During development, the axons of neurons in the mammalian central nervous system lose their ability to regenerate after injury. In order to study the regeneration process, we used a laser dissector system based on a sub-nanosecond pulsed UVA laser to inflict a partial damage to the axon of mouse hippocampal neurons. The use of such laser gives the possibility to deliver very low average power to the sample to be ablated. Therefore, the collateral damage due to temperature rise was reduced and for the first time, we were able to manipulate the neurite of cultured mouse neurons during the first days in vitro with sub-cellular precision. Force spectroscopy measurements were performed in parallel during and after the partial ablation of the neurite, by the use of a bead attached to the neurite membrane and held in an optical trap. The sub-piconewton and millisecond resolution of the force spectroscopy measurements allowed to quantify the damage inflicted to the process and to monitor the viscoelastic properties of the axonal membrane during regeneration. The reorganization and regeneration of the axon was documented by long-term (24-48 hours) bright-field live imaging using an optical microscope equipped with a custom-built cell culture incubator.
The Koh Young Best Paper Award 2012
Integration of Optical Manipulation and Electrophysiological Tools to Modulate and Record Activity in Neural Networks. F. Difato, L. Schibalsky, F. Benfenati, and A. Blau. International Journal of Optomechatronics, 2011, 5(3), 191-216.
Corresponding Author: Difato F.