Image Analysis in Gen5 Software to Analyze Drug Response in Patient-Derived Organoids Using Multiplexed Live-Cell Imaging

To begin, image the fluorescently-labeled PDOs using the Cytation 5 live cell imaging system. Then, launch the Gen5 software. Navigate to Experiments and click Open in the Task Manager.

Open the desired experiment and select Plate, followed by View in the toolbar. Then, switch the data dropdown menu to Z Projection. Double-click on a chosen well.

Select Analyze. Click, I want to set up a new image analysis data reduction step, and click OK.In Analysis settings, set type to Cellular Analysis and detection channel to Z Projection transformed TSF Brightfield. Click Options to open the primary mask and count page.

Check Auto in the threshold box, and click OK to close the popup window. Under Object Selection, define minimum and maximum object sizes to filter out cellular debris or single cells, and select other features as desired. Next, click Apply and the masked objects will be highlighted.

To set up a subpopulation analysis in the cellular analysis toolbar, click on Calculated Metrics. Then, click Select or create object level metrics of interest at the bottom-right corner. From available object metrics, choose desired metrics, such as Circularity, and click Insert.

Click OK to confirm. Next, select Subpopulation Analysis in the cellular analysis toolbar, and click Add to create a new subpopulation. Enter a name for the subpopulation.

In the object metrics, click Add Condition to add a chosen metric. Input desired parameters in the Edit Condition window, and click OK.Select the desired results in the table at the bottom of the window and click OK, followed by Apply. Under Object Details, select the designated subpopulation to view masking.

If satisfied, click OK in the Cellular Analysis window, and click Add Step to apply the analysis to all wells.