In the past, the nonratiometric Fluo dyes have been very useful and popular because they have a large dynamic range (Fmax/Fmin ≈ 100). Asante Calcium Green (ACG) is much brighter than the Fluo dyes with an even larger dynamic range: Fmax/Fmin = 220 (Figure 4.7), where the relevant quantum efficiencies are: Ca2+-free Q = 0.00225, Ca2+-bound Q = 0.495. Figure 4.8 compares the quantum efficiency of Ca2+-bound ACG to fluorescein.
ACG’s high fluorescence quantum efficiency makes it the brightest Ca2+ indicator with the highest fluorescent dynamic range to date. This exceptional brightness and dynamic range allows ACG to report both small and large Ca2+ signals with good signal-to-noise.
TEFLabs plans to offer multiple versions of ACG with a range of Kds. Improved methods of non-invasive cell loading are being developed
DATA
Excitation: 517 nm
Emission: 540 nm
kd: 135 nM
Solubility: H20 (salt form) DMSO (AM form)
Molecular Weight: 1200g/mol (salt) 1400 g/mol (AM)
RESULTS
Table 4.5 summarizes ACG properties.
Figure. 4.7 ACG titration
Figure. 4.8 ACG (Ca2+-bound form) quantum efficiency relative to fluoresceinACG’s high fluorescence quantum efficiency makes it the brightest Ca2+ indicator to date. Its exceptional brightness and dynamic range allows ACG to report both small and large Ca2+ signals with good signal-to-noise. Figure 4.9 shows ACR responses to neuronal Ca2+ transients evoked by depolarizations ranging in duration from 1 to 1000 msec. ACG (K+ salt) in rat vagal sensory neurons in whole-cell patch configuration. Calcium signals evoked by depolarization in rat vagal sensory (nodose ganglion) neurons. 50 μM Asante Ca Green K+ salt was included in the intracellular solution filling a patch electrode. Whole-cell configuration was established to allow loading of the indicator from the patch electrode into the cytosol of the neuron. The holding potential of the neuron was Vm = −70 mV. The neuron received a series of step depolarizations from −70 to +10 mV that ranged in duration from 1 − 1,000 msec. Intracellular Ca2+ signals evoked by the depolarizing steps were recorded as increases in indicator fluorescence. For ease of quantitative comparison, fluorescence signals are reported as fractional change of fluorescence relative to baseline fluorescence intensity (ΔF/F0). (J Kao, University of Maryland)
Figure 4.9: ACG responses to neuronal Ca2+ transients evoked by depolarizations ranging in duration from 1 to 1000 msec. ACG was introduced into rat vagal sensory (nodose ganglion) neurons through a whole-cell patch electrode (electrode filling solution containing 50 µM ACG K+salt). The holding potential of the neuron was Vm = −70 mV. The neuron received a series of step depolarizations from −70 to +10 mV that ranged in duration from 1 − 1000 msec. Intracellular Ca2+ signals evoked by the depolarizing steps were recorded as increases in indicator fluorescence. For ease of quantitative comparison, fluorescence signals are reported as fractional change of fluorescence relative to baseline fluorescence intensity (∆F/F0). [J Kao, Univ. of Maryland Medical School]
TEFLabs plans to offer multiple versions of ACG with a range of Kds. Improved methods of non-invasive cell loading are being developed