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Both FRET systems had a small maximal fluorescence change at saturating glucose (7% and 16%), badan attached at C152 was associated with a 300% maximal fluorescence increase with glucose, though with badan at C182 there was no change.
To determine the maximal fluorescence change [12], trains of 100 action potentials of 62 and 82 Hz were applied (Fig 2B, C).
The maximal fluorescence change was about 9 11% for OpuA (Fig. 2A, Table 1) and 23 24% for OpuAC (Fig. 2B, Table 1).
We used the highest fluorescence plateaus reached to determine the maximal fluorescence change, which in some cases were from the 62 Hz trains since not all cells could always follow the 82 Hz stimulation reliably.
(B ) Cumulative data comparing the effects of 0.5 mM (blue; 61 boutons from 4 fish) and 5 mM (red; 44 boutons from 3 fish) EGTA on the time required to reach 20% maximal fluorescence change in boutons of ω-conotoxin GVIA-treated fish.
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Next, we calculated actual change/maximal possible change ratio.
The maximal fluorescence changes saturated, which either reflected the saturation of the uptake systems, or the intrinsic properties of the reporter.
Fractional fluorescence changes were corrected and normalized with respect to the dilution factors and maximal fluorescence changes, respectively.
The calibration procedure reported in Figure 1 yielded maximal fluorescence changes Δ F/F0 in cell 2, close to cell 1, which rarely exceeded 10%.
With this approach, Perceval ATP sensitivity was similar to that in MIN6 beta cells with a half-maximal fluorescence change at 2.6 mmol/l (data not shown).
The probe has a maximal fractional fluorescence change that is comparable to some of the best organic voltage-sensitive dyes.
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maximal fluorescence intensity
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