Practical demonstration of threshold calibration in autophagy image analysis using Fiji macros and an R evaluation script.
Explains why threshold calibration matters for vacuole selection in image analysis.
Locate processed TIFF files; copy one good, one average, one poor scan into a "calibration" folder.
In Fiji: Plugins → Macros → Run → Autoflux/imageJ macro/calibrateThreshold.Igm. Point at the calibration folder.
For each input image the macro writes a series of masks back into the calibration folder, plus an overview TIFF.
Open EvaluateCalibration.R → Code → Source → choose the calibration folder to generate ThresholdGraph.tiff.
Combine the ThresholdGraph with the masks to pick an upper threshold; the demo lands on a value of 3.
New protocols and pitfalls, in your inbox
A short email when we add notable lab videos and failure cases. No spam, unsubscribe anytime.