3.4.4. Fluorescence Microscopy

CD20-expressing Raji cells were prepared and pre-treated with modified or non-modified RTX as described above (cell-binding studies). Instead of the radioligand, 50 µL (22 nM) of iron protected SulfoCyanine5-[Fe]MAFC-PEG5-Tz was added and the cells were treated according to the cell-binding studies with the <sup>68</sup>Ga-labeled counterparts. After resuspension fluorescence microscopy was performed using a laser (λ = 561 nm) for excitation of the fluorescent dye. The imaging was carried out with a spinning-disc confocal microscopic system (Ultra VIEW VoX, PerkinElmer, Waltham, MA, USA) linked to a Zeiss AxioObserver Z1 inverted microscope (Zeiss, Oberkochen, Germany). Images were acquired with Volocity software (PerkinElmer) utilizing a 63× oil immersion objective (numerical aperture 1.42). The images show z-stacks (*n* = 4; 1 µm spacing). Cell morphology was visualized by adding fluorescently labeled wheat germ agglutinin (Alexa Fluor® 488 WGA, ThermoFischer Scientific, Vienna, Austria).
