Pulsed white light laser

256 × 256 pixel FLIM images were
collected, before and after
peptide addition, in the time domain using the Leica TCS SP5 microscope
coupled with a PicoHarp 300 TCSPC Module (PicoQuant, Berlin, Germany).
CF and di-4-ANEPPDHQ fluorescence was acquired using excitation
at 470 nm from the pulsed White Light Laser (Leica Microsystem) in
the range 500–650 nm. Laurdan fluorescence was acquired under
two-photon excitation using λ_exc = 780 nm in two
channels: 410–460 nm (blue channel) and 480–540 nm (green
channel).

All microscopy experiments were carried out on a Sp8x confocal laser scanning microscope equipped with a 405 nm diode laser and a pulsed white light laser (Leica microsystems) using 10× Air, 20× Air, and 100× Oil magnification objectives. For the binding assays and the 2D internalization assay, microscope settings were: 50% laser power, 6% shutter intensity for the 405 nm laser line, and 5% for the 594 nm laser line, respectively. Images were acquired consecutively to prevent cross talk between dye excitations or bleeding of emissions in different channels. The scan speed was 200 Hz and the fluorescent signal was visualized using hybrid detectors in BrightR mode, with line accumulation and frame averaging set to three. Settings for the 3D internalization assays were identical, but the shutter intensities were 3% for the 405 nm laser line and 10% for the 594 nm laser line, or 485 nm laser line for doxorubicin excitation. Images were acquired and processed using LasX software, version 1.8.1.13759 (Leica, Wetzlar, Germany).