→The auto threshold calculation method can fail if fed inappropriate information: fix typos, clarify, add a little detail
== The auto threshold calculation method can fail if fed inappropriate information ==
That means it does not like images with high offset / high flat background
, which adds a constant number to the "real proportional" intensity relationship to the "concentration" of the dye. If pixels that contain no real signal have large intensity values, the algorithm has no way of knowing that, and can reach a result for the thresholds where one or both of them is below the value of the lowest intensity value present in that channels of the image. This means that ALL of that channel's pixels are considered to be colocalised, then the Manders Coefficients that you get will reflect that aberrant situation. In these cases, the background / offset should be carefully removed/subtracted before running the [[Colocalization Threshold]] plugin. The images below are an example of this situation, using the badly behaved data set: [http://fiji.sc/samples/150707_WTstack.lsm 150707_WTstack.lsm]. Note that the vaules for M1 and tM1 are the same! This should not be the case. You can see the green channel threshold is wrongly set below the intensity where the image data actually starts.