Intracellular diffusion is critical for molecule translocation in cytoplasm and mediates many important cellular processes. Meanwhile, the diffusion dynamics is affected by the heterogeneous cytoplasm. Previous studies on intracellular diffusion are mainly based on two-dimensional (2D) measurements under the assumption that the three-dimensional (3D) diffusion is isotropic. However, the real behaviors of 3D diffusion of molecules in cytoplasm are still unclear. Here, we have built a 3D single-particle tracking (SPT) microscopy and studied the 3D diffusion of quantum dots (QDs) in adherent A549 cells. Notably, we found that the intracellular diffusion of QDs is quasi-2D, with the axial motion being severely confined. Further investigations demonstrated that disrupting the cytoskeleton component or endoplasmic reticulum (ER) does not alter the quasi-2D diffusion pattern, although ER reduces the diffusion rates and slightly relieves the constraint in the axial diffusion. The preferred quasi-2D diffusion is quite robust and attributed to the complex cytoarchitectures in the flat adherent cells. With the aid of 3D SPT method, the quasi-2D diffusion in cells was revealed, shedding new light on the physical nature of cytoplasm.

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