FGF2 and PI(4,5)P ₂ colocalize in areas enriched in cholesterol as analyzed by super-resolution STED microscopy. U2OS FGF2-Halo cells were transfected with GRAM1b-G187L-GFP to visualize membrane areas containing elevated levels of accessible cholesterol. Cells were then treated with doxycycline to induce FGF2-Halo expression 24 h prior to fixation and antibody incubation. (A) Confocal (top row) and STED (lower row) images of FGF2 (in green), PI4,5)P₂ (in red) and GRAM1b-G187L-GFP (in grey). Dual or triple color merged images and cropped images showing a close-up perspective are presented. Areas with FGF2 and PI(4,5)P₂ in close proximity are indicated with arrows. Scale bar = 1 µm. (B and C) Larger magnifications of the three- and two-color STED images shown in panel A with white arrows pointing at pairs of FGF2 (green) and PI(4,5)P₂ (red) in membrane areas with elevated levels of accessible cholesterol (grey; GRAM1b-G187L-GFP cholesterol sensor). For experimental details, see Materials and methods. Scale bar = 1 µm.