Fluorescence intensity in membrane with CellProfiler?

Hello
I have 2 types of images. One image show fluorescence intensity of the membrane and other in mostly cytoplasm. I would like to know if I can analyze the fluorescence intensity and segmented the membrane in the two images. I am new to cell profiller and I am trying to learn to analyze my images.
cytoplasm.tif (1.2 MB) membrane.tif (978.2 KB)

I tried the analysis with the QUIMP plugin on FIJI but the plugin changes my 32-bit image to 8 bit so it creates results with a different scale.

I would like to set up a value for the membrane so then I can analyze all my images with the same membrane size.

Please, see attached the images.
Thanks in advance for any help
Regards

Hi Pablo,

Could I ask what you mean by “a value for the membrane”? Do you mean that you’d like to limit the thickness of the membrane segmentation, or perhaps something else?

Also, could you confirm whether the images you provided are of the same cell? It looks like they’ve already been segmented and the images are of different sizes, it’d be useful to see the whole field instead.

Thanks

thank you for your answer.
yes, value means that I would like to have the same thickness of the membrane. I was using the QUIMP plugin and you can choose values from 0.5 um or bigger.
The images are different cells with 2 different biosensor, one sensor is cytoplasmatic and the other is localized at the membrane. The are already segmented because they are ratio images. The macro that I use to do that makes and ROI around the cell of interest to do the segmentation and do the ratio.

After I have the ratio I would like to measure the intensity of the ratio in the periphery of the cells to see the intensity at the cell cortex.

HI @Pablo_M,

Seems like it would be a whole lot easier to do all the processing in #cellprofiler, rather than switching between different applications.

But, as @DStirling said, without seeing your raw data, it’s difficult to make specific recommendations about how you should go about analysing it.

Thank you very much both for the suggestion, please see attached both images. CFP-YFP for the cytoplasmatic and membrane sensor.

Cytoplasmraw.tif (4.0 MB) membraneraw.tif (4.0 MB)

Hi @Pablo_M,

I don’t understand what you’re showing here - there’s very little correspondence between the membrane and cytosol? It looks like they belong to different cells? Either that or there is a VERY long time lag between acquiring each channel? Did you acquire this on a scanning confocal microscope by any chance?

@Pablo_M,

In addition there are couple of things you might have to make it clear,

  1. Definitely as @djpbarry pointed out, your cytoplasm & membrane looks from a different cells
  2. You have mentioned that you were making an ROI around the cell of interest, in the sample image you have provided were will you draw your ROI in Cytoplasm or membrane.
  3. Just a clarification, Where is your membrane? is it around the cytoplasm?
    It would be great if you could explain your problem little clear, if possible with the screen shot.

Regards,
Lakshmi
Fujifilm Wako Automation (Consultant)
www.wakoautomation.com
For Cellprofiler training or optimised pipeline write to,
lakshmi.balasubramanian.contractor@fujifilm.com

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Thank for the answer. These are two different cell with two different sensors that show different subcelular localisation. One fill all the cell and have cytoplasm localisation, the other sensor localised mainly at the cortex of the cell.

I did some analysis with the QUIMP plying on Fiji. (Please, See the new image attached) In those images you can see the overlay that the plugin does when you want to measure a section fo the cell focusing in the cell cortex.

The Roi it is just a square ROI around the cell that my macros do to analyse the ratio.
These are not the same cell the image are two different cells, I just want to know if cell profiler can measure the intensity fluorescence around the cell cortex.
QUIMP can only analyze 8 bit image and I want to analyze 32 bit images, since that is the format of my ratio image.
I hoped to clarify the doubt.

Thanks for the help

rawcytoplasmoverlay.tif (3.0 MB) rawmembraneoverlay.tif (669.5 KB)

@Pablo_M,

Thanks. Its clear.
There are two ways,

  1. If you have the mask of the cortex or you complete cell from your earlier application you have used, you could use that in Cellprofiler to measure the intensity (Using “MaskObject” module on your Image in which you want to measure the intensity fluorescence)
  2. The other option is to do the complete steps in Cellprofiler, i.e. getting the cell cortex from your membrane channel and your complete cell from your cytoplasm channel and measure the intensities.
    [First segment your cortex & cytoplasm and measure intensity from your fluorescence image]

Regards,
Lakshmi
Fujifilm Wako Automation (Consultant)
www.wakoautomation.com
For Cellprofiler training or optimised pipeline write to,
lakshmi.balasubramanian.contractor@fujifilm.com

Thank you, Lakshmi. I will try to apply your suggestion.
Regards