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.
Articles tagged with: axon regeneration
By depositing a high instant energy in a small focal volume, the probability for multi-photon absorption, and the occurrence of other nonlinear optical effects is increased. Such localized energy deposition may serve for three-dimensionally confined sample ablation (Berns 1981). Using sub-nanosecond pulsed UV laser light instead, the average power at the sample required for optical dissection can be further reduced while, sharply confining the photo-damage to the focal volume. The laser dissector based on a sub-nanosecond pulsed UV-A source, permit to perform three dimensionally confined ablation just like systems based on two-photon absorption processes. However, it has the advantage of delivering extremely low average power (few µW) to the sample, thereby minimizing undesirable thermal effects.By laser dissection and concurrent calcium imaging it is possible to estimate the extracellular solution influx in to the dissected neurite.