Imaging and thresholding methods. Images of roots grown on either Anchor blue (A + B) or Sebio grey (C + D). Red circles highlight regions for which the different thresholding methods yielded contrasting results (Lateral roots vanished, parallel growing roots became one root or the intensity of background noise). Blue circles indicate the removal of droplets and reflections. A: Image taken with diffuse lighting. B: Two images taken with flash light (right/left side) including polarization filters on flash and camera lens and combined to a minimum tonal image. The red channel was used for the conversion into greyscale. C: Image taken with near-infrared front lighting. D: Image taken with near-infrared backlighting. Thresholding was done using the WinRhizo or GiARoots routines. Only the routines resulting in the best separation between root and background are shown.