“…The NA is chosen to reduce the effects of spherical aberration, which can decrease the Strehl ratio as ablation depth is increased, 21 and to avoid self-focusing, which can cause damage before the intended focus or create a tighter focus, and lead to an underestimation of the ablation fluence threshold. 22,23 We ablate a 40 × 40 μm 2 FoV by scanning 512 lines at 1/16 frames per second, causing ∼0.078 μm separation between lines and ∼1.28 μm separation between pulses on the same line, resulting in a ∼20 pulse overlap. An FoV is ablated rather than a single point to better sample the tissue and provide a more accurate representation of l s and F th .…”