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Objective: Adhesion of cells to the extracellular matrix is facilitated by integrin receptors. We recently found that a nanoscale organization of plasma membrane located integrins containing the β1 subunit is responsible for an enhanced radio-resistance in 3D cultured cells over cells grown in 2D. While ionizing radiation is known to have broad effects on the lipid composition of the plasma membrane and their organization in lipid-rafts, it is not clear whether the effects of ionizing radiation on the nanoscale clustering of integrins is lipid-raft dependent. Results: Using single molecule microscopy we can show that β1 integrins colocalize with cholesterol in lipid-rafts. Ionizing radiation, as an extrinsic stressor, causes the separation of β1 integrins from cholesterol lipid raft suggesting that the effects of ionizing radiation on the clustering of β1 integrins are lipid-raft independent.