I know that dF/F =( F(t) - F0)/F0 and that F(t) is the fluorescent value at a given time and F0 is the resting fluorescence value, but for my particular system I am unsure how to calculate the F0 since I cannot start imaging until after calcium signaling starts. When I turn on the scope the cells are already flashing. I take a 30 second video and in about half the cases the cells are flashing the entire time. What should I do to measure F0?
I was thinking just finding 2 or 3 frames between a flash and averaging the pixel intensity there and using that value, but I dont know if this is kosher. Any suggestions?
Hi Andrew,
You should take a look through the literature on calcium indicators to see how most people show the fold change. Here's just one of many examples (see attached, below). In particular, look at figures 3 and 4 and read the methods on how they generated the oscillation graphs. You may also need to account for photobleaching over time, at least for many of the common fluorescent sensors. Most people who image cellular calcium signaling do so for a period much longer than 30 seconds, so you should also probably re-assess your experiment to make sure you are adequately capturing the information you want.
Article A genetically encoded Ca2+ indicator based on circularly per...
It depends on your objectives. If you are interested in the number of flashes, the duration of the flashes, the fraction of responding cells, ... the exact parameter used to define the threshold between on and off is not critical. ( F(t) - F0)/F0 is OK and you can take FO as measured on exhausted cells, non reactive cells ... .
If you want to quantify the free calcium concentration in your cell, the you need to calibrate the calcium indicator response in the cells that you are using. For that you need to have a calcium buffer and to permeabilized the cell membranes for calcium. See for example : Article Study of intra cellular calcium in live cells by fluorescenc...
Hi Andrew. If your cells are firing sparsely you can estimate F0 as the average fluorescence on your trace as your events will contribute less to your average signal than you think. If you have concerns about overestimating your dF/F, you can take the average on the portions of your traces without events.
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