High-energy two-photon laser targeting eliminates neural cells. (A) Neurons were identified in MAPT lungs by eGFP fluorescence with confocal microscopy. Infrared light (820 nm) was focused to target cell bodies or axons. (B) Neurons were imaged pre- and post-ablation. Cells or axons in boxed regions (top) were targeted and are seen at higher magnification post-ablation. (B1) After targeting cell bodies, a ring of autofluorescence is seen, or the cell is no longer visible. (B2) After slicing axons, eGFP is dissipated distal to the cuts (yellow arrow). Targeting a cell confines ablation to that cell: (C) Pre-ablation, a neuron of interest is shown in the X-Y plane with the red circle indicating the ablation focus. (D) The same neuron is shown in the axial plane (red star), with a neuron directly behind it (white arrow). (E) Post-ablation, a ring of autofluorescence marks the destroyed cell (red box). (F) Viewed axially, the ablated cell (red) is seen with the unaffected neuron behind it (white arrow). Scale bar = 20 μm throughout. Bystander cell death was assessed post-ablation in MAPT lung explants treated with Topro3 dead cell indicator: (G) A pre-ablation projected image stack shows cells and axons, with the yellow arrow pointing to future targets and the yellow line to the region of laser focus. (H) A post-ablation image stack was collected immediately following ablation to show that targeted cells are no longer visible. (I) After four hours in culture, the explant was re-treated with Topro3 and the same region imaged. The few targeted cells are permeable to indicator at the ablation site (purple). Scale bar = 30 μm throughout. eGFP, enhanced green fluorescent protein; MAPT, microtubule-associated protein tau eGFP.