title: Calcium rubies: a family of red-emitting functionalizable indicators suitable for two-photon Ca2+ imaging.
abstract: We designed Calcium Rubies, a family of functionalizable BAPTA-based red-fluorescent calcium (Ca(2+)) indicators as new tools for biological Ca(2+) imaging. The specificity of this Ca(2+)-indicator family is its side arm, attached on the ethylene glycol bridge that allows coupling the indicator to various groups while leaving open the possibility of aromatic substitutions on the BAPTA core for tuning the Ca(2+)-binding affinity. Using this possibility we now synthesize and characterize three different CaRubies with affinities between 3 and 22 μM. Their long excitation and emission wavelengths (peaks at 586/604 nm) allow their use in otherwise challenging multicolor experiments, e.g., when combining Ca(2+) uncaging or optogenetic stimulation with Ca(2+) imaging in cells expressing fluorescent proteins. We illustrate this capacity by the detection of Ca(2+) transients evoked by blue light in cultured astrocytes expressing CatCh, a light-sensitive Ca(2+)-translocating channelrhodopsin linked to yellow fluorescent protein. Using time-correlated single-photon counting, we measured fluorescence lifetimes for all CaRubies and demonstrate a 10-fold increase in the average lifetime upon Ca(2+) chelation. Since only the fluorescence quantum yield but not the absorbance of the CaRubies is Ca(2+)-dependent, calibrated two-photon fluorescence excitation measurements of absolute Ca(2+) concentrations are feasible.
authors: UPMC Université Paris 06, Ecole Normale Supérieure, Paris, F-75005 France.
authors: Mayeul
authors: M
authors: Collot
authors: Christina
authors: C
authors: Loukou
authors: Aleksey V
authors: AV
authors: Yakovlev
authors: Christian D
authors: CD
authors: Wilms
authors: Dongdong
authors: D
authors: Li
authors: Alexis
authors: A
authors: Evrard
authors: Alsu
authors: A
authors: Zamaleeva
authors: Laurent
authors: L
authors: Bourdieu
authors: Jean-François
authors: JF
authors: Léger
authors: Nicole
authors: N
authors: Ropert
authors: Jens
authors: J
authors: Eilers
authors: Martin
authors: M
authors: Oheim
authors: Anne
authors: A
authors: Feltz
authors: Jean-Maurice
authors: JM
authors: Mallet
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000737:chemistry / Q000187:drug effects; D002118:Calcium / Q000032:analysis*; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry* / Q000493:pharmacokinetics; D007202:Indicators and Reagents / Q000138:chemical synthesis / Q000737:chemistry* / Q000493:pharmacokinetics; D051379:Mice; D008815:Mice, Inbred Strains; D015394:Molecular Structure; D017785:Photons
chemical_list: D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D004533:Egtazic Acid; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium