Hi Simon,
thanks for submitting the workflow. Your are right suspecting the Image Reader node causing the problem. But it's rather easy to solve: png-images consist of 4 different channels (RGB and one channel to encode transparency). Our Image Reader reads all 4 channels (whereof the 4th channel usually is filled with all 0's) whereas the "Read PNG Images" node omits the 4th channel (the resulting image only has 3 channels). And this "emtpy" 4th channel causes some troubles in the substract background macro. To make this also work with our Image Reader, just select only the first 3 channels (Subset selection-Tab in the Image Reader-dialog (saves memory, because only the selected channels are read), or by using the Image Cropper node). Actually, as the example image is grayscale (i.e. all three color channels are identical), selecting only the first channel is sufficient.
A little tip: to conveniently check the dimensions of an image (e.g. the number of channels), just click with the right mouse button on an image-column header und choose "Metadata" as renderer.
Cheers,
Martin
P.S. I have never tried to load images with the image reader from web-urls. But nice to know that this works as well (thanks to BioFormats ;) ).