Module: IdentifyTertiaryObjects

Identify Tertiary Objects identifies tertiary objects (e.g., cytoplasm) by removing smaller primary objects (e.g. nuclei) from larger secondary objects (e.g., cells), leaving a ring shape.

What is a tertiary object?

In CellProfiler, we use the term object as a generic term to refer to an identifed feature in an image, usually a cellular subcompartment of some kind (for example, nuclei, cells, colonies, worms). We define an object as tertiary when it is identified by using a prior primary and secondary objects for reference. A common use case is when nuclei have been found using IdentifyPrimaryObjects and the cell body has been found using IdentifySecondaryObjects but measurements from the cytoplasm, the region outside the nucleus but within the cell body, are desired. This module may be used to define the cytoplasm as an new object.

What do I need as input?

This module will take the smaller identified objects and remove them from the larger identified objects. For example, "subtracting" the nuclei from the cells will leave just the cytoplasm, the properties of which can then be measured by downstream Measure modules. The larger objects should therefore be equal in size or larger than the smaller objects and must completely contain the smaller objects; IdentifySecondaryObjects will produce objects that satisfy this constraint. Ideally, both inputs should be objects produced by prior Identify modules.

What do I get as output?

A set of tertiary objects are produced by this module, which can be used in downstream modules for measurement purposes or other operations. Because each tertiary object is produced from primary and secondary objects, there will always be at most one secondary object for each primary object. See the section "Available measurements" below for the measurements that are produced by this module.

Note that creating subregions using this module can result in objects with a single label that nonetheless are not contiguous. This may lead to unexpected results when running measurment modules such as MeasureObjectSizeShape because calculations of the perimeter, aspect ratio, solidity, etc. typically make sense only for contiguous objects. Other modules, such as MeasureImageIntensity and MeasureTexture modules, are not affected and will yield expected results.

Available measurements

Image measurements:

Object measurements:

See also IdentifyPrimaryObject and IdentifySecondaryObject modules.


Select the larger identified objects

Select the larger identified objects. This will usually be an object previously identified by a IdentifySecondaryObjects module.

Select the smaller identified objects

Select the smaller identified objects. This will usually be an object previously identified by a IdentifyPrimaryObjects module.

Name the tertiary objects to be identified

Enter a name for the new tertiary objects. The tertiary objects will consist of the smaller object subtracted from the larger object.

Name the outline image

(Used only if the outline image is to be retained for later use in the pipeline)
Enter a name for the outlines of the identified objects. The outlined image can be selected in downstream modules by selecting them from any drop-down image list.

Retain outlines of the tertiary objects?

Select Yes to retain the outlines of the new objects for later use in the pipeline. For example, a common use is for quality control purposes by overlaying them on your image of choice using the OverlayOutlines module and then saving the overlay image with the SaveImages module.

Shrink smaller object prior to subtraction?

Select Yes to shrink the smaller object by 1 pixel before subtracting the objects. this approach will ensure that there is always a tertiary object produced, even if it is only 1 pixel wide.

Select No to subtract the objects directly, which will ensure that no pixels are shared between the primary/secondary/tertiary objects and hence measurements for all three sets of objects will not use the same pixels multiple times. However, this may result in the creation of objects with no area. Measurements can still be made on such objects, but the results will be zero or not-a-number (NaN)