Diagram of the custom-modified epi-luminescence imaging system employed for single-UCNP and spectral imaging.

<p>A wide-field inverted epi-luminescence microscope was modified to allow external fiber-coupled laser illumination. The optical fiber was dithered to average out speckles. The excitation light was configured to uniformly illuminate the field-of-view at the sample plane via a modified Köhler illumination scheme. The sample plane was imaged using an EMCCD camera, optionally mounted with an AOTF for hyperspectral imaging. An adjustable iris diaphragm allowed reduction of the field-of-view to restrict imaging to several single UCNP particles and small clusters.</p

Spectral imaging of UCNPs.

<p>Emission spectra of UCNPs in (A) single, (B) small cluster (designated ‘cluster 2’ in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063292#pone-0063292-g002" target="_blank">Figure 2</a>) and (C) powder form (data points joined by solid lines) captured using hyperspectral epi-luminescence microscopy, overlaid with the ensemble-averaged spectra of UCNP powder captured by a calibrated spectrometer (dashed lines). The corresponding exposure times and EMCCD camera electron-multiplication (EM) gains were (A) 4 sec and 255; (B) 1.5 sec and 44; and (C) 0.014 sec and 9. Since the samples (A) and (B) contained considerably less emitters than the powder sample (C), the excitation intensities at λ_ex = 978 nm were varied, respectively, from 250 W/cm² to 8 W/cm² to accommodate for the large disparity in the emission signals that would otherwise exceed the dynamic range of the EMCCD. The decreased I_ex resulted in an increased green-to-red emission ratio in (C) due to the varied upconversion energy redistribution between the green and red multiplets. Top panel, schematic diagram of NaYF₄:Yb,Er UCNP.</p

Single-UCNP correlative imaging.

<p>(A) TEM and (B) epi-luminescence microscopy images corresponding to the same areas of the sample TEM grid. The distances between the individual (encircled) nanoparticles/clusters, given in (A), were precisely matched to those in (B) to identify the same UCNP constellation. (C) Close-up TEM image of the same area as in (A), where UCNP sites designated ‘cluster 1’, ‘cluster 2’, and ‘single’ correspond to the three sites in (B). The individual UCNPs within ‘cluster 1’ and ‘cluster 2’ were optically unresolvable. “Single” designates a single UCNP particle clearly observable, as a diffraction-limited spot in (B). The excitation wavelength, intensity and exposure time were 978 nm, ∼250 W/cm² and 0.7 s, respectively. The pixel values were converted to photons/second (ph/s) and color-coded according to the look-up color bar in (B).</p

Epi-luminescence imaging of a single UCNP using a “blood-immersion” objective.

<p>(A) A photograph of the hemolyzed blood layer between the objective and cover slip. (B) Absorption spectrum of the hemolyzed blood (red solid curve) and UCNP emission spectrum (green dashed curve). (C) Low-magnification images of the UCNP sample recorded through the eyepiece port using the water- (top) and blood- (bottom) immersion objective. The dried UCNP colloid rims appeared green (top) and red (bottom) due to the green light absorption by blood. (D) Epi-luminescence microscopy image of the UCNP constellation identified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063292#pone-0063292-g002" target="_blank">Figure 2C</a>, imaged using the blood-immersion objective. The single UCNP is clearly observable, although blurred. The EMCCD camera settings and excitation parameters were equivalent to these of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063292#pone-0063292-g002" target="_blank">Figure 2</a>. The pixel values were converted to photons/second (ph/s) and color-coded using the look-up bar in (D).</p

Conversion efficiency, size and morphology of UCNPs synthesized in-house.

<p>(A) Plot of the absolute conversion efficiency (<i>η</i>_uc) [W/W] of the reported upconversion nanoparticle sample versus the excitation intensity at <i>λ</i>_ex = 978 nm measured using a calibrated integrating sphere set-up. <i>η</i>_uc is the ratio of the emitted power integrated over the entire emission spectral range (500–700 nm) to the absorbed power. (B) Size histogram obtained by analyzing the transmission electron microscopy (TEM) images of NaYF₄:Yb,Er UCNPs (330 particles). A typical TEM-image is shown in (C).</p

Theoretical estimation of single-emitter detection sensitivity in skin.

<p>A plot of the optical (confocal) detection contrasts of a single upconversion nanoparticle (UCNP, brown) and organic fluorescence dye (fluorescein, FC, green) versus their depth in skin, as modeled theoretically. The inset shows more detailed quantitative plots of the imaging signal (dashed) and background (dotted) of UCNP and FL versus depth in skin expressed in electrons per second (e⁻/s). The black dotted line demarcates the contrast value of 1. See text for details.</p

<i>In vivo</i> near-infrared fluorescence imaging of Xenolight 750 probes in the rat AVM model.

<p>Rats with an AVM creation were sham treated or irradiated with a 15 Gy marginal dose to the AVM region by Gamma Knife and imaging performed 12 h after conjugate dye injection (25 μg/kg). Representative montages of x-ray (left), fluorescent (centre) and merged (right) images after injection of Xenolight 750 probes: (A) Xenolight-750 isotype control in irradiated animal; (B) Xenolight 750-ICAM-1 probe and; (C) Xenolight 750-VCAM-1 probe, at day 21 after sham (top panels) or radiation (bottom panels). Image J quantitation of fluorescence at day 21 post-irradiation or sham with Xenolight 750-ICAM-1 (D) or Xenolight 750-VCAM-1 (E) probes and Xenolight-750 isotype control probe.</p

ELISA and immunocytochemical analysis of ICAM-1 and VCAM-1 expression in irradiated bEnd.3 cells.

<p>ELISA analysis of surface ICAM-1 (A) and VCAM-1 (D) expression in irradiated bEnd.3 cells normalized to Janus green absorbance to account for changes in cell number. Immunocytochemistry was performed on bEnd.3 cells using CF555-conjugated ICAM-1 or CF640-conjugated VCAM-1 antibodies (red). Staining was quantitated as integrated density using Image J (arbitrary units) for ICAM-1 (B) and VCAM-1 (E). Representative images are shown at 120 h (ICAM-1) (C) or 72 h (VCAM-1) (F) post-radiation at doses of 0–25 Gy. Isotype controls for ICAM-1 (IgG1-CF555) and VCAM-1 (IgG2b-CF640) showed no staining (representative images shown at 25 Gy, 72h). Cells were counterstained with DAPI to visualize nuclei (blue). All images were acquired at a magnification of 200× (scale bar = 100 μm). Values are mean ± SEM, n = 3 for each group.</p

Effect of radiation on bEnd.3 cell morphology and viability.

<p>(A) Representative images of bEnd.3 cells after radiation at doses of 15 and 25 Gy. Scale bar = 20 μm. All images at 200× magnification. (B) Cell viability was determined by trypan blue assay. Values are mean ± SEM, n = 3 for each group.</p

Time course of ICAM-1 and VCAM-1 expression in the rat AVM model.

<p>Xenolight-750 probe binding was examined over a period of 84 days for ICAM-1 (A) and VCAM-1 (B) in control and irradiated (15 Gy) animals (n = 7 per group). Raw MFI (mean fluorescence intensity) was normalized to day 1 in matched animals to reduce inter-animal variation. No significant differences were detected at any time between irradiated and control AVMs. <i>Ex vivo</i> image analysis of Xenolight 750-ICAM-1 (C) and Xenolight 750-VCAM-1 (D) probe binding in excised tissue: common carotid artery (CCA), external jugular vein (EJV) (n = 7).</p