Real-Time In Vitro Imaging of Axonal Branching in Mouse Cortical Neurons

Take a glass-bottom imaging dish containing mouse embryonic cortical neurons.

Place the dish inside the environmental chamber of a differential interference contrast, or DIC, microscope to maintain cell viability during imaging.

DIC microscopy splits polarized light into two beams that traverse the specimen.

Variations in the refractive index across the specimen introduce phase shifts between these beams.

When the beams recombine, their interference generates a high-contrast image, making finer cellular structures visible.

Identify neurons with unbranched axons and use time-lapse imaging to visualize axonal branching.

Treat with netrin-1, an extracellular guidance cue that binds to axonal receptors and triggers localized exocytosis.

Exocytic vesicles deliver membrane components to the sites while the cytoskeleton is reorganized to support membrane expansion.

Under the microscope, these changes appear as small protrusions, marking the onset of axonal branching.

Over time, these protrusions develop into mature axonal branches, illustrating the dynamic process of neuronal development.

At two days in vitro, place the glass bottom imaging dish containing untransfected neurons in a pre-warmed hum