Questions about movies

Hi there,
I am working with “Tiff Stacks/Movies” and have a number of questions.
The movies are time series of fluorescently labelled cells, with multiple cells per frame, that are being identified as objects at their size and intensity measured over time.
i)Is there a frame number count? I cannot seem to find this.
ii)Is therea way to label identified objects with their object number, so you know what they are, other than working off xy co-ords.
iii)Is object numbering consistent over a movie. Will object one always be object 1 even if it dissappears for a few frames…
Cheers,
James

Hi James,

Are you referring to an output measurement in the final spreadsheet? If so, there should be a Group_index column in the per-image table.

In the per-object spreadsheet, each object gets a unique ObjectNumber for that frame (but see the answer to your question below). If you want to actually see the label on the image, I suggest using DisplayDataOnImage and then save the resultant image.

For regular object identification, you will not get persistent object labeling across frames. However, we have a module, TrackObjects, which will attempt to keep track of all objects across the full movie. The result will be an additional column which contains the unique object label which should be constant across the movie until the movie ends or the object disappears whichever is first. There are several types of tracking you can select, so take a look at the options and see which works best for you.

Regards,
-Mark

Thanks Mark,
I found the Frame numbers, I just had to select the correct output option.
I will try object tracking today.
I get the best results when i seed each cell of interest with a “Fake” nucleus. The problem with this approach is that when there is no way I can see to segment secondary objects. This becomes necessary when a cell that is not of interest touches a cell of interest and becomes a part of it. Is there an option i am missing that would enable me to do this?
Thanks for the help,
James

Hi Mark,
I have had some success tracking objects, however, I am losing tracks in some frames due to mis-identification of objects. So I have been trying the LAP tracking method in order to track objects across missing frames. When i do this however, new object numbers are assigned to the objects every 2 frames. This is before any gaps occur.
I have increased the sd for search radius dramatically and the radius limit.
I am really not sure what I am doing wrong as the objects do not move.

Using the overlap method works perfectly except for missing frames. Do you have any suggestions?
Could the problem be the lack of movement? making the sd to small?

Any help would be greatly appreciated.
Cheers,
James

Hi James,
Would you be able to upload your pipeline and either a movie or image sequence (i.e., a set of individual images) where this behavior occurs? The latter is probably best since you can provide the smallest set of frames in which this happens.

As an aside, could you also try using downloading and installing our trunk build from here? This is the latest version of CP since the release which includes all bugfixes made since then. You might find that the bug has already been caught and fixed, and so you continue to use this build. If the problem still persists, then it’s a bona fide bug and we’ll need to dig deeper.

Regards,
-Mark