By James W Putney
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Extra resources for Calcium signaling
With this approach, it is theoretically possible to restrict the effective illumination to a region less than 200 nm from the membrane . The problem with this approach is getting the region of interest to lie flat up next to a coverslip. However, even with highly confined illumination, there is still a problem using diffusible calcium indicators in that they will tend to dampen or dissipate calcium gradients .
The -5N series of indicators are a little slower than the tricarboxylate indicators with BTC substantially slower than the -5N series of indicators. Kinetics for the FF series of indicators have not been determined but they appear to be similar to those of the -5N series. In a number of cases, the lower affinity indicators have seen differences in the amplitude of calcium signals that were not apparent when measured with fura-2. They also report fast events more faithfully than the low-affinity indicators.
First the sum of the on-rate and off-rate should be as large as possible. Second, the concentration of the intracellular indicator should be set to a level where it is always large relative to the equilibrium concentration of the indicator–calcium complex. Finally, in order to satisfy the previous requirements and not to drastically perturb or buffer [Ca2þ]i levels, the affinity of the indicator needs to be relatively weak. The thinking about indicator kinetics has mostly focused on increasing the offrate because on-rate is essentially diffusion-limited.
Calcium signaling by James W Putney