Run Review Guide for Cytoprofiling

The following run review guide provides steps and guidance on how to determine your cytoprofiling run quality and troubleshoot Teton™ and Teton Atlas™ runs and associated metrics.

Run outputs are based on input quality, materials, and conditions. To diagnose issues that can appear, recommended guidance might direct you to pre-run steps. In some cases, you can also filter data to improve data quality. However, in other cases, such as with over-confluent cells, high-quality data recovery might not be feasible.

To limit variability in run output metrics, HeLa is the default context for this run review guide. Use the specified metric thresholds and HeLa example values as reference points to guide your post-run review.

Post-Run Review

1. After a cytoprofiling run completes, use one of the following methods to review the QC metrics before data analysis:
   • Execute Cells2Stats off instrument to generate the MultiQC HTML report.
   • Review the RunStats.json file that is generated when a run completes.
2. Generate visualization data with Cells2Stats.
3. Open the visualization data in CytoCanvas™. Then, visually inspect cell membrane and nuclear segmentation:
   • If segmentation is not as expected, use a different model to resegment.
   • To perform resgementation with a custom developed model, see Performing Resegmentation and Cell Assignment Using Python.
4. After you confirm segmentation, review the following metrics to evaluate run quality.
   • Each metric includes a definition, which run types that it applies to, metric locations within output files, and guidance on how to read the metric. Guidance includes threshold ranges, cell-type variation considerations, and troubleshooting tips if the metric is not within the ideal range.
   • The Metric Thresholds row indicates warnings and errors that do not currently display in ElemBio™ Cloud UI:
     • ⚠️ Warning: Metric is outside the ideal range. The run might still be usable with caution.
     • ❌ Error: Metric indicates a serious issue. Data is likely compromised and requires a re-run.

Run-Level Barcoding and Read Quality Metrics

Cell Assigned Counts per mm²

Field	Details
Definition	The number of assigned polony counts per mm² of segmented cell area. MultiQC reports K per mm²; RunStats.json reports cell assigned counts per mm² (MultiQC × 1000).
Run Applicability	Teton Teton Atlas
Metric Locations	MultiQC (Assigned Counts K/mm²) RunStats.json (AverageAssignedCountsPerMM2)
Metric Thresholds	⚠️ Warning < 100 k/mm² ⚠️ Warning > 500 k/mm²
Cell Type Variation	Small cells that have high expression might saturate polony concentrations.
Guidance	To check expected expression profiles, see The Cell Line Resource. Cells missing many targets might show lower density. If cell assigned counts are > 500 k/mm², select the small-cell recipe during instrument run setup for future runs with the same cell type to lower polony density.

Assigned Rate

Field	Details
Definition	Percentage of total polonies that are assigned to a cellular protein or RNA target, per well and per batch.
Run Applicability	Teton: RNA and protein Teton Atlas: Protein only
Metric Locations	MultiQC (Assigned Reads and Percent Assigned) RunStats.json (PercentAssignedReads)
Metric Thresholds	⚠️ Warning < 65% ❌ Error < 50%
Cell Type Variation	Small, over-confluent, or stacked cells can lower the assignment rate.
Guidance	Over-confluency might cause stacking, which lowers assignment rates. If < 65%, check confluency and whether Cell Assigned Counts per mm² is too high. Also, confirm that the correct Panel.json was used. Omitted barcodes are counted as Unassigned.

Mismatch Rate

Field	Details
Definition	Percentage of assigned reads that are not a perfect match, per well and per batch. Sequences with ≤ 2 mismatches are assigned. Due to large Hamming distance between barcodes, barcodes with one or two mismatches can still be assigned.
Run Applicability	Teton Teton Atlas
Metric Locations	MultiQC (Mismatches and Percent Mismatch) RunStats.json (PercentMismatch, per batch only)
Metric Thresholds	⚠️ Warning > 30% ❌ Error > 50%
Cell Type Variation	Over-confluency and stacking can elevate mismatches. High expression of abundant transcripts or proteins can cause crowding and increase mismatches.
Guidance	If > 30%, review Cell Assigned Counts per mm², Confluency, and confirm the correct cellular protein or RNA target use.

Extracellular Ratio

Field	Details
Definition	Percentage of assigned polonies located outside cell segmentation boundaries, per well.
Run Applicability	Teton Teton Atlas
Metric Locations	MultiQC (Extracellular Ratio) RunStats.json (ExtraCellularRatio)
Metric Thresholds	⚠️ Warning 25-30% ❌ Error > 50%
Cell Type Variation	Some cell types naturally shed more protein and RNA. Low confluency can increase background adhesion.
Guidance	If > 25%, overlay segmentation with the Cell Membrane filter in CytoCanvas to confirm alignment. If segmentation is poor, use an Element or custom model to resegment. For more information, see Resegmentation and Cell Assignment. If segmentation is acceptable, contact Element Technical Support. Cell lysate, expressed proteins, and debris can also contribute to extracellular polonies. Debris might appear as membrane-like clumps without nuclei. Unsupported or excessive surface coatings can also increase signal.

Segmentation and Cell Metrics

Cell Count

Field	Details
Definition	Total number of segmented cells, per well.
Run Applicability	Teton Teton Atlas
Metric Locations	MultiQC (Cell Count) RunStats.json (#Cells, CellCount)
Metric Thresholds	No fixed thresholds. Counts should align with expected seeding density and growth duration.
Cell Type Variation	Cell counts scale with seeding density and cell size.
Guidance	If cell counts are below expectations, confirm segmentation in CytoCanvas and check for peeling. Compare post-fixation Brightfield images to CytoCanvas. If peeling occurred, optimize fixation and surface conditions for future runs.

Confluency

Field	Details
Definition	Percentage of well area covered by cells.
Run Applicability	Teton Teton Atlas
Metric Locations	MultiQC (Confluency, Percent Confluency) RunStats.json (PercentConfluency)
Metric Thresholds	⚠️ Warning < 10% or > 70% (cell-line specific)
Cell Type Variation	50–70% is recommended for adherent cells, such as HeLa. 30–50% is recommended for neuronal cells. For more information on confluency recommendations, see the Teton CytoProfiling User Guide (MA-00053). For more information on supported cell types, see the Element Knowledge Base.
Guidance	Adjust seeding density and culture conditions to optimize confluency. Use Brightfield microscopy to count cells, assess viability, and confirm confluency. The Teton Cell Paint Probe Kit supports fluorescence inspection. The Teton Onboard Cell Paint Imaging Kit enables visualization with AVITI24. Avoid stacking and overgrowth. If < 10%, inspect for large areas with no counts. If > 70%, check for overlapping cells with poor segmentation.

Percent Nucleated Rate

Field	Details
Definition	Percentage of segmented cells with a segmented nucleus, per well.
Run Applicability	Teton Teton Atlas
Metric Locations	MultiQC (Percent Nucleated Cells, Nucleated Cells) RunStats.json (PercentNucleatedCells)
Metric Thresholds	⚠️ Warning < 90% ❌ Error < 75%
Cell Type Variation	Percent nucleated rate is often affected by improper cell paint handling.
Guidance	Confirm that cells have a nucleus as expected. If the percent nucleated rate is low, visually check the nuclear signal and look for non-nucleated segmented regions, which may indicate debris. To mitigate this issue in future experiments, use less stressful conditions to reduce lysis. For runs with this issue, filter out cells with nuclear area equal to 0 in RawCellStats.

Controls Metrics

Positive Control (GAPDH, HeLa)

Field	Details
Definition	Detection of intracellular GAPDH probes, per segmented cell area per well per batch. The GAPDH RNA probe concentration is 40x lower than the NSB negative-control probe concentration. MultiQC reports K per mm²; RunStats.json reports counts (MultiQC x 1000).
Run Applicability	Teton
Metric Locations	MultiQC/Barcoding Control Targets (Positive Control 1) RunStats.json (ControlTargets)
Metric Thresholds	⚠️ Warning < 2 k/mm² (MultiQC values)
Cell Type Variation	Expressions vary by tissue and cell type.
Guidance	Compare expected expression relative to HeLa. Cells with low GAPDH expression might drop below threshold. If counts are low, check RNase inhibitor use and the flow cell storage interval, since RNA degradation reduces signal. To compare GAPDH and NSB counts, multiply GAPDH counts by 40 to account for probe concentration differences.

Non-Specific Binding (NSB, HeLa)

Field	Details
Definition	Background binding density per mm², measured with a non-targeting probe, and calculated per batch per well. Each batch per well includes four probes. MultiQC reports K per mm²; RunStats.json reports counts (MultiQC × 1000).
Run Applicability	Teton
Metric Locations	MultiQC/Barcoding Control Targets (Negative Control 1–4) RunStats.json (ControlTargets)
Metric Thresholds	⚠️ Warning > 4 k/mm² ❌ Error > 10 k/mm² (MultiQC values)
Cell Type Variation	NSB can increase with stacking and overgrowth.
Guidance	Calculate the mean NSB count and compare to the threshold. If NSB is high, review run-level barcoding and read quality metrics, segmentation and cell metrics, and controls metrics. To evaluate surface chemistry issues, check whether the Extracellular Ratio is high or if other indicators of poor surface chemistry are present, such as peeling.