Measure EGFP inside nuclei and surrounding rings

Every input uses the same measurement pipeline

The ratio compares a nucleus with its own local ring

The saved preview labels the physical ring width and its pixel equivalent. Rings exclude nuclear pixels and can be truncated by nearby objects or the image boundary.

Read the well assignments before interpreting the heatmaps

Download the saved well assignments. This layout image is generated from that CSV, not inferred from fluorescence. Special and blank wells remain explicitly labeled; their unspecified doses are not invented.

Controls appear in the well summaries, but only treatment wells with finite doses enter the dose-response table. The normalized fit panels use fitted bottom and top values, not positive-control normalization.

Interpret both maps with the plate assignments and saved well summary; color alone does not establish a treatment effect.

Fit the responding fraction, then inspect the normalized curve

The fit uses a bounded NumPy grid search for an increasing four-parameter Hill curve. The normalized panel subtracts the fitted bottom and divides by the fitted response range.

The estimates are specific to this dataset and segmentation settings, not general compound reference values. Too few finite positive-dose responses or constant responses leave the fit unavailable.

Inspect dose-response values · Inspect fit parameters and status

Keep analysis outputs separate from the published snapshot

cd /d "F:\Pixi_Projects\zen_czi\smartmicroscopy"
pixi run --locked -e default python "examples\translocation\translocation_assay.py" ^
  --input "data\transloc_gfp_dapi_0_2micron.czi" ^
  --plate-layout "data\plate_layout_example.xlsx" ^
  --output-dir "results\translocation\visual-example" ^
  --plot-backend seaborn

CMD uses ^ for continuation, with no trailing spaces. Select an unused output folder to retain earlier results. The command illustrates the CLI; it did not create the snapshot shown above.

Seaborn writes static PNGs. The CLI defaults to Plotly for interactive HTML. Omit --plate-layout only when the built-in assignments match the sample.

The field loop feeds well summaries and dose fits

flowchart TD
    A["main: parse CLI arguments"] -->|call| B["run_analysis: validate parameters and input"]
    B -->|load| C["Load Excel or built-in well assignments"]
    C -->|prepare| D["Load metadata, map fields to wells, check channels and XY scaling"]
    D -->|iterate| E{"Next mapped field?"}
    E -->|yes| F["Read or compute EGFP and DAPI planes"]
    F -->|validate| G["Require matching two-dimensional planes"]
    G -->|segment_nuclei| H["Threshold DAPI, clean foreground, split with watershed"]
    H -->|make_ring_labels| I["Assign nearby background to nuclei and remove label contacts"]
    I -->|measure_scene_objects| J["Measure EGFP nucleus and ring means"]
    J -->|calculate| K["Record nucleus/ring ratio or NaN if invalid"]
    K -->|continue| E
    E -->|no| L["summarize_wells: pool field measurements by well"]
    L -->|dose_response_table| M["Pool valid treatment-cell ratios by drug and dose"]
    M -->|fit_hill| N{"Finite positive-dose responses sufficient and varying?"}
    N -->|yes| O["Bounded grid search for increasing Hill curve"]
    N -->|no| P["Store unavailable fit status and reason"]
    O -->|collect| Q["Return AssayResults tables and preview"]
    P -->|collect| Q
    Q -->|write| R["Export field mapping and optional layout snapshot"]
    R -->|write_analysis_outputs| S["Save CSVs, plots, fit status, and analysis parameters"]

Editable Mermaid flowchart. Validation and read errors stop the analysis; they are not successful empty results.