Two-Photon Luminescent Bone Imaging Using Europium Nanoagents Esther M. Surender, Steve Comby, Brenton L. Cavanagh, Orlaith Brennan, T. Clive Lee, Thorfinnur Gunnlaugsson  Chem  Volume 1, Issue 3, Pages 438-455 (September 2016) DOI: 10.1016/j.chempr.2016.08.011 Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Structural Design of the Nanoparticulate Imaging Agent (A) The 1,3-diketone-sensitizing ligand nta. (B) The synthesized complex 1.Eu.Na. (C) The surface-functionalized AuNP system AuNP-1.Eu.Na. Chem 2016 1, 438-455DOI: (10.1016/j.chempr.2016.08.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 DLS and TEM Characterization of the Size of Functionalized Gold Nanoparticles (A) Volume-distribution profile determined by DLS analysis for AuNP-1.Eu.Na (1 × 10−7 M) in H2O at 298 K. (B) Size-distribution profile calculated from analysis of the TEM images of AuNP-1.Eu.Na (4 × 10−7 M). Chem 2016 1, 438-455DOI: (10.1016/j.chempr.2016.08.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Spectroscopic Determination of 1:1 Ternary-Complex Formation Changes in the normalized Eu(III) emission (λex = 330 nm) of 1.Eu.Na (10 μM, ) and AuNP-1.Eu.Na (0.1 μM) at 612 and 616 nm, respectively, as a function of equivalents of nta in buffered 0.1 M HEPES solution at pH 7.4 (I = 0.1 M NaCl) () and D2O (). Chem 2016 1, 438-455DOI: (10.1016/j.chempr.2016.08.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Phosphorescence Studies Showing the pH Dependence of the Ternary Complex System AuNP-1.Eu.Na-nta in Aqueous Solution (A) The phosphorescent response of AuNP-1.Eu.Na (1.0 × 10−7 M) and nta (20 equiv) as a function of pH in H2O (I = 0.1) at 298 K (λexc = 330 nm). (B) Changes in the integrated Eu(III) emission as a function of pH for both the forward (pH 9.0→3.5, red) and backward (pH 3.5→9.0, blue) titrations, measured between 570 and 720 nm. Chem 2016 1, 438-455DOI: (10.1016/j.chempr.2016.08.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Illustration of Microscratched Bovine Bone Specimens Specimens were immersed in either AuNP-1.Eu.Na or AuNP-1.Eu initially for 20 hr and then for a further 4 hr. Chem 2016 1, 438-455DOI: (10.1016/j.chempr.2016.08.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Microscopy of Microdamaged Bovine Bone Structure Images show microdamaged bovine bone structure after being immersed for 0, 4, and 24 hr in an aqueous solution of AuNP-1.Eu.Na (3.1 × 10−4 M) and then immersed in an aqueous solution of nta (1.5 × 10−4 M). (A) Polarized light images. (B) Epifluorescence images were acquired from a LED source (λexc = 365 nm) and a 520 longpass emission filter. All scale bars represent 250 μm; aqueous solution = buffered 0.1 M HEPES (I = 0.1 M NaCl [pH 7.4]). Chem 2016 1, 438-455DOI: (10.1016/j.chempr.2016.08.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 TPE Fluorescence Microscopy of Microdamaged Bovine Bone Structure Images show microdamaged bovine bone structure after being immersed for 0, 4, and 24 hr in an aqueous solution of AuNP-1.Eu.Na (7.8 × 10−5 M) and then immersed in an aqueous solution of nta (1.5 × 10−4 M) for 30 s. (A) Images taken at the bottom of the microcrack. (B) Images taken at the top of the microcrack. (C) Whole-projection images of the microcrack (z stack). Each image was acquired with a Ti-sapphire laser (λ = 750 nm) and a red-channel emission filter (λem = 565–610 nm). All scale bars represent 150 μm; aqueous solution = buffered 0.1 M HEPES (I = 0.1 M NaCl [pH 7.4]). Chem 2016 1, 438-455DOI: (10.1016/j.chempr.2016.08.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 8 2D TPE Microscopy of Microdamaged Bovine Bone Structure Images show microdamaged bovine bone structure after being immersed for 0, 4, and 24 hr in an aqueous solution of AuNP-1.Eu.Na and then immersed in an aqueous solution of nta (1.5 × 10−4 M) for 30 s. All scale bars represent 85 μm. (A) Concentration of AuNP-1.Eu.Na = 3.1 × 10−4 M. (B) Concentration of AuNP-1.Eu.Na = 1.6 × 10−4 M. Chem 2016 1, 438-455DOI: (10.1016/j.chempr.2016.08.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 9 3D TPE Microscopy of Microdamaged Bovine Bone Structure Images show microdamaged bovine bone structure after being immersed for 0, 4, and 24 hr in an aqueous solution of AuNP-1.Eu.Na and then immersed in an aqueous solution of nta (1.5 × 10−4 M) for 30 s. All scale bars represent 85 μm. (A) Concentration of AuNP-1.Eu.Na = 3.1 × 10−4 M. (B) Concentration of AuNP-1.Eu.Na = 1.6 × 10−4 M. Chem 2016 1, 438-455DOI: (10.1016/j.chempr.2016.08.011) Copyright © 2016 Elsevier Inc. Terms and Conditions

Chem 2016 1, 438-455DOI: (10.1016/j.chempr.2016.08.011) Copyright © 2016 Elsevier Inc. Terms and Conditions