In this example, TopoChip is used as a sample to screen the bio-active surface topography. The goal is to count the number of cells (“Actin_20x” channel) and their nuclei (“Hoechst_20x1” channel) inside the central square (TopoUnit) and to measure the intensity (signal) of the “Scx_20x” channel for each cell.

Figure 759. Overview of the analysis.

Figure 760. Original DIA channel.

At first, the “DIA” channel is segmented using Homogeneous Area. Holes are filled and filtering, thickening and smooth is applied to correctly detect the central square topography unit on which the measurement will be performed. Using a camera ROI would not bring satisfactory results because of the slight tilt of the sample.

Figure 761. DIA segmenting nodes before grouping into a Function.

The whole sequence is then grouped into a single “Function”.

Figure 762. Grouped function.

Figure 763. Segmented DIA channel.

To detect cells, “Actin_20x” channel is thresholded. Because the area of interest is just the central square unit, logical conjunction (And node) is done between the result and the segmented “DIA” image.

Figure 764. Original Actin_20x channel.

Figure 765. Segmented Actin_20x channel shows the examined cells.

Figure 766. Logical AND between DIA and Actin_20x shows the cells inside the area of interest.

To detect the nuclei in the central TopoUnit, bright spots are detected on the “Hoechst_20x1” channel and binary objects from the segmented “DIA” binary result being inside or touching the bright spots are found (Having node).

Figure 767. Nuclei detected on the Hoechst_20x1 channel.

Assuming each cells has one nucleus, watershed algorithm is applied to the result of the Having node and another logical And is done between the watershed result and “Actin_20x” result to properly assign nuclei to the respective cells.

Figure 768. Watershed algorithm finds the cell borders.

Figure 769. Logical And between the watershed result and Actin_20x result.

Finally, object count calculates the number of cells (bright spots) present in the central TopoUnit and Sum Object Intensity calculates the amount of “Scx_20x” signal per each cell.

Figure 770. Number of cells counted by the Object Count node.

Figure 771. Sum Object Intensity for each spot (cell).

Figure 772. Resulting image.