Dear imagej/fiji experts and friends,
About an year ago I asked on the ImageJ mail list a question about Channel Unmixing with Poisson NMF, but received no answer. So I am reposting the questions here with the hope that someone might know what is going on.
Recently I started experimenting with channel unmixing and more precisely my interest was caught by the plugin “Poisson NMF” as it allows to do blind as well as unmixing with known spectra , and even allows for mixed approach (using known spectra as starting point for the deconvolution or keeping known spectra constant and extracting unknown ). My first impressions from the plugin are very good - there is minimum user interaction and thus minimal bias, it’s reasonably fast and produces images of high quality.
I decided to try and extract autofluoresence from the images by adding it as unknown source of fluorescence in addition to all the known spectra I have. The idea was to create e.g. 4-channel image from a 3-channel set (the fourth beeing the autofluorescent portions of the image): I add the known spectra of the dyes as the first three sources and the autofluorescence as the fourth source. Then I specify the channel wavelength boundaries of the three initial channels. However the plugin returned an image with 4 channels but consisting of NaNs.
While experimenting to see what went wrong I figured out that this happens all the time when the sources do not match the number of the initial channels. It doesn’t matter how I will specify the sources spectra (manually, spectral library, ROI or Gaussian) nor if I keep some or all of the known spectra constant.
Have any of you encountered this behavior? Is this a bug or am i doing something wrong?”
Later I noticed that NaNs are returned sometimes even when the number of channels match the preselected number of spectra but could not figure out when this happens.