. Butkevich"'
Search Results
2. Accelerated MINFLUX Nanoscopy, through Spontaneously Fast-Blinking Fluorophores
- Author
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Michael Remmel, Lukas Scheiderer, Alexey N. Butkevich, Mariano L. Bossi, and Stefan W. Hell
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Biomaterials ,General Materials Science ,General Chemistry ,Biotechnology - Abstract
The introduction of MINFLUX nanoscopy allows single molecules to be localized with one nanometer precision in as little as one millisecond. However, current applications have so far focused on increasing this precision by optimizing photon collection, rather than minimizing the localization time. Concurrently, commonly used fluorescent switches are specifically designed for stochastic methods (e.g., STORM), optimized for a high photon yield and rather long on-times (tens of milliseconds). Here, accelerated MINFLUX nanoscopy with up to a 30-fold gain in localization speed is presented. The improvement is attained by designing spontaneously blinking fluorescent markers with remarkably fast on-times, down to 1–3 ms, matching the iterative localization process used in a MINFLUX microscope. This design utilizes a silicon rhodamine amide core, shifting the spirocyclization equilibrium toward an uncharged closed form at physiological conditions and imparting intact live cell permeability, modified with a fused (benzo)thiophene spirolactam fragment. The best candidate for MINFLUX microscopy (also suitable for STORM imaging) is selected through detailed characterization of the blinking behavior of single fluorophores, bound to different protein tags. Finally, optimization of the localization routines, customized to the fast blinking times, renders a significant speed improvement on a commercial MINFLUX microscope.
- Published
- 2023
3. Photoactivatable Large Stokes Shift Fluorophores for Multicolor Nanoscopy
- Author
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Ilya Likhotkin, Richard Lincoln, Mariano L. Bossi, Alexey N. Butkevich, and Stefan W. Hell
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Colloid and Surface Chemistry ,General Chemistry ,Biochemistry ,Catalysis - Abstract
We designed caging-group-free photoactivatable live-cell permeant dyes with red fluorescence emission and ∼100 nm Stokes shifts based on a 1-vinyl-10-silaxanthone imine core structure. The proposed fluorophores undergo byproduct-free one- and two-photon activation, are suitable for multicolor fluorescence microscopy in fixed and living cells and are compatible with super-resolution techniques such as STED (stimulated emission depletion) and PALM (photoactivated localization microscopy). Use of photoactivatable labels for strain-promoted azide-alkyne cycloaddition and self-labeling protein tags (HaloTag, SNAP-tag), and duplexing of imaging channel with another large Stokes shift dye have been demonstrated.
- Published
- 2023
4. Spontaneously blinking fluorophores for accelerated MINFLUX nanoscopy
- Author
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Michael Remmel, Lukas Scheiderer, Alexey N. Butkevich, Mariano L. Bossi, and Stefan W. Hell
- Abstract
Spontaneously blinking fluorophores, a class of molecules switching rapidly between a dark and a brightly emitting state, have emerged as a popular core to build fluorescent markers for super-resolution microscopy. With typical on-times in the order of tens of milliseconds, they are most suitable for STORM and related nanoscopy methods. Recent MINFLUX nanoscopy, however, can localize molecules even within a millisecond and achieve an up to ten times higher localization precision. Here, we present a series of spontaneous blinkers with short on-times (1-3 ms) matching MINFLUX recording time-scales. Our design builds upon a silicon rhodamine fluorescent core with a modified thiophene- or a benzothiophene-fused spirolactam fragment, which shifts the spirocyclization equilibrium toward the dark closed form at physiological conditions, imparting cell permeability. Concurrently, we obtain a highly photostable, short-lived open form with bright red emission. Characterizing the blinking behavior of single fluorophores bound to three different protein tags (antibodies, nanobodies, and HaloTag self-labeling enzyme) allowed us to select the best candidate for MINFLUX microscopy. The short on-times speed up MINFLUX localization by up to 30-fold.
- Published
- 2022
5. Modular Synthetic Approach to Silicon-Rhodamine Homologues and Analogues via Bis-aryllanthanum Reagents
- Author
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Alexey N. Butkevich
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Fluorophore ,Nucleophilic addition ,Letter ,Tandem ,010405 organic chemistry ,Organic Chemistry ,Regioselectivity ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Combinatorial chemistry ,Fluorescence ,0104 chemical sciences ,Rhodamine ,chemistry.chemical_compound ,chemistry ,Reagent ,Functional group ,Physical and Theoretical Chemistry - Abstract
A modular synthetic approach toward diverse analogues of the far-red fluorophore silicon-rhodamine (SiR), based on a regioselective double nucleophilic addition of aryllanthanum reagents to esters, anhydrides, and lactones, is proposed. The reaction has improved functional group tolerance and represents a unified strategy toward cell-permeant, spontaneously blinking, and photoactivatable SiR fluorescent labels. In tandem with Pd-catalyzed hydroxy- or aminocarbonylation, it serves a streamlined synthetic pathway to a series of validated live-cell-compatible fluorescent dyes.
- Published
- 2021
6. Photoactivatable fluorescent dyes with hydrophilic caging groups and their use in multicolor nanoscopy
- Author
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Michael Weber, Elisa D’Este, Lynn M Ostersehlt, Alexey N. Butkevich, Stefan W. Hell, and Angel R. Cereceda Delgado
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Carbamate ,Aqueous solution ,Infrared ,Chemistry ,medicine.medical_treatment ,Communication ,General Chemistry ,Photochemistry ,Biochemistry ,Fluorescence ,Catalysis ,Wavelength ,Colloid and Surface Chemistry ,medicine ,Irradiation ,Stimulated emission - Abstract
We propose a series of fluorescent dyes with hydrophilic carbamate caging groups that undergo rapid photoactivation under UV (≤400 nm) irradiation but do not undergo spurious two-photon activation with high-intensity (visible or infrared) light of about twice the wavelength. The caged fluorescent dyes and labels derived therefrom display high water solubility and convert upon photoactivation into validated super-resolution and live-cell-compatible fluorophores. In combination with popular fluorescent markers, multiple (up to six)-color images can be obtained with stimulated emission depletion nanoscopy. Moreover, individual fluorophores can be localized with precision
- Published
- 2021
7. Triarylmethane Fluorophores Resistant to Oxidative Photobluing
- Author
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Gražvydas Lukinavičius, Alexey N. Butkevich, Stefan W. Hell, and Mariano L. Bossi
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Fluorescence-lifetime imaging microscopy ,Fluorophore ,Spectral stability ,STED microscopy ,Context (language use) ,General Chemistry ,010402 general chemistry ,Photochemistry ,01 natural sciences ,Biochemistry ,Photobleaching ,Fluorescence ,Catalysis ,Article ,0104 chemical sciences ,Rhodamine ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,chemistry - Abstract
Spectral stability of small-molecule fluorescent probes is required for correct interpretation and reproducibility of multicolor fluorescence imaging data, in particular under high (de)excitation light intensities of super-resolution imaging or in single-molecule applications. We propose a synthetic approach to a series of spectrally stable rhodamine fluorophores based on sequential Ru- and Cu-catalyzed transformations, evaluate their stability against photobleaching and photoconversion in the context of other fluorophores using chemometric analysis, and demonstrate chemical reactivity of fluorophore photoproducts. The substitution patterns providing the photoconversion-resistant triarylmethane fluorophores have been identified, and the applicability of nonbluing labels in live-cell STED nanoscopy is demonstrated.
- Published
- 2018
8. Correction to 'Triarylmethane Fluorophores Resistant to Oxidative Photobluing'
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Mariano L. Bossi, Gražvydas Lukinavičius, Alexey N. Butkevich, and Stefan W. Hell
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Colloid and Surface Chemistry ,Chemistry ,General Chemistry ,Computational biology ,Oxidative phosphorylation ,Biochemistry ,Catalysis ,Addition/Correction - Abstract
Spectral stability of small-molecule fluorescent probes is required for correct interpretation and reproducibility of multicolor fluorescence imaging data, in particular under high (de)excitation light intensities of super-resolution imaging or in single-molecule applications. We propose a synthetic approach to a series of spectrally stable rhodamine fluorophores based on sequential Ru- and Cu-catalyzed transformations, evaluate their stability against photobleaching and photoconversion in the context of other fluorophores using chemometric analysis, and demonstrate chemical reactivity of fluorophore photoproducts. The substitution patterns providing the photoconversion-resistant triarylmethane fluorophores have been identified, and the applicability of nonbluing labels in live-cell STED nanoscopy is demonstrated.
- Published
- 2019
9. Cell-permeant large stokes shift dyes for transfection-free multicolor nanoscopy
- Author
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Alexey N. Butkevich, Stefan W. Hell, Gražvydas Lukinavičius, and Elisa D’Este
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Cell Membrane Permeability ,Cell ,Color ,Nanotechnology ,Transfection ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Catalysis ,HeLa ,symbols.namesake ,Colloid and Surface Chemistry ,Stokes shift ,medicine ,Animals ,Stimulated emission ,Fluorescent Dyes ,Microscopy ,Low toxicity ,biology ,010405 organic chemistry ,Chemistry ,STED microscopy ,General Chemistry ,biology.organism_classification ,Fluorescence ,Rats ,0104 chemical sciences ,medicine.anatomical_structure ,symbols ,Biophysics - Abstract
We designed cell-permeant red-emitting fluorescent dye labels with >140 nm Stokes shifts based on 9-iminoanthrone, 9-imino-10-silaxanthone, and 9-imino-10-germaxanthone fluorophores. The corresponding probes selectively targeting mitochondria, lysosomes, and F-actin demonstrate low toxicity and enable stimulated emission depletion (STED) nanoscopy in neurons, human fibroblasts, U2OS, and HeLa cells. In combination with known small Stokes shift dyes, our probes allow live-cell three-color STED nanoscopy of endogenous targets on popular setups with 775 nm STED wavelength.
- Published
- 2017
10. Multicolour nanoscopy of fixed and living cells with a single STED beam and hyperspectral detection
- Author
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Volker Westphal, Maria Loidolt, Steffen J. Sahl, Carola Gregor, Franziska R. Winter, Stefan W. Hell, and Alexey N. Butkevich
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0301 basic medicine ,Diffraction ,Physics ,Multidisciplinary ,Microscopy, Confocal ,business.industry ,STED microscopy ,Hyperspectral imaging ,Image processing ,Fluorescence ,Article ,Matrix decomposition ,03 medical and health sciences ,030104 developmental biology ,Optics ,Microscopy, Fluorescence ,Microscopy ,Image Processing, Computer-Assisted ,business ,Beam (structure) ,Algorithms ,Fluorescent Dyes - Abstract
The extension of fluorescence nanoscopy to larger numbers of molecular species concurrently visualized by distinct markers is of great importance for advanced biological applications. To date, up to four markers had been distinguished in STED experiments featuring comparatively elaborate imaging schemes and optical setups, and exploiting various properties of the fluorophores. Here we present a simple yet versatile STED design for multicolour imaging below the diffraction limit. A hyperspectral detection arrangement (hyperSTED) collects the fluorescence in four spectral channels, allowing the separation of four markers with only one excitation wavelength and a single STED beam. Unmixing of the different marker signals based on the simultaneous readout of all channels is performed with a non-negative matrix factorization algorithm. We illustrate the approach showing four-colour nanoscopy of fixed and living cellular samples.
- Published
- 2017
11. Tandem Suzuki-Miyaura Coupling/Acid-Catalyzed Cyclization between Vinyl Ether Boronates and Vinyl Halides: A Concise Approach to Polysubstituted Furans
- Author
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Janine Cossy, Lieven Meerpoel, Patrick Angibaud, Andrei Corbu, Ian Stansfield, Alexey N. Butkevich, Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), and Université Paris sciences et lettres (PSL)
- Subjects
Vinyl Compounds ,Dihydropyran ,Halide ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Catalysis ,chemistry.chemical_compound ,Halogens ,medicine ,Organic chemistry ,Molecule ,[CHIM]Chemical Sciences ,Physical and Theoretical Chemistry ,Furans ,Molecular Structure ,Tandem ,010405 organic chemistry ,Pinacol ,[CHIM.ORGA]Chemical Sciences/Organic chemistry ,Organic Chemistry ,Vinyl ether ,Boronic Acids ,0104 chemical sciences ,3. Good health ,chemistry ,Cyclization ,Halogen ,Acids ,medicine.drug - Abstract
International audience; Polysubstituted 2-(ω-hydroxyalkyl)furans were prepared by tandem Suzuki-Miyaura coupling/acid-catalyzed cyclization starting from appropriately substituted 3-haloallylic alcohols and dihydrofuran-, dihydropyran-or glycal-derived pinacol boronates.
- Published
- 2013
12. Two-Step One-Pot Synthesis of Benzoannulated Spiroacetals by Suzuki–Miyaura Coupling/Acid-Catalyzed Spiroacetalization
- Author
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Lieven Meerpoel, Pascal Bonnet, Alexey N. Butkevich, Janine Cossy, Andrei Corbu, Ian Stansfield, Patrick Angibaud, Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), and Université Paris sciences et lettres (PSL)
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One-pot synthesis ,Two step ,Deoxyglucose ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Catalysis ,chemistry.chemical_compound ,Acetals ,Acid catalyzed ,Organic chemistry ,Spiro Compounds ,Physical and Theoretical Chemistry ,Alkyl ,chemistry.chemical_classification ,Molecular Structure ,010405 organic chemistry ,Pinacol ,[CHIM.ORGA]Chemical Sciences/Organic chemistry ,Organic Chemistry ,Benzene ,0104 chemical sciences ,Coupling (electronics) ,chemistry ,Cyclization ,Acids - Abstract
International audience; Substituted benzoannulated spiroacetals were prepared from (2-haloaryl)alkyl alcohols and dihydropyranyl or dihydrofuranyl pinacol boronates using a SuzukiÀMiyaura coupling followed by an acid-catalyzed spirocyclization. Application of the reaction to a glycal boronate provides an approach to annulated spiroacetals in enantiopure form.
- Published
- 2012
13. Synthesis of 2H-chromenes and 1,2-dihydroquinolines from aryl aldehydes, amines, and alkenylboron compounds
- Author
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Nicos A. Petasis and Alexey N. Butkevich
- Subjects
Inorganic Chemistry ,chemistry.chemical_compound ,chemistry ,Salicylaldehyde ,Aryl ,Organic Chemistry ,Quinoline ,Materials Chemistry ,Organic chemistry ,Physical and Theoretical Chemistry ,Condensation reaction ,Biochemistry ,Article - Abstract
The one-step reaction of salicylaldehydes with amines and alkenyl boronic acids or alkenyl trifluoroborates to form 2H-chromenes (2H-1-benzopyrans) has been investigated in more detail and new suitable conditions have been identified, including the use of tertiary amines and protic solvents including water. This process was applied to a concise synthesis of a tocopherol analog. The analogous condensation reaction between 2-sulfamidobenzaldehydes and alkenyl trifluoroborates provides an efficient synthesis of 1,2-dihydroquinoline derivatives.
- Published
- 2009
14. Discovery and Preclinical Evaluation of a Novel Class of Cytotoxic Propynoic Acid Carbamoyl Methyl Amides (PACMAs).
- Author
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Roppei Yamada, Xuefei Cao, Alexey N. Butkevich, Melissa Millard, Srinivas Odde, Nick Mordwinkin, Rambabu Gundla, Ebrahim Zandi, Stan G. Louie, Nicos A. Petasis, and Nouri Neamati
- Published
- 2011
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15. MINSTED fluorescence localization and nanoscopy
- Author
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Marcel Leutenegger, Michael Weber, Alexey N. Butkevich, Stefan Stoldt, Stefan W. Hell, Stefan Jakobs, and Tiberiu S. Mihaila
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Diffraction ,Fluorophore ,02 engineering and technology ,01 natural sciences ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Optics ,Microscopy ,Stimulated emission ,030304 developmental biology ,Physics ,0303 health sciences ,010405 organic chemistry ,Super-resolution microscopy ,business.industry ,Resolution (electron density) ,STED microscopy ,021001 nanoscience & nanotechnology ,Fluorescence ,Atomic and Molecular Physics, and Optics ,0104 chemical sciences ,Electronic, Optical and Magnetic Materials ,chemistry ,0210 nano-technology ,business - Abstract
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.
- Full Text
- View/download PDF
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