Fluorescent Dyes:
Introduction - Quantifying calcium - Definitions&References - Protocols&Examples
 

Protocols & Examples

Analysis of a calcium experiments

Any image analysis software can be used to process data off-line and the main features are similar. In this case, data were analyzed using MetaMorph® software, though ImageJ free software was proved to render equivalent results.

Once a stack of suitable bit-dept* is built, image processing starts. Usually cells shape change slightly during measurement, so it is easier to threshold images and use a loose ROI (region of interest). The main steps are resumed below:

  1. Built a stack like this (4,6 MB) using a time sequence (check bit-dept). Use a comfortable colour (pseudocolors allow easier visual analysis).
  2. Threshold the image. Cells should be visible in all planes and just some background noise should be visible (if threshold value is too high, it means that acquiring conditions can be improved).
  3. Draw a loose ROI on each cell (discard those not well defined or unhealthy).
  4. ROIs used to quantify calcium
  5. Measure the thresholded average intensity (the average intensity of the area thresholded).
  6. Arrange data using Excel or any other data processing software, so they can be graphically represented (using self-built macros saves a lot of time when many series of data are processed).
  7. Represent fluorescence (average intensity) of each ROI vs. time (or frame), indicating important points (addition of reagents, etc.).
  8. Fluorescence response of ROIs with the time (Ionomycin is added after 25 s)

  9. Normalized graphic: use the initial frames to calculate F0 for each ROI (F0 will be the average of these values). Represent F/F0 vs. time (or frame) as before.
  10. This kind of representation allows a better comparison. A four to five-fold increase in intensity can be seen for most of the cells studied in this example.

* Be careful when you choose the image dept: some programs just work fine with 8-bits images!

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