A new feature in the v3 BioApplications include an assay parameter entitled "Object Type". This assay parameter adds functionality to investigate Brightfield images where dark objects like edges of cytoplasm can be identified next to the brighter background.

As a default setting in the BioApplication, the value the "Object Type" assay parameter is set to 0, and lets the application consider bright objects on a dark background as potential objects, while a value of 1, causes dark areas within an image to be considered as potential objects.

Table 1 - Settings for Object Type for Assay Parameters.

Examples where objects are identified with different settings:

ObjectTypeChn = 0

Figure 1 - Raw image of SH-SY5Y cells with algorithmic overlays from the Cellomics Neuronal Profiling BioApplication indicating the endothelial cells (orange demarcation) that were removed from the analysis based on their cell body average intensity. Other overlays indicate cell body and neurite demarcation.

Figure 2 - Raw image of HeLa cells with algorithmic overlays from the Cellomics Compartmental Analysis v3.0 BioApplication. Blue overlays indicate nuclei selected for analysis and orange overlays indicated cells that are rejected from the analysis due to user defined criteria.

ObjectTypeChn = 1

Figure 3 - A549 cells imaged with the Brightfield module on the Arrayscan VTI HCS Reader. Dark blue overlays indicate cell boundaries and light blue overlays indicate nuclei as identified in a downstream fluorescent channel using the Spot Detector v3.0 BioApplication.

Figure 4 - c. elegans imaged at 5x magnification with the Brightfield module on the ArrayScan VTI and subsequently analyzed with the Spot Detector V3 BioApplication. Red spots overlay GFP signal detected by the system via fluorescence. Blue mask indicate worm border using the Object Type value set to 1 in channel one using brightfield.

When you decided that you want to use the setting of 1, we suggest a few additional tips and tricks to help you identify objects better.

• TIP – The more contrast in the image you can provide, the easier it will be to identify objects. There are a number of ways this can be done. One common way is to set an offset (Delta Z) when taking a Brightfield image to obtain a focal plane that has more contrast in it.
• TIP – Aggressive settings (10-30) for background correction will help in the object identification process especially when the intensity difference between background and objects are small.

Remember, irrespective of whether you are analyzing bright objects on a dark background or dark objects on a bright background, the strategy for object identification/detection is similar. You will always optimize the threshold value so that it separates objects from background based on intensity.