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Super-resolution microscopy is broadening our in-depth understanding of cellular structure. However, super-resolution approaches are limited, for numerous reasons, from utilization in longer-term intravital imaging. We devised a combinatorial imaging technique that combines deconvolution with stepwise optical saturation microscopy (DeSOS) to circumvent this issue and image cells in their native physiological environment. Other than a traditional confocal or two-photon microscope, this approach requires no additional hardware. Here, we provide an open-access application to obtain DeSOS images from conventional microscope images obtained at low excitation powers. We show that DeSOS can be used in time-lapse imaging to generate super-resolution movies in zebrafish. DeSOS was also validated in live mice. These movies uncover that actin structures dynamically remodel to produce a single pioneer axon in a “top-down” scaffolding event. Further, we identify an F-actin population – stable base clusters – that orchestrate that scaffolding event. We then identify that activation of Rac1 in pioneer axons destabilizes stable base clusters and disrupts pioneer axon formation. The ease of acquisition and processing with this approach provides a universal technique for biologists to answer questions in living animals. 

Sensory axons must traverse a spinal cord glia limitans to connect the brain with the periphery. The fundamental mechanism of how these axons enter the spinal cord is still debatable; both Ramon y Cajal’s battering ram hypothesis and a boundary cap model have been proposed. To distinguish between these hypotheses, we visualized the entry of pioneer axons into the dorsal root entry zone (DREZ) with time-lapse imaging in zebrafish. Here, we identify that DRG pioneer axons enter the DREZ before the arrival of neural crest cells at the DREZ. Instead, actin-rich invadopodia in the pioneer axon are necessary and sufficient for DREZ entry. Using photoactivable Rac1, we demonstrate cell-autonomous functioning of invasive structures in pioneer axon spinal entry. Together these data support the model that actin-rich invasion structures dynamically drive pioneer axon entry into the spinal cord, indicating that distinct pioneer and secondary events occur at the DREZ.