Supplementary Figure 5: Dealing with nonspecific staining.
From: Multi-target spectrally resolved fluorescence lifetime imaging microscopy
U2OS cells were labeled with ATTO 488 phalloidin (green), Alexa Fluor 488 indirect immunofluorescence against giantin (magenta), and EdU (nascent DNA) clicked to ATTO 488 azide (blue). (a) Resulting sFLIM image. Here not only the nucleus is blue as intended, but there are also some vesicle-like spots clearly visible in the cytoplasm. They most likely consist of free ATTO 488 azide. After introduction of a new pattern for this unspecific staining, the yellow “bubbles” are clearly separated from the blue nucleus (b). Optionally one could remove the unspecific signal. Scale bars, 10 µm.
