Object numbers and lsm files

Hello,

I have two questions. First, I am tracking the area and intensity of stained CHO cells over multiple frames. I have a pipeline that seems to be working well but today I noticed that for a set of images with two cells the object number would change over each frame. For example frame 1 would have cell 1, labeled 1, and cell 2, labeled 2, but then the next frame the labeles would switch. Is there a way to keep the object numbers consistent or control how CP numbers objects?

My second question is using lsm files. Currently, I open the lsm files in ImageJ and split them (3 Channels) and save the sequence of frames as multiple TIFF files. Is there a way to do this in CP? I tried using LoadImages in CP to open the lsm file but it only recognizes the first image and not the series of frames.

Thank you so much for your help.
Justin

Hi Justin,

When you say “tracking the area and intensity”, do you mean that you are using the TrackObjects module? Or are you taking each frame as an independent image?

  • If the former, it might be worth taking a look at our pipeline and possibly an example LSM file.
  • If the latter, then CP essentially labels objects by order of their appearance in the image, first by row, then by column, and is not controllable. So I can imagine a case, such as when cells are aligned horizontally, where the cell shifts vertically slightly between frames to cause a different numbering (I don’t know for use with your images, I’,m just guessing)

In any case, if you want consistent numbering and the cells aren’t moving too much, then the TrackObjects module might work for you by assigning a consistent label as the same cell is tracked from frame to frame.

Are you using the latest version of CP? If so, you can use the Input modules to open the LSM files; see Help > Creating a project > Loading image stacks and movies for pointers on this issue.

Regards.
-Mark

Mark,

The tracking is just taking each frame as an independent image. I’ll take a look at the TrackObjects module and the help files for the lsm issue and let you know how it goes. Thanks for the help!

Justin

So after looking at TrackObjects I don’t understand how it can be used to make consistent measurements with MeasureObjectAreaShape if there is no Output for TrackObjects. Shouldn’t MeasureObjectAreaShape use the results from TrackObjects so that cells can be kept consistent?

Thanks,
Justin

MeasureObjectSizeShape takes measurements of each collection of objects in a frame independently of any other frame. You would rely on the TrackObjects module to assign the unique cell label, which is one of the outputs of the module.

Now, one of the tracking methods in TrackObjects is to perform tracking using an object measurement made by an upstream module, e.g., area. In this case, the module will try to match up objects in succeeding frames via virtue of the one with the closest value match with the selected measurement.
-Mark

That makes sense! I got the lsm files to load correctly as well.

Thanks for the help!
Justin