Hi
I am investing the calcium signaling using Fluoro 4 in human iPSC derived motor neurons. While living imaging cells, how could I set the confocal parameters? so as to know at what calcium level the control samples are lying and with respect to control, patient samples are hypo or hyper excited?
Many thanks
Best
I am not 100% clear on what you are asking, so forgive me if I'm answering things that are beside the point.
First of all, do you mean fluo 4, the calcium-sensitive indicator? This dye is best for measuring relative changes in calcium, and not as good for measuring absolute calcium levels. If you want to measure resting calcium inside cells, you should probably use a ratiometric dye such as fura-2. Grynkiewicz et al 1985 provides a good roadmap for how to quantify Ca. It is possible to do with a non-ratiometric dye like fluo-4, but it is more difficult. (See Brenowitz & Regehr 2003 for methodology.)
I'm not sure in your system what would constitute increased or decreased excitation. Differences in resting calcium are not commonly used as a readout of excitation. Rather, people often quantify action potential number or frequency. You can assess that sort of activity using calcium imaging, in which case a ratiometric dye isn't necessary, and fluo-4 is a good choice.
For "confocal parameters", I would say that things like gain and dwell time and resolution and stuff are whatever it takes to get a good, unsaturated image. By "unsaturated", I mean you want to be able to detect increases and decreases in brightness, so you don't want pixels to be at the maximum or minimum brightness values.
For assessing activity associated with action potentials, you would want to monitor fluorescence over an extended period of time. In other words, you want some sort of movie. Confocals can have slow frame rates for whole 2D images, and you don't want to miss calcium rises associated with action potentials. A frame rate on the order of 100 Hz is probably good, and 10 Hz is probably slow. A line or point scan of the cells you are interested in would speed up the temporal resolution.
These are my first thoughts.
-Matthew
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