Sp8 las x software

The samples were imaged with an inverted STED-gated Leica TCS SP8 microscope (Leica, Germany) using a HC PL APO CS2 63x/1.40 oil immersion objective lens. The instrument was equipped with a 405-nm diode for DAPI excitation and a WLL Laser. Blue, green, and red fluorescence emission was collected using 410–460, 505–550, and 560–760 nm wide emission slits in sequential mode, respectively. The pinhole was set to 1.0 Airy unit, giving an optical slice thickness of 0.89 μm. Twelve-bit numerical confocal micrographs were processed using Leica SP8 LAS X software (Version 2.0.1; Leica, Germany). Confocal optical sectioning was performed each 0.3 μm along the z axis.

MCEC and H9c2 cells were seeded in 8-well Ibidi plates at a density of 15,000 cells per well. Cells were left to adhere for 24 h and then incubated with 12 µg/mL or 60 µg/mL of fluorescent SqCsA NPs for 2, 18, and 24 h in a 5% CO₂ humidified incubator at 37 °C. After incubation, wells were washed with PBS, and then, cells were fixed with 4% PFA for 5 min and permeabilized with 0.1% Triton™ X-100 for 3 min. The cytoskeleton of actin was stained with phalloidin for 1 h at room temperature, and nuclei were stained with DAPI contained in the mounting medium 15 min before observation. Images were obtained using an inverted Confocal Laser Scanning Microscope (CLSM) Leica TCS SP8 with an HC PL APO CS2 63×/1.40 oil immersion objective lens. The instrument was equipped with a 405 nm diode for DAPI (nuclei) excitation and a WLL Laser (488 nm excitation for phalloidin-Atto 488 and 542 nm for CholEsteryl BODIPY™ NPs). Blue, green, and red fluorescence emissions were collected respectively with 410–460, 505–550, and 560–760 nm wide emission slits using a sequential mode. The pinhole was set at 1.0 Airy unit giving an optical slice thickness of 0.89 µm. Twelve-bit numerical images were acquired using Leica SP8 LAS X software (Version 3.6; Leica, Wetzlar, Germany)