{ "schemaVersion": 2, "meta": { "name": "Cell uptake 3 channels coloc", "shortDescription": "Uptake assay with two spot channels and brightfield cell.", "description": "Uptake assay with two spot channels and brightfield cell.", "authors": [ "Melanie Schuerz" ], "authorOrganization": "University of Salzburg", "creationTime": "2026-06-11T00:00:00Z", "category": "Colocalization", "tags": [ "assay", "uptake-assay", "colocalization", "brightfield", "3-channel" ] }, "classification": { "classes": [ { "id": { "VALID": 0 }, "color": "#97978f", "name": "Background", "notes": "" }, { "id": { "VALID": 4 }, "color": "#3399FF", "name": "dapi@nucleus", "notes": "", "measure": {} }, { "id": { "VALID": 11 }, "color": "#33FF99", "name": "brightfield@cell-area", "notes": "", "measure": { "areaSize": [ "AVG", "SUM" ] } }, { "id": { "VALID": 12 }, "color": "#66FF33", "name": "brightfield@cells", "notes": "", "measure": { "areaSize": [ "AVG", "SUM" ] } }, { "id": { "VALID": 21 }, "color": "#33CFFF", "name": "ch1@spot", "notes": "", "measure": { "areaSize": [ "AVG" ], "intensitySum": [ "AVG" ], "intensityAvg": [ "AVG" ] } }, { "id": { "VALID": 1 }, "color": "#D2FF00", "name": "ch2@spot", "notes": "", "measure": { "areaSize": [ "AVG" ], "intensitySum": [ "AVG" ], "intensityAvg": [ "AVG" ] } }, { "id": { "VALID": 2 }, "color": "#FF3366", "name": "ch3@spot", "notes": "", "measure": { "areaSize": [ "AVG" ], "intensitySum": [ "AVG" ], "intensityAvg": [ "AVG" ] } }, { "id": { "VALID": 13 }, "color": "#33CFFF", "name": "ch1@in-cell-spots", "notes": "", "measure": { "areaSize": [ "AVG" ], "intensitySum": [ "AVG" ], "intensityAvg": [ "AVG" ] } }, { "id": { "VALID": 3 }, "color": "#33CFFF", "name": "ch2@in-cell-spots", "notes": "", "measure": { "areaSize": [ "AVG" ], "intensitySum": [ "AVG" ], "intensityAvg": [ "AVG" ] } }, { "id": { "VALID": 5 }, "color": "#33CFFF", "name": "ch3@in-cell-spots", "notes": "", "measure": { "areaSize": [ "AVG" ], "intensitySum": [ "AVG" ], "intensityAvg": [ "AVG" ] } }, { "id": { "VALID": 6 }, "color": "#9933FF", "name": "ch1@ in-cell-spots coloc ch2", "notes": "", "measure": { "areaSize": [ "AVG" ], "intensitySum": [ "AVG" ], "intensityAvg": [ "AVG" ] } }, { "id": { "VALID": 14 }, "color": "#9933FF", "name": "ch1@ in-cell-spots coloc ch3", "notes": "", "measure": { "areaSize": [ "AVG" ], "intensitySum": [ "AVG" ], "intensityAvg": [ "AVG" ] } }, { "id": { "VALID": 7 }, "color": "#9933FF", "name": "ch1@ in-cell-spots coloc ch2and3", "notes": "", "measure": { "areaSize": [ "AVG" ], "intensitySum": [ "AVG" ], "intensityAvg": [ "AVG" ] } }, { "id": { "VALID": 8 }, "color": "#9933FF", "name": "ch2@ in-cell-spots coloc ch1", "notes": "", "measure": { "areaSize": [ "AVG" ], "intensitySum": [ "AVG" ], "intensityAvg": [ "AVG" ] } }, { "id": { "VALID": 9 }, "color": "#9933FF", "name": "ch2@ in-cell-spots coloc ch3", "notes": "", "measure": { "areaSize": [ "AVG" ], "intensitySum": [ "AVG" ], "intensityAvg": [ "AVG" ] } }, { "id": { "VALID": 10 }, "color": "#9933FF", "name": "ch2@ in-cell-spots coloc ch1and3", "notes": "", "measure": { "areaSize": [ "AVG" ], "intensitySum": [ "AVG" ], "intensityAvg": [ "AVG" ] } }, { "id": { "VALID": 15 }, "color": "#9933FF", "name": "ch3@ in-cell-spots coloc ch1", "notes": "", "measure": { "areaSize": [ "AVG" ], "intensitySum": [ "AVG" ], "intensityAvg": [ "AVG" ] } }, { "id": { "VALID": 16 }, "color": "#9933FF", "name": "ch3@ in-cell-spots coloc ch2", "notes": "", "measure": { "areaSize": [ "AVG" ], "intensitySum": [ "AVG" ], "intensityAvg": [ "AVG" ] } }, { "id": { "VALID": 17 }, "color": "#9933FF", "name": "ch3@ in-cell-spots coloc ch1and2", "notes": "", "measure": { "areaSize": [ "AVG" ], "intensitySum": [ "AVG" ], "intensityAvg": [ "AVG" ] } }, { "id": { "VALID": 18 }, "color": "#9933FF", "name": "ch1@ in-cell-spots non coloc", "notes": "", "measure": { "areaSize": [ "AVG" ], "intensitySum": [ "AVG" ], "intensityAvg": [ "AVG" ] } }, { "id": { "VALID": 19 }, "color": "#9933FF", "name": "ch2@ in-cell-spots non coloc", "notes": "", "measure": { "areaSize": [ "AVG" ], "intensitySum": [ "AVG" ], "intensityAvg": [ "AVG" ] } }, { "id": { "VALID": 20 }, "color": "#9933FF", "name": "ch3@ in-cell-spots non coloc", "notes": "", "measure": { "areaSize": [ "AVG" ], "intensitySum": [ "AVG" ], "intensityAvg": [ "AVG" ] } } ] }, "plate": { "groupingMode": "NO_GROUPING", "groupingRegex": "", "plateCols": 1, "plateRows": 1, "wellCols": 4, "wellRows": 4, "wellImageOrder": [ 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 ] }, "pipelines": [ { "id": 1, "name": "Nuclei", "description": "1. Set image nucleus channel\n2. Adapt threshold. Recommanded autoTH: Triangle, Moments or Otsu\n3. Define min particle size in \"Classifier\" to filter out small aggregates not connected to Nuclei.", "imageSource": { "channel": 0 }, "enabled": true, "steps": [ { "enabled": true, "command": { "type": "ROLLING_BALL", "radius": 4.0, "ballType": "PARABOLOID", "preSmooth": true } }, { "enabled": true, "command": { "type": "BLUR", "kernelSize": 3 } }, { "enabled": true, "command": { "type": "BLUR", "kernelSize": 3 } }, { "enabled": true, "command": { "type": "THRESHOLD", "thresholds": [ { "method": { "type": "TRIANGLE" }, "minThreshold": 200.0, "maxThreshold": 65535.0, "unit": "BIT", "objectClassId": 4 } ] } }, { "enabled": true, "command": { "type": "WATERSHED", "maximumFinderTolerance": 0.5, "smoothingSigma": 0.0, "minObjectSize": 0 } }, { "enabled": true, "command": { "type": "CONNECTED_COMPONENTS" } }, { "enabled": true, "command": { "type": "EXTRACT_OBJECTS", "maxObjectsBeforeFail": 100000 } }, { "enabled": true, "command": { "type": "CLASSIFY_OBJECTS", "inputClasses": [ { "VALID": 4 } ], "matchHandling": "REMOVE_ALL_CLASSES_IF_NOT_MATCH", "originSegmentation": [], "outputClass": "UNSET", "overlappingWith": "UNSET", "minIntersectionArea": 0.0, "sizeUnit": "px", "minArea": 1000.0, "maxArea": 2147483648.0, "minCircularity": 0.0, "maxCircularity": 1.0, "minSolidity": 0.0, "maxSolidity": 1.0, "minAspectRatio": 0.0, "maxAspectRatio": 2147483648, "minEccentricity": 0.0, "maxEccentricity": 1.0, "minFeret": 0.0, "maxFeret": 2147483648, "allowEdgeTouching": true } } ] }, { "id": 2, "name": "Cell area", "description": "Detects the whole brightfield cell area.\n1. Define Brightfield image channel\n2. Adjust TH if needed\n3. Define min particle size in \"Classifier\" to filter out small aggregates.", "imageSource": { "channel": 1 }, "enabled": true, "steps": [ { "enabled": true, "command": { "type": "EDGE_DETECTION_SOBEL", "kernelSize": 3 } }, { "enabled": true, "command": { "type": "BLUR", "kernelSize": 3 } }, { "enabled": true, "command": { "type": "BLUR", "kernelSize": 3 } }, { "enabled": true, "command": { "type": "BLUR", "kernelSize": 3 } }, { "enabled": true, "command": { "type": "BLUR", "kernelSize": 3 } }, { "enabled": true, "command": { "type": "BLUR", "kernelSize": 3 } }, { "enabled": true, "command": { "type": "BLUR", "kernelSize": 3 } }, { "enabled": true, "command": { "type": "THRESHOLD", "thresholds": [ { "method": { "type": "MIN_ERROR" }, "minThreshold": 0.0, "maxThreshold": 65535.0, "unit": "BIT", "objectClassId": 11 } ] } }, { "enabled": true, "command": { "type": "CONNECTED_COMPONENTS" } }, { "enabled": true, "command": { "type": "EXTRACT_OBJECTS", "maxObjectsBeforeFail": 100000 } }, { "enabled": true, "command": { "type": "CLASSIFY_OBJECTS", "inputClasses": [ { "VALID": 11 } ], "matchHandling": "REMOVE_ALL_CLASSES_IF_NOT_MATCH", "originSegmentation": [], "outputClass": "UNSET", "overlappingWith": "UNSET", "minIntersectionArea": 0.0, "sizeUnit": "px", "minArea": 2000.0, "maxArea": 2147483648.0, "minCircularity": 0.0, "maxCircularity": 1.0, "minSolidity": 0.0, "maxSolidity": 1.0, "minAspectRatio": 0.0, "maxAspectRatio": 2147483648, "minEccentricity": 0.0, "maxEccentricity": 1.0, "minFeret": 0.0, "maxFeret": 2147483648, "allowEdgeTouching": true } } ] }, { "id": 3, "name": "Cells", "description": "Based on the detected nuclei a Voronoi algorithm is used to separete individual cells. The detected cell area is used to find the border of the cells\n1. Measure intensity per cell in your required channels (default Ch0,1 and 2 is measured)", "imageSource": "SCRATCHPAD", "enabled": true, "steps": [ { "enabled": true, "command": { "type": "VORONOI", "centers": { "VALID": 4 }, "centerFilterClasses": [], "mask": { "VALID": 11 }, "maskFilterClasses": [], "outputClass": { "VALID": 12 }, "unit": "PIXELS", "maxRadius": 0.0, "excludeAreasAtTheEdges": false, "excludeAreasWithNoCenter": true } } ] }, { "id": 4, "name": "spot ch1", "description": "1. Select EV image channel\n2. Adapt manual threshold\n\n Approximation notes: $reclassify was approximated via Colocalization", "imageSource": { "channel": 2 }, "enabled": true, "steps": [ { "enabled": true, "command": { "type": "MEDIAN_SUBTRACT", "radius": 3.0 } }, { "enabled": true, "command": { "type": "BLUR", "kernelSize": 3 } }, { "enabled": true, "command": { "type": "BLUR", "kernelSize": 3 } }, { "enabled": true, "command": { "type": "THRESHOLD", "thresholds": [ { "method": { "type": "MANUAL" }, "minThreshold": 200.0, "maxThreshold": 65535.0, "unit": "BIT", "objectClassId": 21 } ] } }, { "enabled": true, "command": { "type": "WATERSHED", "maximumFinderTolerance": 0.5, "smoothingSigma": 0.0, "minObjectSize": 0 } }, { "enabled": true, "command": { "type": "CONNECTED_COMPONENTS" } }, { "enabled": true, "command": { "type": "EXTRACT_OBJECTS", "maxObjectsBeforeFail": 100000 } }, { "enabled": true, "command": { "type": "CLASSIFY_OBJECTS", "originSegmentation": [], "inputClasses": [ { "VALID": 21 } ], "matchHandling": "REMOVE_ALL_CLASSES_IF_NOT_MATCH", "outputClass": "UNSET", "overlappingWith": "UNSET", "minIntersectionArea": 0.0, "sizeUnit": "px", "minArea": 5.0, "maxArea": 2147483648.0, "minCircularity": 0.0, "maxCircularity": 1.0, "minSolidity": 0.0, "maxSolidity": 1.0, "minAspectRatio": 0.0, "maxAspectRatio": 2147483648, "minEccentricity": 0.0, "maxEccentricity": 1.0, "minFeret": 0.0, "maxFeret": 2147483648, "allowEdgeTouching": true } }, { "enabled": true, "command": { "type": "COLOCALIZATION", "classesToColoc": [ { "VALID": 21 }, { "VALID": 12 } ], "filterClasses": [], "classForOverlappingAreas": { "VALID": 13 }, "multiplicity": "MANY_TO_MANY", "sizeUnit": "px", "minColocArea": 0.0 } } ] }, { "id": 5, "name": "spot ch2", "description": "1. Select EV image channel\n2. Adapt manual threshold\n\n Approximation notes: $reclassify was approximated via Colocalization", "imageSource": { "channel": 3 }, "enabled": true, "steps": [ { "enabled": true, "command": { "type": "MEDIAN_SUBTRACT", "radius": 3.0 } }, { "enabled": true, "command": { "type": "BLUR", "kernelSize": 3 } }, { "enabled": true, "command": { "type": "BLUR", "kernelSize": 3 } }, { "enabled": true, "command": { "type": "THRESHOLD", "thresholds": [ { "method": { "type": "MANUAL" }, "minThreshold": 200.0, "maxThreshold": 65535.0, "unit": "BIT", "objectClassId": 1 } ] } }, { "enabled": true, "command": { "type": "WATERSHED", "maximumFinderTolerance": 0.5, "smoothingSigma": 0.0, "minObjectSize": 0 } }, { "enabled": true, "command": { "type": "CONNECTED_COMPONENTS" } }, { "enabled": true, "command": { "type": "EXTRACT_OBJECTS", "maxObjectsBeforeFail": 100000 } }, { "enabled": true, "command": { "type": "CLASSIFY_OBJECTS", "originSegmentation": [], "inputClasses": [ { "VALID": 1 } ], "matchHandling": "REMOVE_INPUT_CLASS_IF_NOT_MATCH", "outputClass": "UNSET", "overlappingWith": "UNSET", "minIntersectionArea": 0.0, "sizeUnit": "px", "minArea": 5.0, "maxArea": 2147483648.0, "minCircularity": 0.0, "maxCircularity": 1.0, "minSolidity": 0.0, "maxSolidity": 1.0, "minAspectRatio": 0.0, "maxAspectRatio": 2147483648, "minEccentricity": 0.0, "maxEccentricity": 1.0, "minFeret": 0.0, "maxFeret": 2147483648, "allowEdgeTouching": true } }, { "enabled": true, "command": { "type": "COLOCALIZATION", "classesToColoc": [ { "VALID": 1 }, { "VALID": 12 } ], "filterClasses": [], "classForOverlappingAreas": { "VALID": 3 }, "multiplicity": "MANY_TO_MANY", "sizeUnit": "px", "minColocArea": 0.0 } } ] }, { "id": 6, "name": "spot ch3", "description": "1. Select EV image channel\n2. Adapt manual threshold\n\n Approximation notes: $reclassify was approximated via Colocalization", "imageSource": { "channel": 4 }, "enabled": true, "steps": [ { "enabled": true, "command": { "type": "MEDIAN_SUBTRACT", "radius": 3.0 } }, { "enabled": true, "command": { "type": "BLUR", "kernelSize": 3 } }, { "enabled": true, "command": { "type": "BLUR", "kernelSize": 3 } }, { "enabled": true, "command": { "type": "THRESHOLD", "thresholds": [ { "method": { "type": "MANUAL" }, "minThreshold": 200.0, "maxThreshold": 65535.0, "unit": "BIT", "objectClassId": 2 } ] } }, { "enabled": true, "command": { "type": "WATERSHED", "maximumFinderTolerance": 0.5, "smoothingSigma": 0.0, "minObjectSize": 0 } }, { "enabled": true, "command": { "type": "CONNECTED_COMPONENTS" } }, { "enabled": true, "command": { "type": "EXTRACT_OBJECTS", "maxObjectsBeforeFail": 100000 } }, { "enabled": true, "command": { "type": "CLASSIFY_OBJECTS", "originSegmentation": [], "inputClasses": [ { "VALID": 2 } ], "matchHandling": "REMOVE_ALL_CLASSES_IF_NOT_MATCH", "outputClass": "UNSET", "overlappingWith": "UNSET", "minIntersectionArea": 0.0, "sizeUnit": "px", "minArea": 5.0, "maxArea": 2147483648.0, "minCircularity": 0.0, "maxCircularity": 1.0, "minSolidity": 0.0, "maxSolidity": 1.0, "minAspectRatio": 0.0, "maxAspectRatio": 2147483648, "minEccentricity": 0.0, "maxEccentricity": 1.0, "minFeret": 0.0, "maxFeret": 2147483648, "allowEdgeTouching": true } }, { "enabled": true, "command": { "type": "COLOCALIZATION", "classesToColoc": [ { "VALID": 2 }, { "VALID": 12 } ], "filterClasses": [], "classForOverlappingAreas": { "VALID": 5 }, "multiplicity": "MANY_TO_MANY", "sizeUnit": "px", "minColocArea": 0.0 } } ] }, { "id": 7, "name": "Coloc", "description": "Colocalisation is set to 0.1 (0.1-1) as default and may be changed.\n\nWith these settings you will find the following groups:\n- @spots: contains spots outside the cells\n- @in-cell-spots: includes all spots that are within the cell(coloc and non-coloc spots)\n- @ in-cell-spot non coloc: spots only detected in the one respective channel\n- 2 channel coloc spots (e.g. ch1@in-cell-spots coloc ch2) includes all spots in cell that have channel 1 and 2 and also includes spots that might have ch3\n- 3 channel coloc spots: in-cell-spots recognized in ch1, 2 and 3\n Approximation notes: $colocalization N-way matrix approximated as pairwise Colocalization steps (each non-Undefined entry paired with the first other non-Undefined entry) $colocalization N-way matrix approximated as pairwise Colocalization steps (each non-Undefined entry paired with the first other non-Undefined entry) $colocalization N-way matrix approximated as pairwise Colocalization steps (each non-Undefined entry paired with the first other non-Undefined entry) $colocalization N-way matrix approximated as pairwise Colocalization steps (each non-Undefined entry paired with the first other non-Undefined entry)", "imageSource": "SCRATCHPAD", "enabled": true, "steps": [ { "enabled": true, "command": { "type": "COLOCALIZATION", "classesToColoc": [ { "VALID": 13 }, { "VALID": 3 } ], "filterClasses": [], "classForOverlappingAreas": { "VALID": 6 }, "multiplicity": "MANY_TO_MANY", "sizeUnit": "px", "minColocArea": 0.0 } }, { "enabled": true, "command": { "type": "COLOCALIZATION", "classesToColoc": [ { "VALID": 3 }, { "VALID": 13 } ], "filterClasses": [], "classForOverlappingAreas": { "VALID": 8 }, "multiplicity": "MANY_TO_MANY", "sizeUnit": "px", "minColocArea": 0.0 } }, { "enabled": true, "command": { "type": "COLOCALIZATION", "classesToColoc": [ { "VALID": 13 }, { "VALID": 5 } ], "filterClasses": [], "classForOverlappingAreas": { "VALID": 14 }, "multiplicity": "MANY_TO_MANY", "sizeUnit": "px", "minColocArea": 0.0 } }, { "enabled": true, "command": { "type": "COLOCALIZATION", "classesToColoc": [ { "VALID": 5 }, { "VALID": 13 } ], "filterClasses": [], "classForOverlappingAreas": { "VALID": 15 }, "multiplicity": "MANY_TO_MANY", "sizeUnit": "px", "minColocArea": 0.0 } }, { "enabled": true, "command": { "type": "COLOCALIZATION", "classesToColoc": [ { "VALID": 3 }, { "VALID": 5 } ], "filterClasses": [], "classForOverlappingAreas": { "VALID": 9 }, "multiplicity": "MANY_TO_MANY", "sizeUnit": "px", "minColocArea": 0.0 } }, { "enabled": true, "command": { "type": "COLOCALIZATION", "classesToColoc": [ { "VALID": 5 }, { "VALID": 3 } ], "filterClasses": [], "classForOverlappingAreas": { "VALID": 16 }, "multiplicity": "MANY_TO_MANY", "sizeUnit": "px", "minColocArea": 0.0 } }, { "enabled": true, "command": { "type": "COLOCALIZATION", "classesToColoc": [ { "VALID": 13 }, { "VALID": 3 } ], "filterClasses": [], "classForOverlappingAreas": { "VALID": 7 }, "multiplicity": "MANY_TO_MANY", "sizeUnit": "px", "minColocArea": 0.0 } }, { "enabled": true, "command": { "type": "COLOCALIZATION", "classesToColoc": [ { "VALID": 3 }, { "VALID": 13 } ], "filterClasses": [], "classForOverlappingAreas": { "VALID": 10 }, "multiplicity": "MANY_TO_MANY", "sizeUnit": "px", "minColocArea": 0.0 } }, { "enabled": true, "command": { "type": "COLOCALIZATION", "classesToColoc": [ { "VALID": 5 }, { "VALID": 13 } ], "filterClasses": [], "classForOverlappingAreas": { "VALID": 17 }, "multiplicity": "MANY_TO_MANY", "sizeUnit": "px", "minColocArea": 0.0 } } ] } ] }