Table 2.
Parameters used for each high content imaging bio-application in HCS studio software.
| High Content Imaging Colocalization Analysis Parameters | High Content Imaging Morphology Analysis Parameters | High Content Imaging Target Activation Analysis Parameters | High Content Imaging Spot Detection Analysis Parameters | ||||
|---|---|---|---|---|---|---|---|
| Condition | Value | Condition | Value | Condition | Value | Condition | Value |
| Ch1: SmoothFactor | 1 | Ch1: SmoothFactor | 1 | Ch1: SmoothFactor | 1 | Ch1: SmoothFactor | 1 |
| Ch1: Thresholding (Fixed) | 500 to 700 | Ch1: Thresholding (Fixed) | 800 | Ch1: Thresholding (Fixed) | 300 | Ch1: Thresholding (Fixed) | 200 |
| BackgroundCorrectionCh1 | 50 | Object.BorderObject.Ch1 | Y | BackgroundCorrectionCh1 | 28 | BackgroundCorrectionCh1 | 255 |
| Object.Ch1.Average Intensity.Ch1 | 0 to 6,000 | BackgroundCorrectionCh1 | Y | Object.Ch1.Average Intensity.Ch1 | 518.44 to 65,535 | ObjectAreaCh1 | 1312.74 to 21262.58 |
| Ch2: SmoothFactor | 3 | Ch1: Exposure Method | Fixed | Ch2: Exposure Parameters | Fixed (0.1) | Object.Ch1.Average Intensity.Ch1 | 0 to 65,535 |
| Ch2: Thresholding (Auto or Fixed) | 500 to 850 | ObjectAreaCh1 | 1223.46 to 19288.87 | Ch2: Thresholding (Fixed) | 500 to 850 | ObjectSegmentationCh1 | −500 |
| BackgroundCorrectionCh2 | −255 | ObjectShapeP2ACh1 | 0 to 1,000 | BackgroundCorrectionCh2 | 50 | SpotDetectRadiusCh1 | 12 |
| Ch2: Segmentation (Intensity) | −300 | ObjectShapeLWRCh1 | 0 to 1,000 | Object.Average Intensity.Ch2 | 0 to 23809.66 | Ch2: SmoothFactor | 3 |
| ObjectAreaCh2 | 53.52 to 1170.72 | Object.Ch1.Average Intensity.Ch1 | 0 to 6,000 | Ch3: SmoothFactor | 2 | Ch2: Thresholding (Fixed) | 150 |
| Object.Average Intensity.Ch2 | 0 to 4062.65 | Ch2: Exposure Method | Fixed | Ch3: Thresholding (Fixed) | 400 | BackgroundCorrectionCh2 | 25 |
| Ch3: SmoothFactor | 2 | Ch2: Thresholding (Fixed) | NA | Ch3: Segmentation (Intensity) | 70 | Ch2: Segmentation (Intensity) | −450 |
| Ch3: Thresholding (Fixed) | 400 | Object.BorderObject.Ch2 | Y | Object.Average Intensity.Ch3 | 0 to 65,535 | SpotAreaCh2 | 1 to 625 |
| BackgroundCorrectionCh3 | −255 | BackgroundCorrectionCh2 | Y | Object.Average Intensity.Ch2 | 0 to 32,767 | ||
| Ch3: Segmentation (Intensity) | −3000 | CircModifierCh2 | 2 | ||||
| ObjectAreaCh3 | 22.09 to 8935.92 | SpotDetectRadiusCh2 | 11 | ||||
| Object.Average Intensity.Ch3 | 0 to 65,535 | Ch3: SmoothFactor | 2 | ||||
| ROI.A.Mask Ch | Channel 1 (DAPI) | Ch3: Thresholding (Fixed) | 200 | ||||
| ROI.A.Target_I | Channel 2 (NF-κB) | BackgroundCorrectionCh3 | 27 | ||||
| ROI.B.Mask Ch | Channel 3 (CMDR) | SpotAverageIntensity.Ch3 | 215.72 to 65,535 | ||||
| ROI.B.Target_I | Channel 2 (NF-κB) | CircModifierCh3 | 3 | ||||
| ROI.B.Exclude | Channel 1 (DAPI) | SpotDetectRadiusCh3 | 8 | ||||
| RejectBorderObjects | Y | ||||||