Vertebrate brains have a dual structure, composed of (i) axons that can be well-captured with graph-theoretical methods and (ii) axons that form a dense matrix in which neurons with precise connections operate. A core part of this matrix is formed by axons (fibers) that store and release 5-hydroxytryptamine (5-HT, serotonin), an ancient neurotransmitter that supports neuroplasticity and has profound implications for mental health. The self-organization of the serotonergic matrix is not well understood, despite recent advances in experimental and theoretical approaches. In particular, individual serotonergic axons produce highly stochastic trajectories, fundamental to the construction of regional fiber densities, but further advances in predictive computer simulations require more accurate experimental information. This study examined single serotonergic axons in culture systems (co-cultures and monolayers), by using a set of complementary high-resolution methods: confocal microscopy, holotomography (refractive index-based live imaging), and super-resolution (STED) microscopy. It shows that serotonergic axon walks in neural tissue may strongly reflect the stochastic geometry of this tissue and it also provides new insights into the morphology and branching properties of serotonergic axons. The proposed experimental platform can support next-generation analyses of the serotonergic matrix, including seamless integration with supercomputing approaches.