IMAGING REAL-TIME NEURITE OUTGROWTH AND CYTOSKELETAL REORGANIZATION WITH AN ATOMIC-FORCE MICROSCOPE

An atomic force microscope was used to image the morphology and struct ural reorganization of rat NIH/3T3 fibroblasts and PC-12 cells growing in petri dishes. NIH/3T3 fibroblasts had a uniform morphology and an extensive cytoskeletal network. Cell thickness varied from similar to 2-3 mu m above the nucleus to similar to 20-30 nm over the distal proc esses, and cytoskeletal fibers as small as 30 nm wide were observed. I maging over an extended period of time showed a limited degree of cyto skeletal reorganization. Localized force dissection did not induce sig nificant retraction of cellular processes and immediate cell death. Di fferentiating PC-12 cells with a neuronal phenotype had a nonuniform m orphology, abundant cytoskeletal elements, neuritic processes, and gro wth cones. The cell thickness varied from similar to 5-8 mu m over the nucleus to similar to 100-500 nm over the neuritic processes; growth cones similar to 50-700 nm wide and end structures similar to 30-150 n m wide were visible. Repeated imaging showed reorganization of the gro wth cone, especially the appearance and disappearance of beadlike feat ures and fibrous organization. Thus an atomic force microscope can be used for high-resolution real-time studies of the dynamic subcellular mechanisms that drive cell behavior.