Synthesis and properties of Asante Calcium Red—A novel family of long excitation wavelength calcium indicators |
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Authors: | Krzysztof L Hyrc Akwasi Minta P Rogelio Escamilla Patrick PL Chan Xenia A Meshik Mark P Goldberg |
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Institution: | 1. The Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO 63110, USA;2. Center for the Investigation of Membrane Excitability Diseases, Washington University School of Medicine, St. Louis, MO 63110, USA;3. Alafi Neuroimaging Laboratory, Washington University School of Medicine, St. Louis, MO 63110, USA;4. TEF Labs, Inc. , Austin, TX 78747, USA |
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Abstract: | Although many synthetic calcium indicators are available, a search for compounds with improved characteristics continues. Here, we describe the synthesis and properties of Asante Calcium Red-1 (ACR-1) and its low affinity derivative (ACR-1-LA) created by linking BAPTA to seminaphthofluorescein. The indicators combine a visible light (450–540 nm) excitation with deep-red fluorescence (640 nm). Upon Ca2+ binding, the indicators raise their fluorescence with longer excitation wavelengths producing higher responses. Although the changes occur without any spectral shifts, it is possible to ratio Ca2+-dependent (640 nm) and quasi-independent (530 nm) emission when using visible (<490 nm) or multiphoton (∼780 nm) excitation. Therefore, both probes can be used as single wavelength or, less dynamic, ratiometric indicators. Long indicator emission might allow easy Ca2+]i measurement in GFP expressing cells. The indicators bind Ca2+ with either high (Kd = 0.49 ± 0.07 μM; ACR-1) or low affinity (Kd = 6.65 ± 0.13 μM; ACR-1-LA). Chelating Zn2+ (Kd = 0.38 ± 0.02 nM) or Mg2+ (Kd ∼ 5 mM) slightly raises and binding Co2+ quenches dye fluorescence. New indicators are somewhat pH-sensitive (pKa = 6.31 ± 0.07), but fairly resistant to bleaching. The probes are rather dim, which combined with low AM ester loading efficiency, might complicate in situ imaging. Despite potential drawbacks, ACR-1 and ACR-1-LA are promising new calcium indicators. |
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Keywords: | Confocal microscopy Dissociation constant Emission spectra Excitation spectra Fluorescence Intracellular free calcium concentration Magnesium ions Multiphoton microscopy Ratio imaging Neurons Zinc ions |
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