*2.7. Photoacoustic Imaging (PAI)*

PAI was performed with a Vevo® Lazer 2000 (Fujifilms Visualsonics, Amsterdam, The Netherlands) using a 10 ns pulse laser (680–900 nm) with an energy fluence of 20 mJ/cm2 and 21 MHz central

frequency. These conditions were maintained for the in vitro and in vivo experiments. For in vitro PAI, 200,000 cells were suspended in 100 μL PBS and mixed with 100 μL warm agar (Sigma, Diegem, Belgium). This solution was added and solidified in a bigger agar block. During in vivo PAI, ultrasound imaging is used to determine the tumor location, after which PAI was performed to validate nanostar injection.
