Characterization of neurite dystrophy after trauma by high speed structured illumination microscopy and lattice light sheet microscopy

•A novel means of transecting neurites mounted on a lattice light sheet microscope is presented.•A novel means of quantifying neurite dystrophy in a structured illumination microscopy image is presented.•The neurites of human induced pluripotent stem cell-derived neurons have immature actin cytoskeletal organization.•Transection rapidly perturbs neurite shape but does not affect actin architecture.•2 channel, 3D lattice light sheet microscopy can detect brief, unpredictable events long after trauma and place change in the actin cortex in the context of overall change in neurite shape. Unbiased screening studies have repeatedly identified actin-related proteins as one of the families of proteins most influenced by neurotrauma. Nevertheless, the status quo model of cytoskeletal reorganization after neurotrauma excludes actin and incorporates only changes in microtubules and intermediate filaments. Actin is excluded in part because it is difficult to image with conventional techniques. However, recent innovations in fluorescent microscopy provide an opportunity to image the actin cytoskeleton at super-resolution resolution in living cells. This study applied these innovations to an in vitro model of neurotrauma. New methods are introduced for traumatizing neurons before imaging them with high speed structured illumination microscopy or lattice light sheet microscopy. Also, methods for analyzing structured illumination microscopy images to quantify post-traumatic neurite dystrophy are presented. Human induced pluripotent stem cell-derived neurons exhibited actin organization typical of immature neurons. Neurite dystrophy increased after trauma but was not influenced by jasplakinolide treatment. The F-actin content of dystrophies varied greatly from one dystrophy to another. In contrast to fixation dependent methods, these methods capture the evolution of the actin cytoskeleton over time in a living cell. In contrast to prior methods based on counting dystrophies, this quantification scheme parameterizes the severity of a given dystrophy as it evolves from a local swelling to an almost-perfect spheroid that threatens to transect the neurite. These methods can be used to investigate genetic factors and therapeutic interventions that modulate the course of neurite dystrophy after trauma.