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Specific protein labeling with caged fluorophores for dual-color imaging and super-resolution microscopy in living cells

We present new fluorophore-conjugates for dual-color photoactivation and super-resolution imaging inside live mammalian cells. These custom-designed, photo-caged Q-rhodamines and fluoresceins are cell-permeable, bright and localize specifically to intracellular targets. We utilized established ortho...

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Autores principales: Hauke, Sebastian, von Appen, Alexander, Quidwai, Tooba, Ries, Jonas, Wombacher, Richard
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Royal Society of Chemistry 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5351804/
https://www.ncbi.nlm.nih.gov/pubmed/28451202
http://dx.doi.org/10.1039/c6sc02088g
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author Hauke, Sebastian
von Appen, Alexander
Quidwai, Tooba
Ries, Jonas
Wombacher, Richard
author_facet Hauke, Sebastian
von Appen, Alexander
Quidwai, Tooba
Ries, Jonas
Wombacher, Richard
author_sort Hauke, Sebastian
collection PubMed
description We present new fluorophore-conjugates for dual-color photoactivation and super-resolution imaging inside live mammalian cells. These custom-designed, photo-caged Q-rhodamines and fluoresceins are cell-permeable, bright and localize specifically to intracellular targets. We utilized established orthogonal protein labeling strategies to precisely attach the photoactivatable fluorophores to proteins with subsequent activation of fluorescence by irradiation with UV light. That way, diffusive cytosolic proteins, histone proteins as well as filigree mitochondrial networks and focal adhesion proteins were visualized inside living cells. We applied the new photoactivatable probes in inverse fluorescence recovery after photo-bleaching (iFRAP) experiments, gaining real-time access to protein dynamics from live biological settings with resolution in space and time. Finally, we used the caged Q-rhodamine for photo-activated localization microscopy (PALM) on both fixed and live mammalian cells, where the superior molecular brightness and photo-stability directly resulted in improved localization precisions for different protein targets.
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spelling pubmed-53518042017-04-27 Specific protein labeling with caged fluorophores for dual-color imaging and super-resolution microscopy in living cells Hauke, Sebastian von Appen, Alexander Quidwai, Tooba Ries, Jonas Wombacher, Richard Chem Sci Chemistry We present new fluorophore-conjugates for dual-color photoactivation and super-resolution imaging inside live mammalian cells. These custom-designed, photo-caged Q-rhodamines and fluoresceins are cell-permeable, bright and localize specifically to intracellular targets. We utilized established orthogonal protein labeling strategies to precisely attach the photoactivatable fluorophores to proteins with subsequent activation of fluorescence by irradiation with UV light. That way, diffusive cytosolic proteins, histone proteins as well as filigree mitochondrial networks and focal adhesion proteins were visualized inside living cells. We applied the new photoactivatable probes in inverse fluorescence recovery after photo-bleaching (iFRAP) experiments, gaining real-time access to protein dynamics from live biological settings with resolution in space and time. Finally, we used the caged Q-rhodamine for photo-activated localization microscopy (PALM) on both fixed and live mammalian cells, where the superior molecular brightness and photo-stability directly resulted in improved localization precisions for different protein targets. Royal Society of Chemistry 2017-01-01 2016-09-05 /pmc/articles/PMC5351804/ /pubmed/28451202 http://dx.doi.org/10.1039/c6sc02088g Text en This journal is © The Royal Society of Chemistry 2016 http://creativecommons.org/licenses/by-nc/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 3.0 Unported License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Chemistry
Hauke, Sebastian
von Appen, Alexander
Quidwai, Tooba
Ries, Jonas
Wombacher, Richard
Specific protein labeling with caged fluorophores for dual-color imaging and super-resolution microscopy in living cells
title Specific protein labeling with caged fluorophores for dual-color imaging and super-resolution microscopy in living cells
title_full Specific protein labeling with caged fluorophores for dual-color imaging and super-resolution microscopy in living cells
title_fullStr Specific protein labeling with caged fluorophores for dual-color imaging and super-resolution microscopy in living cells
title_full_unstemmed Specific protein labeling with caged fluorophores for dual-color imaging and super-resolution microscopy in living cells
title_short Specific protein labeling with caged fluorophores for dual-color imaging and super-resolution microscopy in living cells
title_sort specific protein labeling with caged fluorophores for dual-color imaging and super-resolution microscopy in living cells
topic Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5351804/
https://www.ncbi.nlm.nih.gov/pubmed/28451202
http://dx.doi.org/10.1039/c6sc02088g
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