Limits...
CaRuby-Nano: a novel high affinity calcium probe for dual color imaging.

Collot M, Wilms CD, Bentkhayet A, Marcaggi P, Couchman K, Charpak S, Dieudonné S, Häusser M, Feltz A, Mallet JM - Elife (2015)

Bottom Line: The great demand for long-wavelength and high signal-to-noise Ca(2+) indicators has led us to develop CaRuby-Nano, a new functionalizable red calcium indicator with nanomolar affinity for use in cell biology and neuroscience research.In addition, we generated CaRuby-Nano dextran conjugates and an AM-ester variant for bulk loading of tissue.We tested the new indicator using in vitro and in vivo experiments demonstrating the high sensitivity of CaRuby-Nano as well as its power in dual color imaging experiments.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biomolecules, UPMC Université Paris 06, Ecole Normale Supérieure, Paris, France.

ABSTRACT
The great demand for long-wavelength and high signal-to-noise Ca(2+) indicators has led us to develop CaRuby-Nano, a new functionalizable red calcium indicator with nanomolar affinity for use in cell biology and neuroscience research. In addition, we generated CaRuby-Nano dextran conjugates and an AM-ester variant for bulk loading of tissue. We tested the new indicator using in vitro and in vivo experiments demonstrating the high sensitivity of CaRuby-Nano as well as its power in dual color imaging experiments.

Show MeSH
Comparison of CaRuby structures.DOI:http://dx.doi.org/10.7554/eLife.05808.004
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4379494&req=5

fig1s1: Comparison of CaRuby structures.DOI:http://dx.doi.org/10.7554/eLife.05808.004

Mentions: To increase the affinity of CaRuby, we modified the structure of the probe (Figure 1A), focusing on the Ca2+ chelating BAPTA moiety, as increasing the electron density of BAPTA lowers its KD for calcium (Tsien, 1980). We introduced an oxygen atom on one of the aromatic rings of BAPTA by a SNAr reaction. This oxygen also serves as a link for the azido side arm, which was repositioned in the new CaRuby variant (Figure 1—figure supplement 1). Additionally, the fluorophore, which is commonly placed para to the nitrogen of the BAPTA, has an affinity-lowering effect due to its electron withdrawing nature and was therefore placed at a meta position in order to reduce its effect on the ligating nitrogen. These modifications resulted in a CaRuby variant with sub-micromolar affinity (‘CaRuby-Nano’). In cuvette calibration experiments CaRuby-Nano was found to have a KD of 258 ± 8 nM, with a 50-fold (±2) increase of fluorescence on binding [Ca2+] (Figure 1B–C) and a maximum quantum yield of 0.45 (Figure 1—figure supplement 2,3). In addition to being suitable for single photon excitation, CaRuby-Nano also exhibits effective two-photon excitation over a large wavelength band (Figure 1—figure supplement 4).10.7554/eLife.05808.003Figure 1.Chemical and photophysical properties of CaRuby-Nano.


CaRuby-Nano: a novel high affinity calcium probe for dual color imaging.

Collot M, Wilms CD, Bentkhayet A, Marcaggi P, Couchman K, Charpak S, Dieudonné S, Häusser M, Feltz A, Mallet JM - Elife (2015)

Comparison of CaRuby structures.DOI:http://dx.doi.org/10.7554/eLife.05808.004
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4379494&req=5

fig1s1: Comparison of CaRuby structures.DOI:http://dx.doi.org/10.7554/eLife.05808.004
Mentions: To increase the affinity of CaRuby, we modified the structure of the probe (Figure 1A), focusing on the Ca2+ chelating BAPTA moiety, as increasing the electron density of BAPTA lowers its KD for calcium (Tsien, 1980). We introduced an oxygen atom on one of the aromatic rings of BAPTA by a SNAr reaction. This oxygen also serves as a link for the azido side arm, which was repositioned in the new CaRuby variant (Figure 1—figure supplement 1). Additionally, the fluorophore, which is commonly placed para to the nitrogen of the BAPTA, has an affinity-lowering effect due to its electron withdrawing nature and was therefore placed at a meta position in order to reduce its effect on the ligating nitrogen. These modifications resulted in a CaRuby variant with sub-micromolar affinity (‘CaRuby-Nano’). In cuvette calibration experiments CaRuby-Nano was found to have a KD of 258 ± 8 nM, with a 50-fold (±2) increase of fluorescence on binding [Ca2+] (Figure 1B–C) and a maximum quantum yield of 0.45 (Figure 1—figure supplement 2,3). In addition to being suitable for single photon excitation, CaRuby-Nano also exhibits effective two-photon excitation over a large wavelength band (Figure 1—figure supplement 4).10.7554/eLife.05808.003Figure 1.Chemical and photophysical properties of CaRuby-Nano.

Bottom Line: The great demand for long-wavelength and high signal-to-noise Ca(2+) indicators has led us to develop CaRuby-Nano, a new functionalizable red calcium indicator with nanomolar affinity for use in cell biology and neuroscience research.In addition, we generated CaRuby-Nano dextran conjugates and an AM-ester variant for bulk loading of tissue.We tested the new indicator using in vitro and in vivo experiments demonstrating the high sensitivity of CaRuby-Nano as well as its power in dual color imaging experiments.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biomolecules, UPMC Université Paris 06, Ecole Normale Supérieure, Paris, France.

ABSTRACT
The great demand for long-wavelength and high signal-to-noise Ca(2+) indicators has led us to develop CaRuby-Nano, a new functionalizable red calcium indicator with nanomolar affinity for use in cell biology and neuroscience research. In addition, we generated CaRuby-Nano dextran conjugates and an AM-ester variant for bulk loading of tissue. We tested the new indicator using in vitro and in vivo experiments demonstrating the high sensitivity of CaRuby-Nano as well as its power in dual color imaging experiments.

Show MeSH