The photothermal performances of BGNPs were evaluated by measuring the temperature increase during NIR laser irradiation on 96-well plate. A fully automated experimental setup in collaboration with Crisel Instruments (Rome, Italy), integrating a (i) NIR laser source, (ii) XY micropositioning stage, and (iii) thermal camera, was used to perform the treatment.

CW fiber-coupled infrared diode lasers (MDL-F-808; CNI Optoelectronics; Changchun, China) with a nominal power of 2.2 W and emission wavelength of 808 nm were used. A multimode fiber with a core diameter of 400 μm was used to couple the laser with a collimating spherical lens (FOC-01-B, CNI Optoelectronics; Changchun, China), producing a spot size of 6.5 mm at a distance of 10 cm, matching the single well diameter (for 96-well plate). The laser irradiation comes from the top and is perpendicular to the multiwell plate.

The positioning of the plate under the laser spot was performed using the XY micropositioning OptiScan® stage (Prior Scientific, Cambridge, UK), including the XY stage, controller, joystick, and plate holder. The stage was remotely controlled with the Micromanager control software.

Real-time temperature changes were recorded by thermal images acquired with an Optris Xi 400 camera (Optris, Berlin, Germany) coupled with a 18° × 14° lens (f = 20 mm) at a framerate of 27 Hz. Data acquisition and analysis were performed with Optrix PIX Connect software.

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