1030‐nm Ultrashort‐Pulse Laser Irradiation Is Associated With a Delayed Reduction in Cellular Metabolic Activity in A549 Cells

ABSTRACT Objectives This exploratory study aimed to assess acute and delayed changes in relative CCK‐8‐derived cellular metabolic activity across selected 1030‐nm ultrashort‐pulse laser parameters in A549 lung adenocarcinoma cells and to characterize cell‐death and oxidative‐stress responses under an empirically selected high‐effect condition. Methods A549 cells were irradiated under different power density, repetition rate, and pulse width conditions, and CCK‐8‐derived metabolic activity was assessed at 0, 9, and 24 h after irradiation. Direct cell counting was additionally performed under the selected 12.5 W/cm 2 power‐density condition. Under an empirically selected high‐effect condition within the tested ranges, cell‐death phenotypes were evaluated by Annexin V‐FITC/PI flow cytometry, and transcriptomic changes were explored by RNA sequencing. Temporal changes in DCF fluorescence were descriptively examined by repeated fluorescence imaging of the same individual cells at predefined time intervals. NAC pretreatment was used to examine whether reactive oxygen species (ROS) scavenging attenuated the irradiation‐associated DCF fluorescence response. Results Higher power densities were associated with lower relative CCK‐8‐derived metabolic activity, particularly at 0 h, while the largest delayed reduction from 0 to 24 h occurred at 12.5 W/cm 2 . At this power density, direct cell counting showed a progressive decrease in cell number over 24 h after irradiation. Under fixed total irradiation energy, higher repetition rates and longer pulse widths were also associated with lower metabolic activity within the tested ranges. Based on relatively preserved 0 h activity together with a comparatively large 0–24 h decline, 12.5 W/cm 2 , 200 kHz, and 1500 fs were empirically selected for exploratory characterization. Under this condition, Annexin V‐positive and PI‐positive fractions increased, RNA sequencing identified irradiation‐associated transcriptomic changes, and single‐cell imaging showed increased DCF fluorescence that was attenuated by NAC pretreatment. Conclusion Within the tested ranges, 1030‐nm ultrashort‐pulse laser irradiation was associated with parameter‐related changes in relative CCK‐8‐derived metabolic activity in A549 cells. Direct cell counting under the 12.5 W/cm 2 power‐density condition also showed a progressive reduction in cell number over 24 h. An empirically selected representative high‐effect condition showed a comparatively large delayed reduction in metabolic activity, together with changes in Annexin V/PI‐defined cell‐death phenotypes and irradiation‐associated transcriptomic responses. In addition, repeated DCFH‐DA imaging revealed time‐dependent increases in single‐cell DCF fluorescence, which were reduced by NAC pretreatment. These preliminary descriptive findings provide a basis for further mechanistic investigation of ultrashort‐pulse laser–cell interactions.