Issue 1, 2017

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

Abstract

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.

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

Supplementary files

Article information

Article type
Edge Article
Submitted
12 May 2016
Accepted
01 Sep 2016
First published
05 Sep 2016
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY-NC license

Chem. Sci., 2017,8, 559-566

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

S. Hauke, A. von Appen, T. Quidwai, J. Ries and R. Wombacher, Chem. Sci., 2017, 8, 559 DOI: 10.1039/C6SC02088G

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