Videomicroscopy of the Article "Combating resilient Acanthamoeba castellanii cysts: Ultrasonic frequency-dependent effects on viability and excystment"

2.3. Videomicroscopy 2.3.1. Imaging for low-frequency ultrasound treatment After low-frequency ultrasound treatment, A. castellanii cysts were seeded into a µ-slide 8-well plate (ibidi®) at a density of 2.5 × 104 cells per well. These were cultured in PYG medium at room temperature. Images were captured using an Andor Revolution XD imaging system, featuring a CSUX1 Yokogawa spinning disk unit and an Andor Ixon + 897 back-illuminated EMCCD camera (16 µm2 pixel size). This system was mounted on an Olympus inverted IX81S1F-ZDC microscope equipped with a 40x objective lens (NA 0.95). IQ3 acquisition software (Andor Technology) was used to acquire approximately 1,800 images, each 512x512 pixels, over a 16-hr period. 2.3.2. Imaging for high-frequency ultrasound treatment For cells exposed to high-frequency ultrasound, 50 µL of the post-treatment cell suspension was added to a µ-slide 8-well plate (ibidi®) and supplemented with 250 µL of PYG medium. Cells were then incubated at room temperature for 48 h. Images were subsequently acquired every three minutes for 16 h using a Nikon CrestOptics X-Light V3 Spinning Disk Confocal microscope with a 40× objective lens. Nikon Imaging Software was used to capture 2720 × 2720 pixel images.