Is there any kind person who can learn me to use cellprofiler to quantify lipid droplets stained with Bodipy? Thank you very much. i tried but wioth any results
Can you specify what was the issue in your work that you cannot quantify lipid droplets?
There were quite many studies that demonstrate good CP segmentation of lipid droplets in different stains (Bodipy, Nile Red, even with just brightfield).
You may also have some clues from this discussion:
Unfortunately I cannot understand where I have to download the pipeline and what I have to do. I really do not understand every step, there is always something wrong. Is there any manual?
Thank you a lot
You may be interested in our tutorials page; many of them are unfortunately a bit on the older side but they’ll at least get you started.
Thank you I’ll read everything, I hope that it was more intuitive…not for me…
I read and tried again without any good results. maybe I used an old pipeline…Is there any pipeline to detect and quantify bodipy (green) DNA (DAPI) and other proteins (red)?
Thank you very much
These are the results of my pipeline, but I guess no correct identification of nuclei, cells and lipid droplets was done. what are the mistakes?
Can you please upload your original images of bodipy , DNA and protein, together with your .cpproject pipeline here so we can have a look together?
Meanwhile, you can also update your CP to 2.2.0 version.
Given these pretty colorful images, one of the first step to do is to split the color into 3 layers, blue for nuclei, green for lipid droplet, and the orange for proteins.
(Even better, when you export the images out of your microscope, don’t merge all layers into one, the color is pseudo color anyway).
Then you would have 3 separate channels to do segmentation for each component of the cells.
I made a demonstrative pipeline here, please fine tune if needed. bodipy.cpproj (680.1 KB)
(also please update your CellProfiler to 2.2)
Thank you very much! Ok tomorrow I’ll try as you suggested. I upgrated the software.
Please carefully check the channels after you convert ColorToGray. We aim to have Lipid droplet in the OriGreen channel, protein in OriRed channel and OriBlue for nuclei.
The screenshot you uploaded shows a wrong match - up.
Once you have the correct channels, the EnhanceOrSuppressFeatures can then help each components stands out: for example here is the nuclei
Here is the lipid droplets
Identify lipid droplets with correct channel
You can add MeasureObjectSizeShape to measure the sizes of lipid droplets in pixel unit.
To convert from pixels to micrometer:
- Try to check with ImageJ > Image Properties > to see the pixel size of your image.
- If your images don’t contain this information, you would need to look at the settings of your microscope (the software that accompanies and control your microscope). It may also be termed “configuration” or “calibration” .
For e.g. pixel size = 0.245 x 0.25 micron
bodipy.cpproj (677.7 KB)