Laser ablation of infected cells and quantification of volume change after ablation

A549 cells were seeded on a glass bottom 35 mm petri dish (WPI, FlouroDish) and infected the next day with AdV-C2-GFP-V to achieve 20% infected cells. 24 h pi, the full medium was exchanged to full medium supplemented with Hoechst (250 ng/ml). Both imaging and laser ablation were performed on a TCS SP8 multiphoton microscope (Leica) using an Insight DS+ Dual (680-1,300 nm & 1,041 nm) ultrafast NIR laser tuned to 800 nm and operating at 1% of its maximal power of 1.3 W. Ablation was conducted at 20% of the maximal power at 36 h pi. Prior to ablation, a Z-stack was acquired in both nuclear and viral channels, which was then fed into a custom Python tool. The script generated a maximum projection and fed it into the pre-trained CNN to predict the future lytic or nonlytic phenotype. After the ablation, the Hoechst signal of the nuclei was imaged every minute up to 5. The acquired Z-stacks were segmented into nuclear and exerted material volumes using Arivis and Fiji software packages (Schindelin et al., 2012).