examining the cryopreserved sections of skin with multi-photon microscopy (MPM, Zeiss 510 Meta, Germany). To track the delivery of fluorescent chemicals from coated microprojections in the skin, we used MPM to produce a series of images at successive skin depths, and then repeated the procedure at later time points. The projection penetration depth for each projection set was determined by image analysis. The delivery of DiD from 130 projections was measured for each ear with a total of 4 ears analysed (i.e. n = 4). The morphology of projections was examined before and after application to the skin with Scanning Electron Microscopy (SEM, JEOL 6400) to qualitatively confirm the coating and release. Observation of projection penetration in skin, using Cyro-SEM Cryo-SEM was employed to visualise the penetration of projections in skin. A patch was applied to the skin by a custom spring based applicator device at 1.96 m/s. With the projections in place, the skin and patch assembly was frozen in liquid nitrogen for 10 seconds. To avoid condensation accumulation on the skin, the array was removed in a cryo-preparation chamber, under vacuum. Then the skin was cut into pieces in the vacuum chamber and the intersection of the skin was observed under SEM (Phillips XL30) at room temperature. Three patches were also applied onto three mouse ears at a consistent and repeatable velocity, by a custom spring-based applicator device at 1.96 m/s. The number of projections which pierced stratum corneum was counted. Histology and immunostaining Four Cy3 labeled FluvaxH coated patches were applied to the inner earlobe of anaesthetized female 6–8 week old C57BL/6 mice (one patch per ear) with a custom spring based applicator device at 1.96 m/s. After application, each Nanopatch was kept in place on the skin for 2 minutes. Mice were subsequently sacrificed and ears excised and the skin was fixed in 2% paraformaldehyde in 0.1 M phosphate buffer at room temperature for 10 minutes. The tissue was then cryopreserved and then sectioned to 10 mm wide and counterstained with Hoechst 33342. A stock solution of 10 mg/ml Hoechst 33342 in DMSO was prepared. A working solution was made by a 1:10,000 dilution in TBS for nuclei staining. Tissue sections were treated with the working solution of Hoechst 33342 solution and incubated for 10–20 minutes at room temperature. Finally, the tissue sections were rinsed for 3615 min- utes in TBS. The tissue sections were immunostained with mouse anti-MHC II-FITC (eBioscience, San Diego, CA USA) at 1:500 for 1 hour at room temperature. The sections were then rinsed for 3615 minutes in TBS. The tissue was then mounted with Prolong Anti-fade Gold (Invitrogen, Carlsbad, CA USA). Whole ears were split at the dermis-cartilage junction where the epidermis and dermis from the application side were retained for staining. The split-ear tissue was fixed in 2% paraformaldehyde in 0.1M phosphate buffer at room temperature for 30 minutes. The tissue was then washed 3615 minutes in TBS prior to permeabi- lization with 0.1% Triton-X 100 in 0.1M phosphate buffer at room temperature for 15 minutes. The tissue was then washed again 3615 minutes in TBS. The tissues were immunostained with mouse anti-MHC II-FITC (eBioscience, San Diego, CA USA) at 1:200 for 1 hour at room temperature. The sections were then rinsed for 3615 minutes in TBS. The tissue was then mounted with Prolong Anti-fade Gold (Invitrogen, Carlsbad, CA USA). We have defined the targeting of antigen directly to an APC as: (a) the antigen co-localized with the APC (i.e. the color yellow), and/or Skin Targeted Vaccinations (b) the antigen in direct contact with the APC. We define this contact as there not being any ‘empty’ voxels between the fluorescent signals from the antigen and the APC. The voxel size is 0.82 mm 6 0.82 mm 6 1.8 mm (x, y, z), defining the resolution of this direct contact event. Use of Coomassie Blue R-250 as the tracer dye to determine the vaccine dose delivered We measured the dose of FluvaxH antigen delivered to the skin using Coomassie Blue R 250 dye as a tracer. Correspondingly, the mass of delivered FluvaxH antigen was determined by the ratio of FluvaxH to Coomassie Blue R-250 contained in the coating solution. Five Nanopatches were coated with Fluvax 2008H, Coomassie Blue R-250 dye and methylcellulose. With the same applicators, the coated Nanopatches were applied to mouse ears (n = 5). Then the skin sites were gently and thoroughly cleaned with a cotton swab moistened with physiological saline immedi- ately following patch removal. Subsequently, the skin from the patched area was cut and then homogenized in 1 ml of 70% ethanol at 55uC for 2 hours under stirring to extract the Coomassie Blue in the skin. Finally the absorbance (at lmax = 592 nm) of the eluate for the 5 samples was measured and the percentage of Coomassie Blue delivered to the skin from each NanopatchTM was calculated to be 6.46%60.84% (Mean6 SEM) of Coomassie Blue applied to each mouse ear. Using scanning electron microscopy images of coated Nanopatches, we then examined the coating morphology, and found the coating thickness was uniform across a given NanopatchTM and repeatable between Nanopatches. This, for example, is demonstrated by the uniform coating thickness at the middle of the cylindrical part of projections (2.1060.18 mm; n = 5). Furthermore, uniformity of coating to projections was independently confirmed by consistent fluorescence intensity on the projections – measured by confocal microscopy images of Nanopatches coated with a fluorescent dye (rhodamine-dextran; used as a surrogate of vaccine for coating). This collective work validated the assumption that a consistent and repeatable portion of FluvaxH antigen was delivered to skin by each coated NanopatchTM. Vaccination of mice C57BL/6 female mice (groups of five) were vaccinated once by applying Nanopatches dry coated with Fluvax 2008H to the inner lobe of each ear (two Nanopatches per mouse) and held in place for 2 min for the vaccine to diffuse into the epidermis/dermis. A range of doses were tested across different experimental groups. Additional groups of mice were vaccinated by needle and syringe in the caudal thigh muscle. The doses shown are the total HA amounts of the three viral strains combined delivered under the skin of the three different strains present in Fluvax 2008H. Following the single vaccination, all mice were bled at 3 weeks and also at 2 months. The sera were separated and stored frozen at 220uC till assays were performed. All animal experiments were conducted according to the University of Queensland animal ethics regulations. Serological analyses ELISA was performed as previously described [42]. Briefly, the ELISA plates (Nunc Maxisorp) were coated with the commercial trivalent split virion Fluvax2008H. The vaccine was diluted at a concentration equivalent to 3 mg/ml total HA in 0.1M sodium bicarbonate buffer and 50ml of this solution was added to each well of the ELISA plates and incubated overnight at 4uC. The plates were blocked with 4mg/ml BSA and used to determine the titers of antigen specific IgG induced. The color development was PLoS ONE | www.plosone.org 9 April 2010 | Volume 5 | Issue 4 | e10266