Figure 2.

Nanosecond-Pulsed Laser Beam-Based NOD Achieved High-Throughput Transfection of MCO-II-mCherry Plasmids in HEK Cells

(A) Immunostained fluorescence image of HEK cells in a Petri dish transfected with MCO-II-mCherry using NOD (+ plasmids, + fGNRs, + 1,064-nm laser) using total laser dose of 5 mJ/mm² (10 mJ/pulse, 10 Hz, 10 s, 200-mm² area). (B) Zoomed image of the region marked by the square in (A). (C) Immunostained fluorescence image of staining control. (D) ×3 zoomed image of the region marked by the square in (C). (E) Plot of fluorescence intensity of reporter (mCherry) along a line drawn across the transfected cell, showing membrane-specific expression. (F) Quantitative comparison of reporter fluorescence in membrane and cytoplasm. (G) Quantitative comparison of mCherry expression in NOD-transfected cells and controls: (1) − plasmid, − fGNRs, − 1,064-nm laser; (2) − primary antibody; and (3) − 1,064-nm Laser. Primary antibody, anti-mCherry; secondary antibody, Alexa Fluor 488 (green). Nuclear staining with DAPI (blue). Mean ± SD. (H) Transfection efficiency (measured as number of fluorescent cells/total cells) at different NOD laser beam parameters (i.e., energy/pulse and exposure time), with total dose ranging from 2.5 to 15 mJ/mm². ∗p < 0.05 with respect to the “no laser” and “no fGNRs” controls. Mean ± SD.