Fv1000mpe multiphoton microscopy system

Cells were grown on glass chamber slides (Thermo Fisher, Rochester, NY, USA) to 60% to 70% confluence, washed with PBS, and fixed with 4% (w/v) paraformaldehyde. Cells were washed with PBS and incubated at a 1:1000 dilution in PBS of a 1 mg/ml stock solution of Nile Red (Sigma-Aldrich) in acetone for 10 min at room temperature. Cell nuclei were counterstained and mounted with ProLong Gold Antifade reagent (Life Technologies, Grand Island, NY). A 40X LUMPLFL water immersion objective on an Olympus FV1000MPE multiphoton microscopy system (Olympus, Center Valley, PA) with a 690–1040 nm tunable MaiTai DeepSea femtosecond laser (Spectra-Physics, Santa Clara, CA) was used to acquire three dimensional (3D) image stacks from various fields of view in the sample. DAPI stained nuclei and Nile Red stained lipid droplets were simultaneously illuminated with incident laser light of 810 nm and detected at 455 nm (blue channel, DAPI) and 640 nm (red channel, Nile Red) respectively. The number and size of lipid droplets per cell were quantified using in- house software. Approximately 20–40 cells per field of view from at least six different fields of view were analyzed for each cell line.

To detect the 3D binding and uptake of targeted and non-targeted nanoparticles by 4T1 cells and assess their internalization process, two-photon laser scanning microscopy (TPLSM) was used.The FV1000MPE Multiphoton Microscopy System (Olympus^®, Hamburg) was equipped with a Mai TaiDeepSee pulsed Ti:Sapphire laser and a 25 × water dipping objective with a numerical aperture of 0.95. The excitation wavelength was set to 800 nm with 15% power for the image acquisition. This wavelength allowed excitation of all probes. For the detection of fluorescence, signal one photo multipliertube per dye was used and the filters adjusted correspondingly to emission spectra. Image acquistion was executed with Kalman filtering, the following processing and 3D analysis were performed with the Imaris 7.4 software (Bitplane^®, Zurich).