Figure 4

Fluorochromes have different lifetimes when coupled to antibodies or to phalloidin. (a) Confocal image of Abberior STAR 635P coupled to an antibody delineating vimentin (cyan) and to phalloidin (actin staining; red) and subjected to phasor based lifetime separation. (b) Confocal image of ATTO 647N coupled to phalloidin (actin staining, red) and an antibody (nuclear pores, cyan. Both confocal images show good separation of the two stained structures. Scale bars 2 µm. (c, d) Phasor plots for the complete raw images of (a, b). The actin coupled dyes have longer lifetimes (left cloud, red circle) compared to their antibody-conjugated versions (right cloud, cyan circle). (e, f) Phasor plots of respective STED images show that the lifetime differences are much reduced, due to the depletion effect causing a shorter average lifetime. Therefore, despite inclusion of a background component (green circle at G = 1, S = 0), separation of STED images was not satisfying and resulting images are not shown. Cyan and red circles indicate the best possible positions for separation of the two species. In (e, f) the right-hand clouds contain the pixels with antibody label derived photons, but they also contain substantial amounts of actin label derived photons. To compensate for this the circles were positioned next to the clouds.