SIM Imaging of Nano-Drug Delivery

SIM imaging was performed
using a customer three-color system built around an Olympus IX71 microscope
stage, as previously described.^{40 (link),46 (link)} Laser wavelengths of
488 nm (iBEAMSMART- 488, Toptica), 561 nm (OBIS 561, Coherent), and
640 nm (MLD 640, Cobolt) were used to excite the fluorescence in the
samples. A 60 Å∼/1.2 numerical aperture (NA) water immersion
lens (UPLSAPO 60XW, Olympus) focused the structured illumination pattern
onto the sample. This lens captured the samples’ fluorescent
emission light before being imaged onto an sCMOS camera (C11440, Hamamatsu).
Raw images were acquired with HCImage software (Hamamatsu). MCF7 cells
were treated with PP75 coacervate-like nano system and free Dox (both
with 6.4 μg/mL Dox concentration) for various time periods and
then stained with LysotrackerTM according to the protocol provided
by Thermofisher before they were imaged by SIM. Reconstruction of
the SIM images with LAG SIM Resolution-enhanced images were reconstructed
from the raw SIM data with LAG SIM, a custom plugin for Fiji/ImageJ
available in the Fiji Updater. LAG SIM provides an interface to the
Java functions provided by fair SIM.^{47 (link)}