Fig. 5.
High precision correlation applied to the location of adenovirus particles in vitrified cells. (A) Fluorescent cryo microscopy image of plunge-frozen U2OS cells infected with green fluorescent Ad5-488 viruses. Merged fluorescent image after channel alignment using TetraSpeck microsphere fluorescence. Upper row of insets shows magnified images of the RGB channels and merge for the Ad5-488 virus marked by the red square. The lower row of insets shows magnified images of TetraSpeck number 3 illustrating the alignment accuracy (σA=11.8 nm) across the three FM channels. The Ad5-488 signal marked by the red square (σE=75 nm) in the region of interest (ROI) was targeted for cryoET. (B) Medium magnification cryoEM projection image of the ROI shown in (A). The correlation between (A) and (B) was performed using the FluoSpheres numbered 4, 5 and 6 (σC=43.9 nm) as reference points. The subarea selected for tomography is indicated by the red square. (C) Computational slice through tomogram for the subarea indicated in (B) shows an adenovirus particle in an endosome, surrounded by actin filaments. The overall correlation error (σT=87.5 nm) is plotted as radius of the red dashed circle centred on the calculated coordinates of the targeted Ad5-488 signal. Scale Bars: (A, B) 1 μm; (C) 100 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).