Nonlinear optical interactions are intrinsically confined to the focal region of a focused laser beam. This leads to inherent 3D spatial resolution, but also to the possibility of localized targeted photophysics and photochemistry. In parallel to its application in imaging, multi-photon absorption was targeted to the manipulation of the biological sample under investigation. In the nervous system, the opportunity provided by multi-photon nanosurgery to selectively injure single elements of neuronal networks is a valuable tool for dissecting the rules of brain remodeling. The photoionization mechanism relies on a combination of multi-photon ionization and tunneling; the interaction of a high-energy photon and a molecule results in the release of a quasi-free electron in the medium. Neurons irradiated with a focused, controlled femtosecond energy dose were characterized with time lapse 3D two-photon imaging. Electrons are considered “quasi-free” if they have sufficient kinetic energy to be able to move without being captured by local potential energy barriers.