Nucleolar colocalization of 16.4.1-GFP and Rev-CFP. HeLa 16.4.1-GFP cells and control HeLa GFP cells were transiently transfected with a Rev-CFP expression plasmid. Nuclei were counterstained with Hoechst 33342. 3D-Maximum image projections of Z-stacks were deconvolved and processed by multichannel unmixing, applying identical parameters to all images (see Material and Methods). Left images represent the GFP-channel (green), middle images the CFP-channel (pseudocolored red) and right images either an interference contrast photo (A) or a single z-slice of a phase contrast image (B). (A) Exemplary images of over 25 analyzed cells demonstrating nucleolar colocalization of 16.4.1-GFP and Rev-CFP. The left image shows HeLa-16.4.1-GFP cells either without Rev-CFP (white arrow), or with concurrent Rev-CFP expression (red arrow). The nucleolar 16.4.1-GFP signal is only visible in cells coexpressing Rev-CFP. Cells with extremely high expression levels of Rev-CFP were excluded from analysis. Scale bars: 10 μm. (B) Controls for separation of GFP and CFP and signals by multichannel unmixing. Images of HeLa cells stably expressing GFP (HeLa-GFP) either alone or together with Rev-CFP are shown in the upper and lower panels, respectively. The intracellular distribution of the GFP signal is not influenced by coexpression of Rev-CFP. Conversely, a nuclear/nucleolar CFP signal is detected only in HeLa-GFP cells coexpressing Rev-CFP. These results confirm that multichannel unmixing eliminates spectral crosstalk between CFP and GFP channels. Scale bars: 10 μm.
Kramer-Hämmerle et al. BMC Cell Biology 2005 6:20 doi:10.1186/1471-2121-6-20