Sample preparation for live cell imaging
The human lung cancer cell line A549 was obtained from the American Type Culture Collection (ATCC, Rockville, MD). A549 cells are maintained in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum (Invitrogen) at 37Â°C in a humidified 5% CO2 air incubator. Cells are cultured on uncoated glass bottom dishes (P35Gâ€1.0â€14â€C, MatTek Cooperation). First we image the control cells without adding EPA to the growth medium. For the uptake study, we add 25Î¼M EPA into the growth medium, and cells are incubated for 24h in the 37Â°C incubator. After that, cells were taken out of the incubator and imaged under the microscope within 30min.
Sample preparation for tissue imaging & drug delivery
Tissue from wildâ€type, white mice are obtained from Dr. S. Kesari and coworkers at Dana Farber Cancer Institute (Boston, USA). Directly after sacrificing, the brain is removed from skull, and ear is removed. Tissue is transported in iced phosphate buffer and imaged within one hour after sacrificing. Brain slices are cut with a razor blade and sandwiched between two coverslips. Thin mouse ear can be imaged whole. No further sample preparation is needed.
For drug delivery experiments with DMSO and RA, mouse ear skin from white, wildâ€type mice was used. For DMSO delivery experiments, approximately 25 Î¼l of a 20/80 v/v water/DMSO (Sigma Aldrich) mixture is pipetted onto the 5 x 5 mm piece of skin. The ear tissue is then incubated at 37Â° C and saturating humidity for one hour. The ~1â€2 mm thick ear tissue is placed between two #1 coverslips and imaged using SRL at 670 cmâ€1 for DMSO and 2845 cmâ€1 for the endogenous skin lipids.
For retinoic acid delivery experiments, we found that penetration could be optimized by use of a commercially available sonication device that is marketed for transdermal drug delivery in human patients (SonoPrep, Sontra Medical Corp., Franklin, MA). We followed the manufacturer instructions to sonicate the mouse ear tissue. The tissue was removed from the animal and placed in the disposable foam target ring. The ear was cleaned with the recommended skin preparation pad (containing glycerin, methylâ€paraben, propylparaben, benzyl alcohol, potassium sorbate, DMDM hydantoin and water) and the sonicator was placed over the sample. Coupling medium was applied to the sample, followed by a 30 s sonication period.
After sonication, the excess coupling medium was blotted from the sample and 25 Î¼l of a 2% retinoic acid in myritol (Sigma Aldrich) solution was pipetted onto the skin. Five minutes after drug application, the skin was blotted dry, placed between two #1 coverslips and imaged using SRL at 1570 cmâ€1 for retinoic acid.
Spontaneous Raman spectra are acquired with a confocal Raman microspectrometer (LabRam HR800, Jobin Yvon) under 5mW 633nm Heâ€Ne laser illumination, with 100x 0.9 NA air objective (MPlan, Olympus) and 10 minutes integration time. The spectrometer/CCD was calibrated with a characteristic line of Si at 520cmâ€1.