Tracking in Volocity 4

A quick look at tracking in Volocity 4

In my quest for enlightenment with the Volocity 4 Quantitation product I am going to use the new tools for a tracking experiment.  Tracking is done from the Measurements view where I have all the tools to do the job in one place.

Here is my time resolved data, which I am viewing from the Measurements tab.

The first task, as always when tracking data, is to find the objects as accurately as possible in as many timepoints as possible. To find objects I need a protocol.

I’ve played through all the timepoints in this dataset and can see that the whole image gets darker over time, it looks like despite my best efforts there was some bleaching during the experiment.  My experience tells me that finding objects by their absolute intensity value will not be successful in all the timepoints, so I’m going to use a task to initially find the objects which is independent of absolute intensity value, "Find Objects Using SD Intensity".  Using the mean of the intensities in each timepoint and selecting standard deviations about that mean I will have an adapting selection. Clever, eh?

The objects I am trying to track are brighter than the rest of the cell and the background so I am setting a lower limit of 2 standard deviations above the mean and no upper limit.

Now, those objects towards the bottom have been selected as one object, Volocity has given them all one color.  I can separate them with the "Separate Touching Objects" task.

That’s improved the look of those objects at the bottom.  I need to check this protocol a bit more closely now.  First I can use the inspection tool to move around in the XZ and YZ planes to check throughout the volume.  Then I can check that this protocol will find objects in different timepoints.  Using the Navigation palette I can switch to the last timepoint in the sequence without leaving the Measurements view.

That’s nice, I haven’t left the Measurements view so, as soon as I switched to the last timepoint the protocol was applied and I can see the selection.  Some of these objects look extremely small, too small to be my objects of interest, so I’m going to put a minimum size restriction in the protocol.  Using the "Volume" column of the measurements I can determine an appropriate size limit.

I can go back to the first timepoint to check that this change still works there, and my protocol for finding objects is complete.

I can now add the "Track Objects" task.  It goes in at the bottom of the list.  I click on the icon of the cog or gear wheel to see the secondary dialog which has all the configuration information in it.

I’ve talked about tracking before in my Tips, and this dialog has the same options that I’ve discussed in previous notes.  I’ll set this up and see what results I get.

At first nothing happens but tracking is a time based measurement so I need to measure more than one timepoint at a time.

In the Measurements menu I can turn on "Measure All Timepoints" to give the tracking task something to work on.

As soon as I do that the progress bar appears in the image preview and after waiting for all the timepoints to be processed I can see all my tracks.

That’s a little too much information for even Dr Vipoir to assimilate all at once.  I need to check some of these tracks in more detail to see if I need to make adjustments in the Track Objects task.

In the measurements table I now have a series of object measurements and tracks, I need to scroll down to see the tracks at the bottom.  Or I can look at just the tracks by using the quick filter at the top of the table.

Now I can pick one track at a time to show just that track in the image preview.  Using the Navigation palette to move through timepoints while watching the feedback of the track I can check that I am satisfied with the job that Volocity has done.

In the view above I have zoomed into the image and have used the "View options…" in the Measurements menu to only show the object in the current timepoint so that I can see moving along the track.

Once I’m happy with the "Track Objects" task I’m going to make a measurement item of all this information and work with it further. Making a measurement item stores these measurements because at the moment they are changing every time I change the protocol.

Selecting "Make Measurement Item…" from the Measurements menu I’m going to make a new measurement item measuring all the timepoints.

 

In the library I now have a Measurement item called "Tracking" which contains the measurements of the objects and the tracks.

I can open this item to view the table of raw measurements and work with filters to hide data I’m not interested in but most excitingly I can click straight onto the Chart tab to see a graphical representation of just the tracks.  In this situation I don’t have to do any analysis first.

This is representing all the tracks as relative to the same starting position.  This shows me whether there is any pattern to the direction of object movement.

Choosing "Edit Chart…" from the Chart menu or clicking on the chart icon at the top of the window I can configure this chart.

Here I’ve given my chart a title and changed the plot to show the absolute positions by unchecking "Plot tracks relative to their starting position".

Here I’ve shown the tracks as vectors, or straight lines between the object start position and the end position, switching back to show the tracks as relative to their starting position.

Wow there’s such a lot there.  Thanks for sticking with me right to the end!  I hope it’s been helpful in getting you started with tracking in Volocity 4.  Next time I’ll be looking at batch processing and then there’s the new Analysis view …..it’s so exciting!