Calcium Rubies: a family of red-emitting functionalizable indicators for two-photon Ca2+ imaging. - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Journal of the American Chemical Society Année : 2012

Calcium Rubies: a family of red-emitting functionalizable indicators for two-photon Ca2+ imaging.

Christian D Wilms
  • Fonction : Auteur
Dongdong Li
Alexis Evrard
  • Fonction : Auteur
Alsu I Zamaleeva
  • Fonction : Auteur
Laurent Bourdieu
Jens Karl Eilers
  • Fonction : Auteur
Martin Oheim

Résumé

Calcium Rubies, a family of functionalizable BAPTA-based red-fluorescent calcium (Ca2+) indicators, were designed and synthesized as new tools for intracellular Ca2+ imaging. The attachment of a side arm on the ethylene glycol bridge makes it possible to link the indicator to various groups while leaving open the possibility of aromatic substitutions on the BAPTA core for tuning the Ca2+ binding affinity. Using this approach it has been possible to characterize three different CaRubies having affinities between 3 and 22 µM. Using click chemistry, we demonstrate high-yield linkage of the azido form of their arm to PEG molecules that can be used, e.g. the stoichiometric design of ratiometric, FRET-based indicators. The long excitation and emission wavelengths of CaRubies allow otherwise challenging multi-color experiments, e.g., when combining Ca2+ uncaging or optogenetic stimulation with Ca2+ imaging. We illustrate this capacity by the detection with CaRubies of blue-light-evoked Ca2+ transients in cultured astrocytes expressing CatCh, a light-sensitive Ca2+-translocating channelrhodopsin, linked to yellow fluorescent protein for the identification of transfected cells. Using time-correlated single-photon counting, we measured fluorescence lifetimes for all CaRubies and show a roughly tenfold increase in the average lifetime upon Ca2+ chelation. Since only the fluorescence quantum yield of the CaRubies is Ca2+-dependent, calibrated measurements of absolute Ca2+ concentrations are possible with single-wavelength two-photon fluorescence excitation.

Domaines

Chimie organique
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Dates et versions

hal-00723345 , version 1 (09-08-2012)

Identifiants

Citer

Mayeul Collot, Christina Loukou, Aleksey V Yakovlev, Christian D Wilms, Dongdong Li, et al.. Calcium Rubies: a family of red-emitting functionalizable indicators for two-photon Ca2+ imaging.. Journal of the American Chemical Society, 2012, 134 (36), pp.14923-14931. ⟨10.1021/ja304018d⟩. ⟨hal-00723345⟩
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