C apochromat 40 1.2 w objective

3D image z-stacks were collected on an inverted laser scanning confocal microscope (LSM5 Pascal, Carl Zeiss, Thornwood, NY, USA) using either a Plan-Neofluar 10×/0.3, Plan-Neofluar 40×/0.75 or C-Apochromat 40×/1.2 W objective (Carl Zeiss, Thornwood, NY, USA). The EGFP reporter was excited with the 488 nm laser line using the FITC filter and all other imaging parameters were as described in Kasemeier-Kulesa et al. (2005) (link). Images were collected, processed and analyzed using AIM software (Carl Zeiss, Thornwood, NY, USA). Statistical analysis was performed using the Student's t-test.

The confocal micrographs were obtained using a Zeiss LSM 780 laser confocal microscope equipped with a C-Apochromat 40×/1.2 W objective. The DAPI and DiI-C18 were visualized with Zeiss filter sets 49 (excitation 320–380 nm, emission 420–470 nm) and 15 (excitation 600–650 nm, emission 670–720), respectively.

For the treatment of MCF-10A cells with FL or PEG-EL, the liposomal formulations with or without anthracyclines were diluted 1:50 (v/v) with PBS and incubated with cells seeded on a high-precision microscopy glass (ThermoFisher Scientific, Waltham, MA, USA) for 10 min at 37 °C and 5% CO₂. After washing with PBS, cells were either covered with cell culture medium and imaged live or fixed with 3.7% paraformaldehyde and stained with NucBlue for fixed cells (ThermoFisher Scientific, Waltham, MA, USA). The confocal laser scanning microscope LSM880 equipped with a C-Apochromat 40×/1.2 W objective was used for imaging (all Carl Zeiss, Jena, Germany). The anthracyclines ACL and DOX were imaged by excitation with an argon ion laser at 488 nm and an emission bandwidth of 499–597 nm. The dye DiR was imaged by excitation at 633 nm and an emission bandwidth of 651–758 nm. Data with anthracyclines were presented as maximum intensity projections of z-stacks for better visualization of the anthracycline and fusion (i.e., DiR) signal. Further, the incorporation of the membrane tracer dye DiR after treatment with both liposomal formulations was examined. Here, NucBlue was imaged by excitation at 405 nm and an emission bandwidth of 446–499 nm for nucleus tracking in addition to DiR imaging.