ABB: Add Channels 4i

Add Channels 4i is a customized application-specific building block (ABB) for the commercial Harmony software (Revvity Inc.) that is used on an Opera Phenix High-Content Screening System (Revvity Inc.).

This ABB has been developed by Achim Kirsch (Revvity Inc.) for the image analysis procedure of the Cell Painting PLUS (CPP) method described in von Coburg et al, Nat. Commun., 2025:

"Cell Painting PLUS: Expanding the multiplexing capacity of Cell Painting-based phenotypic profiling using iterative staining-elution cycles"

https://doi.org/10.1038/s41467-025-58765-8

In this article, the Add Channels 4i ABB (v1.0.4) has been used to overlay and align the individual fluorescence images that were acquired using an Opera Phenix from the two consecutive CPP staining cycles 1 and 2 into a single multi-channel stack for subsequent extraction of phenotypic feature data of single cells using the Harmony software (v4.8).

Application of the Add Channels 4i ABB:

In general, this ABB works on image data sets, which contain repeated measurements of the same sample. Typically, this is used with re-staining experiments (e.g., 4i method for antibody staining, CPP method for dye staining), where a first staining is washed away, and a different staining is recorded. This enables experiments with more different stainings with a given number of channels. Typically, such measurements are created with the "Combine Measurement" function in Harmony. For this it is important that all channel settings are identical (focus height might be changed). On an Opera Phenix please also ensure, that the "Channel Sequence" is also identical. The ABB will correlate one image from timepoint zero with one image of a different timepoint, in order to determine the xy-shift between the two measurements. It will then correct additional channels so that structures in the added images correctly overlap the structures in the image from timepoint zero. In addition, the ABB will also return a region, that can be used as Region-of-Interest (ROI) for further analyses in Harmony. This NewROI marks the area of the image, where all channels from all added timepoints overlap. If you do an Evaluation of a plate, only data from timepoint zero will have meaningful information, but you will have to select all needed timepoints for the analysis. The names of the additional image channels can be defined. "Output Image A" will contain the image from "Channel for Matching".

Semantic versioning of the Add Channels 4i ABB:

Version x.y.z

- x => Major change => architectural change maintaining applicability for new Harmony release with largely new infrastructure

- y => Minor change => improvements or additional functionality

- z => Revision => bug fix / patch

Available Add Channels 4i ABB versions:

- v1.y.z => for Harmony version 4.8 and 4.9

- v2.y.z => for Harmony version 5.2

Revisions and releases of the Add Channels 4i ABB:

2023-07-04

- Release of v1.0.4

=> use for Harmony version 4.8 and 4.9

=> used in von Coburg et al, Nat. Commun., 2025 (https://doi.org/10.1038/s41467-025-58765-8)

2024-07-31

- Added: MaxShift as an input. This is needed in case a large shift occurs.

- New: Handling of situations, where some time points show AF errors. Thus, there is no data for those timepoints and the system returns black images with no final overlap area.

- Added: ShiftX, ShiftY and Area as properties for the ROI.

- Fixed: when the ABB was called multiple times, it shifted the overlap region every time, so that the region ended up wrong. Now, for each shift it makes a mask that is shifted and then applied to the Overlap region. Thus, the ROI can be any shape.

2024-08-12

- Fixed: in case alignment fails, it now returns empty ROI and blank images

- Fixed: adding missing property values when alignment could not be done.

2024-08-12

- Release of v2.0.5

=> use for Harmony version 5.2

2024-08-13

- Release of v1.0.7

=> use for Harmony version 4.8 and 4.9