Morsch, M., Radford, R.A., Don, E.K., Lee, A., Hortle, E., Cole, N.J., Chung, R.S. (2017) Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy. Journal of visualized experiments : JoVE. (120).
Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.