Application 3D count
Version 1.0
2/13/16
Application: 3D tracking/counting of objects using Integrated Morphometry and Analysis
This application can be used in place of Track Objects to track new objects as they appear in the image
and follow them until they leave the image set. This application could also be used to count the
number of objects in a Z-series in which an object may appear in more than one Z plane and you wish
to count it only once.
The strategy used for this procedure is to have a “last image” in which the grey value of an object is
equal to the overall object number. By looking to see if an object overlaps anything on the “last
image” you can tell if you have a new object (no overlap) or not. If you do not have a new object, the
grey value of what was overlapped is your overall object number.
Required Software: MetaMorph Complete or MetaMorph Basic (version 4.5 or later) and the Colocalization and Overlay fluorescence option (Part #40059).
Required Dropins: mmvar, journal, IMA, colocal, lpalldir, rgnseq
The journals should be installed in C:\MM\app\mmproc\journals.
Required customizations:
1.
You must determine the threshold level to use for your sample. Some samples that
photobleach may require preprocessing using Equalize light.
2.
You must set up an IMA state file that classifies out objects that you do not want. You may
wish to use size or shape parameters to get rid of non-adherent “blur” objects. You will
have to turn on measurement of the centroid X and centroid Y parameters as well as others
you wish to measure.
3.
You will have to customize the main journal to loop through the specific directory in which
your images are found.
Main journal
 Turn off all measurement preferences.
 Zero the object count (the variable maximum) and the plane number (the variable plane).
 Loop for all images in a directory the journal Go through images in the directory.
 Close the images last image, and next last image.
Go through images in the directory
This journal is used to increment the plane number and call either Plane 1 or EachPlane2.
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Increment the plane number (plane).
Flatten background to remove affects of shading.
If you are working with the first plane run the journal Plane 1, otherwise run the journal
EachPlane2.
Plane1
This journal is used to threshold the images, open the data log, and treat each object in the image as a new object
in the journal first region journal loop.
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Interactively threshold the images.
Record the threshold into variables (threshmin, threshmax, threshstate).
Make 2 blank copies of the starting image – last image, next last image.
Set the threshold on next last image to a minimum of 1.
Interactively open a Data log and put a header on the first line.
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Application 3D count
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Version 1.0
2/13/16
Load an IMA state file with classifiers to exclude uninteresting objects.
Measure the image and create regions around all objects.
Transfer the regions to next last image.
Loop for all regions the journal first region journal loop.
Reselect the starting image and clear the regions from it.
first region journal loop
This journal increments the object number, paints the object with its object number and logs the data.
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Increment the object count (maximum) and set the object number
(objectnumber)
Paint the region with a grey value equal to the object number.
Measure the centroid of the object.
Log the plane, object number and centroid on a single line (plane, object,
centroidX, centroidY) {by calling log data.jnl}
Each plane2
This journal sets the threshold of the image, and starts the analysis of the current image by going through each of
the regions and calling the journal region journal loop.
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Set the threshold of the image to the previous threshold used (threshmin, threshmax,
threshstate).
Clear all regions from the active image and next last image.
Overwrite last image with next last image and clear next last image.
Measure the current image; create regions around all objects.
Set the threshold on next last image to a minimum of 1.
If there are any regions on the image Run the journal Go through regions
Select the starting image again.
Go through regions
 Transfer all of the regions from the current image to next last image.
 Loop for all regions in next last image the journal region journal loop.
Region journal loop
This journal determines whether this is a new object or an object we already knew about
by looking at the colocalization between the current image and last image. It then calls
the journal New Object or Overlap.
 Paint the region in next last image above the threshold minimum.
 Pause data logging and measure the area of colocalization between next last
image and last image using the region of interest. Resume data logging.
 If there is any colocalization run the journal Overlap, otherwise run the journal
New Object.
Overlap
This journal gets object number using the average grey value of the colocalization area in the image
last image and paints next last image with the object number. It then logs the plane, object number
and centroid.
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Set the Object number (objectnumber) based on the grey value of the overlap
area in the image last image.
Paint the region with a grey value equal to the object number.
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Measure the centroid of the object.
Log the plane, object number and centroid on a single line (plane, object,
centroidX, centroidY) {by calling log data.jnl}
New Object
This journal increments the total number of objects and paints next last image with the object
number. It then logs the plane, object number and centroid.
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Increment the total number of objects and set the new object number to that new
value (maximum, objectnumber).
Paint the region with a grey value equal to the object number.
Measure the centroid of the object.
Log the plane, object number and centroid on a single line (plane, object,
centroidX, centroid) {by calling log data.jnl}
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