Troubleshooting Low Cell Segmentation

The Teton^TM and Teton Atlas^TM cytoprofiling assays allow for on board segmentation using 3 Cell Paint markers: cell membrane, nucleus and actin. AVITI24^TM employs on-board segmentation models for the following cells: HeLa, HUVEC, PC-3, Hep-G2, MCF-7, HCT-116, Jurkat and SH-Sy5y. We recommend selecting the cell type that most closely matches your cell of interest, or you can select “Other” with a specified um diameter to apply a general segmentation model.

Once the run has completed, perform a visual inspection in CytoCanvas^TM to ensure the segmentation model has been properly applied to your samples. To do this, run Cells2Stats using the --visualization-only argument to first generate your visualization files. Then, review the visualization files in CytoCanvas. Visually inspect that you have clearly drawn boundaries around the nucleus and cellular membranes.

Poor segmentation could be a result of several factors which are listed below.

• Poor Cell Paint Intensities: Teton Reagent A and B are used to apply the cell membrane and nuclear membrane probes. If the intensities are low, you may want to adjust the time that the cells are in contact with Reagent A/B to adjust signal to desired level. We would recommend titrating using one of our onboard optimization kits, Teton Custom Antibody Screening Kit or the Teton Onboard Cell Paint Imaging Kit, or our off-instrument kit, Teton Cell Paint Probe Kit. With these kits, Reagent A and B are pre-mixed. The timing with the best signal with one of these optimization kits can be used for a Teton or Teton Atlas run. An example time course could be as follows –
  

  Example probe titration using a 12-well slide kit
  	1	2
  A	2 minutes	30 minutes
  B	5 minutes	20 minutes
  C	10 minutes	15 minutes
  D	15 minutes	10 minutes
  E	20 minutes	5 minutes
  F	30 minutes	2 minutes

• Overlapping Cells: Cells that overlap or grow in small colonies will be more difficult to segment. When seeding cells, ensure they form a single cell suspension. For some cell types, this may require additional washes and manual pipetting to fully dissociate the cells. Review our cell culture guidance to ensure that the cells are evenly distributed across the wells. Consider adjusting seeding densities or fixing at a lower confluency to ensure a monolayer distribution.
• Inappropriate Segmentation Model Used: The sample may be sufficiently different than the initial model selected on instrument resulting in poor segmentation. If this is the case, you can follow our Online Help tutorial Performing Resegmentation and Cell Assignment Using Python. This tutorial leverages CellPose, which is also utilized onboard the instrument for segmentation. We provide our pre-trained models in our cytoprofiling package on GitHub. CellPose also allows you to define your own model.
• High Background Intensity: Sufficient cell death or debris can lead to an increase of background intensity which could impair segmentation. Higher background intensity may be seen for certain surface types such as Matrigel and collagen. When working with these custom coatings, ensure the concentration and surface coating incubation time is correct. When passaging cells, ensure you are washing, centrifuging, and resuspending cells sufficiently to remove debris before seeding cells on the slide. This may require an additional wash and centrifugation step. Test treatments by running an IC50 to ensure the treatments are not overly aggressive and lead to excessive cell death.