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Long-Term, Single-Molecule Imaging of Proteins in Live Cells with Photoregulated Fluxional Fluorophores


Eördögh, Ádám; Martin, Annabell; Rivera‐Fuentes, Pablo (2022). Long-Term, Single-Molecule Imaging of Proteins in Live Cells with Photoregulated Fluxional Fluorophores. Chemistry - A European Journal, 28(71):e202202832.

Abstract

Single-molecule localization microscopy (SMLM) can reveal nanometric details of biological samples, but its high phototoxicity hampers long-term imaging in live specimens. A significant part of this phototoxicity stems from repeated irradiations that are necessary for controlled switching of fluorophores to maintain the sparse labeling of the sample. Lower phototoxicity can be obtained using fluorophores that blink spontaneously, but controlling the density of single-molecule emitters is challenging. We recently developed photoregulated fluxional fluorophores (PFFs) that combine the benefits of spontaneously blinking dyes with photocontrol of emitter density. These dyes, however, were limited to imaging acidic organelles in live cells. Herein, we report a systematic study of PFFs that culminates in probes that are functional at physiological pH and operate at longer wavelengths than their predecessors. Moreover, these probes are compatible with HaloTag labeling, thus enabling timelapse, single-molecule imaging of specific protein targets for exceptionally long times.

Keywords: HaloTag; fluorescent probes; live-cell imaging; photophysics; super-resolution microscopy

Abstract

Single-molecule localization microscopy (SMLM) can reveal nanometric details of biological samples, but its high phototoxicity hampers long-term imaging in live specimens. A significant part of this phototoxicity stems from repeated irradiations that are necessary for controlled switching of fluorophores to maintain the sparse labeling of the sample. Lower phototoxicity can be obtained using fluorophores that blink spontaneously, but controlling the density of single-molecule emitters is challenging. We recently developed photoregulated fluxional fluorophores (PFFs) that combine the benefits of spontaneously blinking dyes with photocontrol of emitter density. These dyes, however, were limited to imaging acidic organelles in live cells. Herein, we report a systematic study of PFFs that culminates in probes that are functional at physiological pH and operate at longer wavelengths than their predecessors. Moreover, these probes are compatible with HaloTag labeling, thus enabling timelapse, single-molecule imaging of specific protein targets for exceptionally long times.

Keywords: HaloTag; fluorescent probes; live-cell imaging; photophysics; super-resolution microscopy

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:07 Faculty of Science > Department of Chemistry
Dewey Decimal Classification:540 Chemistry
Scopus Subject Areas:Physical Sciences > Catalysis
Physical Sciences > Organic Chemistry
Uncontrolled Keywords:General Chemistry, Catalysis, Organic Chemistry
Language:English
Date:20 December 2022
Deposited On:09 Jan 2023 09:41
Last Modified:28 Apr 2024 01:42
Publisher:Wiley-VCH Verlag
ISSN:0947-6539
OA Status:Green
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.1002/chem.202202832
PubMed ID:36125781
Project Information:
  • : FunderH2020
  • : Grant ID801572
  • : Project TitleHDPROBES - Photoactivatable Sensors and Blinking Dyes for Live-Cell, Single-Molecule Localization Microscopy
  • Content: Published Version
  • Language: English
  • Licence: Creative Commons: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)