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Stefan W Hell - One of the best experts on this subject based on the ideXlab platform.

  • masked Rhodamine Dyes of five principal colors revealed by photolysis of a 2 diazo 1 indanone caging group synthesis photophysics and light microscopy applications
    Chemistry: A European Journal, 2014
    Co-Authors: Vladimir N Belov, Gyuzel Yu Mitronova, Mariano L Bossi, Vadim P Boyarskiy, Elke Hebisch, Claudia Geisler, Kirill Kolmakov, Christian A Wurm, Katrin I Willig, Stefan W Hell
    Abstract:

    Caged Rhodamine Dyes (Rhodamines NN) of five basic colors were synthesized and used as "hidden" markers in subdiffractional and conventional light microscopy. These masked fluorophores with a 2-diazo-1-indanone group can be irreversibly photoactivated, either by irradiation with UV- or violet light (one-photon process), or by exposure to in- tense red light (l ~ 750 nm; two-photon mode). All Dyes pos- sess a very small 2-diazoketone caging group incorporated into the 2-diazo-1-indanone residue with a quaternary carbon atom (C-3) and a spiro-9H-xanthene fragment. Initial- ly they are non-colored (pale yellow), non-fluorescent, and absorb at l = 330-350 nm (molar extinction coefficient (e) � 10 4 M � 1 cm � 1 ) with a band edge that extends to about l = 440 nm. The absorption and emission bands of the uncaged derivatives are tunable over a wide range (l = 511-633 and 525-653 nm, respectively). The unmasked Dyes are highly colored and fluorescent (e = 3-8 � 10 4 M � 1 cm � 1 and fluores- cence quantum yields (f) = 40-85 % in the unbound state and in methanol). By stepwise and orthogonal protection of carboxylic and sulfonic acid groups a highly water-soluble caged red-emitting dye with two sulfonic acid residues was prepared. Rhodamines NN were decorated with amino-reac- tive N-hydroxysuccinimidyl ester groups, applied in aqueous buffers, easily conjugated with proteins, and readily photo- activated (uncaged) with l = 375-420 nm light or intense red light (l = 775 nm). Protein conjugates with optimal de- grees of labeling (3-6) were prepared and uncaged with l = 405 nm light in aqueous buffer solutions (f= 20-38 %). The photochemical cleavage of the masking group generates only molecular nitrogen. Some 10-40 % of the non-fluores- cent (dark) byproducts are also formed. However, they have low absorbance and do not quench the fluorescence of the uncaged Dyes. Photoactivation of the individual molecules of Rhodamines NN (e.g., due to reversible or irreversible transition to a "dark" non-emitting state or photobleaching) provides multicolor images with subdiffractional optical res- olution. The applicability of these novel caged fluorophores in super-resolution optical microscopy is exemplified.

  • red emitting Rhodamine Dyes for fluorescence microscopy and nanoscopy
    Chemistry: A European Journal, 2010
    Co-Authors: Kirill Kolmakov, Vladimir N Belov, Jakob Bierwagen, Christian Ringemann, Veronika Muller, Christian Eggeling, Stefan W Hell
    Abstract:

    Fluorescent markers emitting in the red are extremely valuable in biological microscopy since they minimize cellular autofluorescence and increase flexibility in multicolor experiments. Novel Rhodamine Dyes excitable with 630 nm laser light and emitting at around 660 nm have been developed. The new Rhodamines are very photostable and have high fluorescence quantum yields of up to 80 %, long excited state lifetimes of 3.4 ns, and comparatively low intersystem-crossing rates. They perform very well both in conventional and in subdiffraction-resolution microscopy such as STED (stimulated emission depletion) and GSDIM (ground-state depletion with individual molecular return), as well as in single-molecule-based experiments such as fluorescence correlation spectroscopy (FCS). Syntheses of lipophilic and hydrophilic derivatives starting from the same chromophore-containing scaffold are described. Introduction of two sulfo groups provides high solubility in water and a considerable rise in fluorescence quantum yield. The attachment of amino or thiol reactive groups allows the Dyes to be used as fluorescent markers in biology. Dyes deuterated at certain positions have narrow and symmetrical molecular mass distribution patterns, and are proposed as new tags in MS or LC-MS for identification and quantification of various substance classes (e.g., amines and thiols) in complex mixtures. High-resolution GSDIM images and live-cell STED-FCS experiments on labeled microtubules and lipids prove the versatility of the novel probes for modern fluorescence microscopy and nanoscopy.

Christopher J Chang - One of the best experts on this subject based on the ideXlab platform.

  • activity based ratiometric fret probe reveals oncogene driven changes in labile copper pools induced by altered glutathione metabolism
    Proceedings of the National Academy of Sciences of the United States of America, 2019
    Co-Authors: Clive Yiksham Chung, Jessica M Posimo, Tiffany Tsang, Julianne M Davis, Donita C Brady, Christopher J Chang
    Abstract:

    Copper is essential for life, and beyond its well-established ability to serve as a tightly bound, redox-active active site cofactor for enzyme function, emerging data suggest that cellular copper also exists in labile pools, defined as loosely bound to low-molecular-weight ligands, which can regulate diverse transition metal signaling processes spanning neural communication and olfaction, lipolysis, rest–activity cycles, and kinase pathways critical for oncogenic signaling. To help decipher this growing biology, we report a first-generation ratiometric fluorescence resonance energy transfer (FRET) copper probe, FCP-1, for activity-based sensing of labile Cu(I) pools in live cells. FCP-1 links fluorescein and Rhodamine Dyes through a Tris[(2-pyridyl)methyl]amine bridge. Bioinspired Cu(I)-induced oxidative cleavage decreases FRET between fluorescein donor and Rhodamine acceptor. FCP-1 responds to Cu(I) with high metal selectivity and oxidation-state specificity and facilitates ratiometric measurements that minimize potential interferences arising from variations in sample thickness, dye concentration, and light intensity. FCP-1 enables imaging of dynamic changes in labile Cu(I) pools in live cells in response to copper supplementation/depletion, differential expression of the copper importer CTR1, and redox stress induced by manipulating intracellular glutathione levels and reduced/oxidized glutathione (GSH/GSSG) ratios. FCP-1 imaging reveals a labile Cu(I) deficiency induced by oncogene-driven cellular transformation that promotes fluctuations in glutathione metabolism, where lower GSH/GSSG ratios decrease labile Cu(I) availability without affecting total copper levels. By connecting copper dysregulation and glutathione stress in cancer, this work provides a valuable starting point to study broader cross-talk between metal and redox pathways in health and disease with activity-based probes.

  • activity based ratiometric fret probe reveals oncogene driven changes in labile copper pools induced by altered glutathione metabolism
    bioRxiv, 2019
    Co-Authors: Clive Yiksham Chung, Jessica M Posimo, Tiffany Tsang, Julianne M Davis, Donita C Brady, Christopher J Chang
    Abstract:

    Copper is essential for life, and beyond its well-established ability to serve as a tightly-bound, redox-active active site cofactor for enzyme function, emerging data suggest that cellular copper also exists in labile pools, defined as loosely bound to low molecular weight ligands, which can regulate diverse transition metal signaling processes spanning neural communication and olfaction, lipolysis, rest-activity cycles, and kinase pathways critical for oncogenic signaling. To help decipher this growing biology, we report a first-generation ratiometric fluorescence resonance energy transfer (FRET) copper probe, FCP-1, for activity-based sensing of labile Cu(I) pools in live cells. FCP-1 links fluorescein and Rhodamine Dyes through a tris[(2-pyridyl)methyl]amine (TPA) bridge. Bioinspired Cu(I)-induced oxidative cleavage decreases FRET between fluorescein donor and Rhodamine acceptor. FCP-1 responds to Cu(I) with high metal selectivity and oxidation-state specificity and facilitates ratiometric measurements that minimize potential interferences arising from variations in sample thickness, dye concentration, and light intensity. FCP-1 enables imaging of dynamic changes in labile Cu(I) pools in live cells in response to copper supplementation/depletion, differential expression of the copper importer CTR1, and redox stress induced by manipulating intracellular glutathione levels and GSH/GSSG ratios. FCP-1 imaging reveals a labile Cu(I) deficiency induced by oncogene-driven cellular transformation that promotes fluctuations in glutathione metabolism, where lower GSH/GSSG ratios decrease labile Cu(I) availability without affecting total copper levels. By connecting copper dysregulation and glutathione stress in cancer, this work provides a valuable starting point to study broader crosstalk between metal and redox pathways in health and disease with activity-based probes.nnSignificanceCopper is a required metal nutrient for life, yet its altered homeostasis is associated with many diseases. Thus, to develop new methods to help decipher copper biology, we present an activity-based ratiometric FRET probe that exploits a biomimetic, copper(I)-dependent cleavage reaction to enable imaging of loosely-bound, labile copper pools in cells with metal and oxidation state selectivity and a self-calibrating ratiometric response. Application of this technology to cellular models of cancer reveals that oncogene-driven changes in the metabolism of glutathione, a major cellular redox buffer, leads to a labile copper(I) deficiency. This work establishes the relevance of copper dysregulation to cancer metabolism and presages further opportunities for activity-based sensing in studies of metal biology.

Vladimir N Belov - One of the best experts on this subject based on the ideXlab platform.

  • masked Rhodamine Dyes of five principal colors revealed by photolysis of a 2 diazo 1 indanone caging group synthesis photophysics and light microscopy applications
    Chemistry: A European Journal, 2014
    Co-Authors: Vladimir N Belov, Gyuzel Yu Mitronova, Mariano L Bossi, Vadim P Boyarskiy, Elke Hebisch, Claudia Geisler, Kirill Kolmakov, Christian A Wurm, Katrin I Willig, Stefan W Hell
    Abstract:

    Caged Rhodamine Dyes (Rhodamines NN) of five basic colors were synthesized and used as "hidden" markers in subdiffractional and conventional light microscopy. These masked fluorophores with a 2-diazo-1-indanone group can be irreversibly photoactivated, either by irradiation with UV- or violet light (one-photon process), or by exposure to in- tense red light (l ~ 750 nm; two-photon mode). All Dyes pos- sess a very small 2-diazoketone caging group incorporated into the 2-diazo-1-indanone residue with a quaternary carbon atom (C-3) and a spiro-9H-xanthene fragment. Initial- ly they are non-colored (pale yellow), non-fluorescent, and absorb at l = 330-350 nm (molar extinction coefficient (e) � 10 4 M � 1 cm � 1 ) with a band edge that extends to about l = 440 nm. The absorption and emission bands of the uncaged derivatives are tunable over a wide range (l = 511-633 and 525-653 nm, respectively). The unmasked Dyes are highly colored and fluorescent (e = 3-8 � 10 4 M � 1 cm � 1 and fluores- cence quantum yields (f) = 40-85 % in the unbound state and in methanol). By stepwise and orthogonal protection of carboxylic and sulfonic acid groups a highly water-soluble caged red-emitting dye with two sulfonic acid residues was prepared. Rhodamines NN were decorated with amino-reac- tive N-hydroxysuccinimidyl ester groups, applied in aqueous buffers, easily conjugated with proteins, and readily photo- activated (uncaged) with l = 375-420 nm light or intense red light (l = 775 nm). Protein conjugates with optimal de- grees of labeling (3-6) were prepared and uncaged with l = 405 nm light in aqueous buffer solutions (f= 20-38 %). The photochemical cleavage of the masking group generates only molecular nitrogen. Some 10-40 % of the non-fluores- cent (dark) byproducts are also formed. However, they have low absorbance and do not quench the fluorescence of the uncaged Dyes. Photoactivation of the individual molecules of Rhodamines NN (e.g., due to reversible or irreversible transition to a "dark" non-emitting state or photobleaching) provides multicolor images with subdiffractional optical res- olution. The applicability of these novel caged fluorophores in super-resolution optical microscopy is exemplified.

  • red emitting Rhodamine Dyes for fluorescence microscopy and nanoscopy
    Chemistry: A European Journal, 2010
    Co-Authors: Kirill Kolmakov, Vladimir N Belov, Jakob Bierwagen, Christian Ringemann, Veronika Muller, Christian Eggeling, Stefan W Hell
    Abstract:

    Fluorescent markers emitting in the red are extremely valuable in biological microscopy since they minimize cellular autofluorescence and increase flexibility in multicolor experiments. Novel Rhodamine Dyes excitable with 630 nm laser light and emitting at around 660 nm have been developed. The new Rhodamines are very photostable and have high fluorescence quantum yields of up to 80 %, long excited state lifetimes of 3.4 ns, and comparatively low intersystem-crossing rates. They perform very well both in conventional and in subdiffraction-resolution microscopy such as STED (stimulated emission depletion) and GSDIM (ground-state depletion with individual molecular return), as well as in single-molecule-based experiments such as fluorescence correlation spectroscopy (FCS). Syntheses of lipophilic and hydrophilic derivatives starting from the same chromophore-containing scaffold are described. Introduction of two sulfo groups provides high solubility in water and a considerable rise in fluorescence quantum yield. The attachment of amino or thiol reactive groups allows the Dyes to be used as fluorescent markers in biology. Dyes deuterated at certain positions have narrow and symmetrical molecular mass distribution patterns, and are proposed as new tags in MS or LC-MS for identification and quantification of various substance classes (e.g., amines and thiols) in complex mixtures. High-resolution GSDIM images and live-cell STED-FCS experiments on labeled microtubules and lipids prove the versatility of the novel probes for modern fluorescence microscopy and nanoscopy.

Kirill Kolmakov - One of the best experts on this subject based on the ideXlab platform.

  • masked Rhodamine Dyes of five principal colors revealed by photolysis of a 2 diazo 1 indanone caging group synthesis photophysics and light microscopy applications
    Chemistry: A European Journal, 2014
    Co-Authors: Vladimir N Belov, Gyuzel Yu Mitronova, Mariano L Bossi, Vadim P Boyarskiy, Elke Hebisch, Claudia Geisler, Kirill Kolmakov, Christian A Wurm, Katrin I Willig, Stefan W Hell
    Abstract:

    Caged Rhodamine Dyes (Rhodamines NN) of five basic colors were synthesized and used as "hidden" markers in subdiffractional and conventional light microscopy. These masked fluorophores with a 2-diazo-1-indanone group can be irreversibly photoactivated, either by irradiation with UV- or violet light (one-photon process), or by exposure to in- tense red light (l ~ 750 nm; two-photon mode). All Dyes pos- sess a very small 2-diazoketone caging group incorporated into the 2-diazo-1-indanone residue with a quaternary carbon atom (C-3) and a spiro-9H-xanthene fragment. Initial- ly they are non-colored (pale yellow), non-fluorescent, and absorb at l = 330-350 nm (molar extinction coefficient (e) � 10 4 M � 1 cm � 1 ) with a band edge that extends to about l = 440 nm. The absorption and emission bands of the uncaged derivatives are tunable over a wide range (l = 511-633 and 525-653 nm, respectively). The unmasked Dyes are highly colored and fluorescent (e = 3-8 � 10 4 M � 1 cm � 1 and fluores- cence quantum yields (f) = 40-85 % in the unbound state and in methanol). By stepwise and orthogonal protection of carboxylic and sulfonic acid groups a highly water-soluble caged red-emitting dye with two sulfonic acid residues was prepared. Rhodamines NN were decorated with amino-reac- tive N-hydroxysuccinimidyl ester groups, applied in aqueous buffers, easily conjugated with proteins, and readily photo- activated (uncaged) with l = 375-420 nm light or intense red light (l = 775 nm). Protein conjugates with optimal de- grees of labeling (3-6) were prepared and uncaged with l = 405 nm light in aqueous buffer solutions (f= 20-38 %). The photochemical cleavage of the masking group generates only molecular nitrogen. Some 10-40 % of the non-fluores- cent (dark) byproducts are also formed. However, they have low absorbance and do not quench the fluorescence of the uncaged Dyes. Photoactivation of the individual molecules of Rhodamines NN (e.g., due to reversible or irreversible transition to a "dark" non-emitting state or photobleaching) provides multicolor images with subdiffractional optical res- olution. The applicability of these novel caged fluorophores in super-resolution optical microscopy is exemplified.

  • red emitting Rhodamine Dyes for fluorescence microscopy and nanoscopy
    Chemistry: A European Journal, 2010
    Co-Authors: Kirill Kolmakov, Vladimir N Belov, Jakob Bierwagen, Christian Ringemann, Veronika Muller, Christian Eggeling, Stefan W Hell
    Abstract:

    Fluorescent markers emitting in the red are extremely valuable in biological microscopy since they minimize cellular autofluorescence and increase flexibility in multicolor experiments. Novel Rhodamine Dyes excitable with 630 nm laser light and emitting at around 660 nm have been developed. The new Rhodamines are very photostable and have high fluorescence quantum yields of up to 80 %, long excited state lifetimes of 3.4 ns, and comparatively low intersystem-crossing rates. They perform very well both in conventional and in subdiffraction-resolution microscopy such as STED (stimulated emission depletion) and GSDIM (ground-state depletion with individual molecular return), as well as in single-molecule-based experiments such as fluorescence correlation spectroscopy (FCS). Syntheses of lipophilic and hydrophilic derivatives starting from the same chromophore-containing scaffold are described. Introduction of two sulfo groups provides high solubility in water and a considerable rise in fluorescence quantum yield. The attachment of amino or thiol reactive groups allows the Dyes to be used as fluorescent markers in biology. Dyes deuterated at certain positions have narrow and symmetrical molecular mass distribution patterns, and are proposed as new tags in MS or LC-MS for identification and quantification of various substance classes (e.g., amines and thiols) in complex mixtures. High-resolution GSDIM images and live-cell STED-FCS experiments on labeled microtubules and lipids prove the versatility of the novel probes for modern fluorescence microscopy and nanoscopy.

Gyuzel Yu Mitronova - One of the best experts on this subject based on the ideXlab platform.

  • masked Rhodamine Dyes of five principal colors revealed by photolysis of a 2 diazo 1 indanone caging group synthesis photophysics and light microscopy applications
    Chemistry: A European Journal, 2014
    Co-Authors: Vladimir N Belov, Gyuzel Yu Mitronova, Mariano L Bossi, Vadim P Boyarskiy, Elke Hebisch, Claudia Geisler, Kirill Kolmakov, Christian A Wurm, Katrin I Willig, Stefan W Hell
    Abstract:

    Caged Rhodamine Dyes (Rhodamines NN) of five basic colors were synthesized and used as "hidden" markers in subdiffractional and conventional light microscopy. These masked fluorophores with a 2-diazo-1-indanone group can be irreversibly photoactivated, either by irradiation with UV- or violet light (one-photon process), or by exposure to in- tense red light (l ~ 750 nm; two-photon mode). All Dyes pos- sess a very small 2-diazoketone caging group incorporated into the 2-diazo-1-indanone residue with a quaternary carbon atom (C-3) and a spiro-9H-xanthene fragment. Initial- ly they are non-colored (pale yellow), non-fluorescent, and absorb at l = 330-350 nm (molar extinction coefficient (e) � 10 4 M � 1 cm � 1 ) with a band edge that extends to about l = 440 nm. The absorption and emission bands of the uncaged derivatives are tunable over a wide range (l = 511-633 and 525-653 nm, respectively). The unmasked Dyes are highly colored and fluorescent (e = 3-8 � 10 4 M � 1 cm � 1 and fluores- cence quantum yields (f) = 40-85 % in the unbound state and in methanol). By stepwise and orthogonal protection of carboxylic and sulfonic acid groups a highly water-soluble caged red-emitting dye with two sulfonic acid residues was prepared. Rhodamines NN were decorated with amino-reac- tive N-hydroxysuccinimidyl ester groups, applied in aqueous buffers, easily conjugated with proteins, and readily photo- activated (uncaged) with l = 375-420 nm light or intense red light (l = 775 nm). Protein conjugates with optimal de- grees of labeling (3-6) were prepared and uncaged with l = 405 nm light in aqueous buffer solutions (f= 20-38 %). The photochemical cleavage of the masking group generates only molecular nitrogen. Some 10-40 % of the non-fluores- cent (dark) byproducts are also formed. However, they have low absorbance and do not quench the fluorescence of the uncaged Dyes. Photoactivation of the individual molecules of Rhodamines NN (e.g., due to reversible or irreversible transition to a "dark" non-emitting state or photobleaching) provides multicolor images with subdiffractional optical res- olution. The applicability of these novel caged fluorophores in super-resolution optical microscopy is exemplified.

  • Masked Rhodamine Dyes of Five Principal Colors Revealed by Photolysis of a 2-Diazo-1-Indanone Caging Group: Synthesis, Photophysics, and Light Microscopy Applications
    'Wiley', 2014
    Co-Authors: Belov, Vladimir N., Gyuzel Yu Mitronova, Bossi, Mariano Luis, Boyarskiy, Vadim P., Hebisch Elke, Geisler Claudia, Kolmakov Kirill, Wurm, Christian A., Willig Katrin, Hell, Stefan W.
    Abstract:

    Caged Rhodamine Dyes (Rhodamines NN) of five basic colors were synthesized and used as “hidden” markers in subdiffractional and conventional light microscopy. These masked fluorophores with a 2-diazo-1-indanone group can be irreversibly photoactivated, either by irradiation with UV- or violet light (one-photon process), or by exposure to intense red light (λ∼750 nm; two-photon mode). All Dyes possess a very small 2-diazoketone caging group incorporated into the 2-diazo-1-indanone residue with a quaternary carbon atom (C-3) and a spiro-9H-xanthene fragment. Initially they are non-colored (pale yellow), non-fluorescent, and absorb at λ=330–350 nm (molar extinction coefficient (ε)≈104 M−1 cm−1) with a band edge that extends to about λ=440 nm. The absorption and emission bands of the uncaged derivatives are tunable over a wide range (λ=511–633 and 525–653 nm, respectively). The unmasked Dyes are highly colored and fluorescent (ε= 3–8×104 M−1 cm−1 and fluorescence quantum yields (ϕ)=40–85 % in the unbound state and in methanol). By stepwise and orthogonal protection of carboxylic and sulfonic acid groups a highly water-soluble caged red-emitting dye with two sulfonic acid residues was prepared. Rhodamines NN were decorated with amino-reactive N-hydroxysuccinimidyl ester groups, applied in aqueous buffers, easily conjugated with proteins, and readily photoactivated (uncaged) with λ=375–420 nm light or intense red light (λ=775 nm). Protein conjugates with optimal degrees of labeling (3–6) were prepared and uncaged with λ=405 nm light in aqueous buffer solutions (ϕ=20–38 %). The photochemical cleavage of the masking group generates only molecular nitrogen. Some 10–40 % of the non-fluorescent (dark) byproducts are also formed. However, they have low absorbance and do not quench the fluorescence of the uncaged Dyes. Photoactivation of the individual molecules of Rhodamines NN (e.g., due to reversible or irreversible transition to a “dark” non-emitting state or photobleaching) provides multicolor images with subdiffractional optical resolution. The applicability of these novel caged fluorophores in super-resolution optical microscopy is exemplified.Fil: Belov, Vladimir N.. Max Planck Institute for Biophysical Chemistry ; AlemaniaFil: Mitronova, Gyuzel Yu.. Max Planck Institute for Biophysical Chemistry ; AlemaniaFil: Bossi, Mariano Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Boyarskiy, Vadim P.. St. Petersburg State University; RusiaFil: Hebisch, Elke. Max Planck Institute for Biophysical Chemistry ; AlemaniaFil: Geisler, Claudia. Max Planck Institute for Biophysical Chemistry ; AlemaniaFil: Kolmakov, Kirill. Max Planck Institute for Biophysical Chemistry ; AlemaniaFil: Wurm, Christian A.. Max Planck Institute for Biophysical Chemistry ; AlemaniaFil: Willig, Katrin I.. Max Planck Institute for Biophysical Chemistry ; AlemaniaFil: Hell, Stefan W.. Max Planck Institute for Biophysical Chemistry ; Alemani