Cell area measurements on phase contrast time-lapse images

Hi all,

I’m looking for advice on how to automatise cell area measurements on my data sets which are phase contrast images from a 12 hr timelapse.

So far this has been done by hand but it is very time consuming and not exactly precise and I am looking for ways to try and make it automatic and more precise/unbiased. Before switching all data acquisition to fluorescent images and repeating a number of experiments I want to try and find a way to work with these datasets.

I have so far tried using ImageJ to play around with detecting cell edges but it hasn’t worked well. Is there a way to maybe create a pipeline in Cell Profiler or other software? What kind of image processing functions should I be looking at?


p1_t0.tif (2.8 MB)

Hi @Anna_c,

I think it would be a lot easier for people on the forum to help you if you uploaded an example image.


I’ve updated the post above, thanks!

In general, ilastik is my go-to software recommendation for segmenting phase contrast images- I believe it also has tracking functionality as well.


Seconding Ilastik for phase contrast images, followed by either plugging it into CellProfiler , or a KNIME workflow using the Trackmate node to follow single segmented cells.


Hi @Anna_c,
Welcome to the forum!
I also found this post that you may find it helpful:
Cell segmentation in phase contrast

Could you walk me through how you segment the images?
From what I’ve seen ilastic requires a segmentation image - how do I obtain this without manually drawing on each t-slice and each cell and creating a mask?


If you use ilastik’s “Pixel Classification + Object Classification” workflow, you can first annotate cells then identify them- maybe someone from the ilastik team can comment here on how best to do this + tracking?

If you want to use ilastik just to call “cell area vs background”, then call and track cells in CellProfiler, we have a tutorial for that.

Hi there, @Anna_c,

before I go into an ilastik-based answer, I’ll have to ask (and please excuse my ignorance here, I am not a biologist…), cell area measurement means you want to know the area of each cell, right?


Hi Dominik,

Yes, I want to measure the area of each cell and then be able to track that cell through each time point while the cell area changes. So then the data would include a measurement of each cell at each time point. I’d also like to be able to exclude the cells that are on the border of the image and those that are move out of the image - but this I can later do manually so ideally I’d need to be able to tell which measurement is from which cell in each image.
Is that something that can be done with either softwares?


If you follow the ilastik + CellProfiler tutorial I posted, you can do this pretty easily- use ilastik to classify the image into its component parts (Cells vs background, or cell edges vs cell centers vs background, etc), then load into CellProfiler, which can then segment, find the area of, and track the cells. You can filter by if cells touch the edge (and throw them out if so), and by minimum track length (so if a cell leaves partway through it won’t be counted). It will also give you X Y center of each cell so that you can do any sort of post-hoc filtering you like.