When you click on a specific plate in the ScreenAssist, you see a detailed breakdown of that plate’s performance. This view contains several plots and summary panels not visible in the Overview. Together they allow you to diagnose plate-level issues at the well, row, column, and scan level.

Key Plate Assay Metrics Panel

What it shows: A summary card panel at the top of the plate view displaying the most important quality metrics for that individual plate, each with a pass/marginal/fail status indicator. Metrics displayed include: 

• Spectral Shift Ratio Z-Prime and Robust Z-Prime 

• 650 nm Fluorescence CV of Reference and Control 

• Poor Reads (% well scans above fail threshold) 

• Neutral RSDE 

• Titration Response Amplitude (Δ Ratio) 

• Titration Signal-to-Noise (S/N)
   

What to look for: Any metric flagged as Marginal (amber) or Fail (red) should be investigated using the plots below. The panel provides an at-a-glance summary; the plots explain why a metric has that status.
 

Tip: A plate can fail on one metric while passing all others. Always review which specific metric is failing before deciding whether to reject or re-screen a plate.

Plot 1: Spectral Shift Ratio Plate Heatmap

What it shows: A spatial map of the entire plate where each well is colored according to its Spectral Shift ratio value. The plate layout (rows A–P, columns 1–48 for a 384-well or 1536-well plate) is shown with a color scale from low ratio (typically lighter/yellow) to high ratio (darker/blue). Control wells and reference wells are visually distinguishable by their position and color.

What to look for:

• The reference wells (neutral control) should all display a consistent, uniform color across their positions, any outlier wells that deviate strongly in color indicate a problem well.

• The positive control wells should display a consistently different (typically darker/higher ratio) color to the reference wells, confirming the assay signal window is present.

• Compound wells will show a range of colors; the majority should match the reference color (non-binders), with a small proportion shifted toward the control color (putative binders/hits).

• Spatial patterns to watch for:   

  • Edge effects: wells at the plate perimeter showing systematically different ratios to interior wells.   

  • Row or column stripes: a full row or column with a different color, indicating a dispensing or incubation artefact.   

  • Isolated dark spots: individual wells with extreme ratio values, typically autofluorescent or quenching compounds.

Common patterns and causes:

Plot 2: 650nm Fluorescence Plate Heatmap

Plot 3: Reference/Control Separation Plot

What it shows: A scatter plot of the Spectral Shift ratio for every well on the plate, plotted against well index (x-axis). Reference wells and positive control wells are highlighted. Horizontal dashed lines indicate the the ± 3σ and ±3 RSDE from the mean or median of the Spectral Shift ratio of the reference and control populations, visually representing the assay window.

What to look for: 

• The reference wells should cluster tightly and show low scatter indicates a tight, reproducible reference population (low RSDE). 

• The control wells should cluster consistently above the reference line, with the gap between the two median lines representing the assay signal window. 

• Outlier reference or control wells (dots far from their respective cluster) are clearly visible here and can be identified by their well index for follow-up investigation. 

• The width of each cluster (vertical spread) corresponds directly to the standard deviation of that population: narrow clusters = high Z’; wide or overlapping clusters = low Z’. 

• A consistent gap between the two dashed lines across all well indices confirms a uniform assay window across the full plate. If the gap narrows toward the edges (smiles or frowns present), this may indicate edge effects.

Common patterns and causes:

Plot 4: Well Scans Overview

What it shows: A scatter plot of the median well scan score for each well across the plate (well index on x-axis, median scan score on y-axis). Wells are color-coded by population: Reference, Control, Ligand/compound. A horizontal threshold line indicates the minimum acceptable scan quality score.

What to look for: 

• The majority of wells should have scan scores above the threshold line. 

• Wells above the threshold are counted toward the Poor Reads metric. 

• The control and reference wells should show consistently low scan scores. High scan scores on control wells indicate a problem with those specific wells (not the compounds), which will likely degrade RZ’ and SSMD. 

• A band of low-scoring wells at a specific well index range (e.g., a particular column) points to a dispensing or plate issue in that region.

Plot 5: Well Scans (Individual Scan Profiles)

What it shows: A plot of the raw fluorescence scan profiles (fluorescence intensity vs. position along the capillary in mm) after selection of individual wells in plot 4. Curves are shown for 650 nm and 670 nm fluorescence emission channels. Each curve represents one well scan, a good scan produces a smooth, symmetrical bell-shaped curve; a poor scan shows irregularities.

What to look for:

• Healthy scans produce smooth, well-defined, symmetrical flat plateaus centered in the capillary. The 650 nm and 670 nm curves should be closely matched in shape.

• Poor scans appear as flat lines (no signal = empty wells), asymmetric or jagged peaks (particulates or bubbles in the wells), or double peaks (two distinct populations of fluorophore, often indicating aggregation).

• Overlapping 650 nm and 670 nm curves that track together confirm that the dye is behaving normally. A large separation in peak height between the two channels on specific wells indicates an anomalous Spectral Shift ratio for those wells.

• This plot is most useful for diagnosing the root cause of poor reads or outlier wells identified in the Well Scans Overview.

Common scan shapes and causes:

Plot 6: Median Spectral Shift Ratio Per Columns

What it shows: A line chart of the median Spectral Shift ratio plotted against plate column number (x-axis). The median Spectral Shift ratio is shown for the Low control (reference), if present Medium control, and High control (positive control) populations per row, with error bars.

What to look for:

• Both lines should be flat and horizontal across all rows. A gradient from top to bottom (rows A to P, or 1–32) indicates a row-dependent spatial effect, such as a dispense gradient across the plate during incubation.

• Together with the Median Spectral Shift Ratio Along Rows plot (Section 6.7), this pair of plots is the primary diagnostic for spatial artefacts and edge effects on a plate. If both show flat lines, the assay is spatially uniform. If either shows a trend, further normalization (e.g., B-score) may be required.

Plot 7: Median Spectral Shift Ratio Per Rows

What it shows: The same concept as Section 6.7, but plotted against plate row number (x-axis). It is shown separately for the Low control (reference), if present Medium control and High control (positive control) populations. Error bars indicate the spread within each column’s control wells.

What to look for: 

• Both lines should be flat and horizontal across all columns. Any systematic trend (e.g., a gradient from left to right) indicates a column-dependent spatial effect on the assay signal. 

• The gap between the two or three lines (high control vs. medium control vs. low control) should remain constant across columns. A narrowing gap on specific columns indicates a locally reduced assay window. 

• Columns at the plate edges showing deviation from the central columns are a classic edge effect signature.

Plot 8: Positive Control Titration Curve

What it shows: The dose-response (titration) curve for the up to two positive control interactions measured on this specific plate. The x-axis shows ligand concentration (M, log scale); the y-axis shows the Spectral Shift ratio. Individual data points (dots) represent replicate measurements at each concentration. The fitted K_d curve (solid line) and EC₅₀ fit (dashed line) are overlaid.

What to look for: 

• The data points should follow a smooth, sigmoidal dose-response curve moving from the unbound Spectral Shift ratio (at low ligand concentration) to a plateau at the fully bound ratio (at saturating ligand concentration). - The plateau at high concentration (the “bound” state) should be clearly distinct from the baseline at low concentration (the “unbound” state). The difference between these plateaus is the Response Amplitude (Δ Ratio), the pass threshold is ≥ 0.018. 

• Tight replicates (small vertical scatter of dots at each concentration) indicate high assay precision and contribute to a reliable K_d/EC₅₀ fit. - Outlier points far from the fitted curve indicate problematic wells at those concentrations (e.g., compound precipitation at high concentrations, pipetting error, or autofluorescence). 

• The S/N ratio of the fit (reported in the Key Metrics panel) reflects how cleanly the curve rises above the noise level, a pass requires S/N ≥ 5, excellent is ≥ 12.
   

Common patterns and causes:

Acceptance thresholds:

Plate Assay Metrics Summary Panels

What it shows: A structured summary panel at the bottom of the plate view, reporting the key numerical metrics for the plate split into three sections: Assay (Z-prime, Robust Z-prime, SSMD), Reference (650 nm fluorescence CV, mean Spectral Shift ratio, Spectral Shift ratio standard deviation, median Spectral Shift ratio, Spectral Shift ratio RSDE), and Control (650 nm fluorescence CV, mean Spectral Shift ratio, Spectral Shift ratio standard deviation, median Spectral Shift ratio, Spectral Shift ratio RSDE).

What to look for:

• Use this table to record and archive the exact numerical values for each plate for your validation report.

• Cross-reference the Reference RSDE here with the Neutral RSDE acceptance criteria (≤ 0.008 = pass).

• Compare the mean vs. median Spectral Shift ratio for both reference and control populations. If they differ substantially, this confirms outlier influence (robust metrics preferred in this case).

• The Spectral Shift ratio standard deviation for reference and control directly feeds into the classical Z’ calculation, elevated values here will depress Z’

   

Interpreting the Individual Plate View: A Suggested Workflow

When reviewing a plate that has been flagged with a marginal or failing metric, follow this sequence:

1. Start with the Key Plate Assay Metrics panel: identify which metric(s) are failing.

2. Check the Spectral Shift Ratio Plate Heatmap:  look for spatial patterns (edge effects, column/row artefacts, isolated outliers).

3. Check the 650 nm Fluorescence Plate Heatmap: assess whether the raw fluorescence is consistent across wells, and look for spatial patterns to identify technical artifacts that may confound the ratio-based readout.

4. Check the Reference/Control Separation plot: confirm whether the assay window is present and identify any outlier control or reference wells by well index.

5. Check Well Scans Overview and Well Scans: for any flagged wells, inspect the raw scan profile to understand the root cause (bubble, precipitate, blockage, etc.).

6. Check Median Spectral Shift Ratio Along Rows and Columns: confirm or rule out spatial/edge effects.

7. Check the Titration Curve: assess response amplitude and S/N; look for hook effects or poor replicate precision.

8. Review the Plate Assay Metrics Summary: record exact values and compare mean vs. median to assess outlier impact.