Calcium flux measurements

Dear Community,

I am looking to analyze ratiometric calcium flux data and am wondering whether Cell Profiler is a suitable program to perform this type of analysis with? Basically I have about 20 views with 100 cells. I image the cells for about 60 time frames at 340 and 380nm. So ideally I’d like to apply a mask to the fluorescent images, have Cell Profiler integrate the fluorescence intensity over each cell and over all 60 time frames. I’d then like to take the ratio and plot the data. Is this possible with Cell Profiler and would you recommend using it for such an application?

It would be great if you could share your insights on this matter (and maybe flow processes if you have any). Thanks so much and many kind regards :smile:

Hi,

Provided the cells are not moving, this should be readily doable. You can create one pipeline to create a binary mask for each time-lapse sequence (perhaps thresholding the first frame), and another pipeline to load it and apply it to each image in the sequence, using IdentifyPrimAutomatic to identify the masked objects. Alternately, if you feel sure that your cells can be consistently identified using IdentifyPrimAutomatic for each frame, you may not need a mask at all.

Afterward, use MeasureObjectIntensity to measure the intensity from each cell for each wavelength (integrated intensity is one of the features measured). Then use CalculateMath to obtain the ratio of the two integrated intensities for each frame.

If the cells are moving, you will need a TrackObjects module to track the location of the cell and match them up frame to frame.

I should mention that support for CP 1.0 will be limited, as most of our development efforts are focused on CP 2.0; some of these procedures are easier in CP 2.0 and CP 1.0 pipelines are still compatible. If you’re willing, it it a try.

Regards,
-Mark