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1.
Figure 1

Figure 1. From: Self-luminescing BRET-FRET near infrared dots for in vivo lymph node mapping and tumor imaging.

Schematic of self-luminescing BRET-FRET near infrared (NIR) polymer nanoparticles. The biochemical energy generated from the Luc8-catalyzed oxidation of coelenterazine transfers initially to the MEH-PPV polymer and is then relayed to doped NIR775 dye to produce NIR emission. An amphiphilic polymer, PS-PEG-COOH, coats the nanoparticle to improve water solubility and biocompatibility. Tumor targeting ligands such as cyclic RGD peptides are conjugated to the nanoparticle surface for in vivo cancer imaging.

Liqin Xiong, et al. Nat Commun. ;3:1193-1193.
2.
Figure 3

Figure 3. From: Self-luminescing BRET-FRET near infrared dots for in vivo lymph node mapping and tumor imaging.

Fluorescence and bioluminescence imaging of lymph nodes in mice. (a) Fluorescence imaging of a mouse following sacrifice and necropsy 24 h after the tail-vein injection of RET1IR NPs (~20 μg). Superficial skin was removed before imaging but peritoneum was left intact. Autofluorescence is coded in green and NPs signal in red; NL, neck lymph nodes; AX, axillary lymph node; LT, lateral thoracic lymph node; IN, inguinal lymph node; L, left; R, right. (b) Fluorescence image of lymph nodes excised from the mouse in (a): 1-4, NL; 5, AX (Left); 6, LT (Left); 7, LT (Right); 8, AX (Right); 9, IN (Left), 10 IN (Right). (c) Bioluminescence and (d) fluorescence imaging of lymphatic basins in a mouse 10 mins after the injection of RET2IR NPs (~2 μg) intradermally in the forepaws. (e) Bioluminescence imaging of lymphatic basins in a mouse with injection of RET2IR NPs (~2 μg) intradermally in the forepaws. All bioluminescence images were acquired with 10 s exposure time; PO, popliteal lymph node; LU, lumbar lymph node; L, left; R, right.

Liqin Xiong, et al. Nat Commun. ;3:1193-1193.
3.
Figure 4

Figure 4. From: Self-luminescing BRET-FRET near infrared dots for in vivo lymph node mapping and tumor imaging.

Fluorescence imaging of U87MG cells in vitro and in vivo with RET1IR@cRGD NPs. (a-c) Live imaging of U87MG cells in vitro incubated with RET1IR@cRGD NPs (4 μg) for (a) 2.5 h and (b) 24 h, or (c) incubated with RET1IR NPs without cRGD for 24 h. Scale bar: 20 μm; excitation: 480/30 nm, dichroic beamsplitter: Q570LP, emission: D755/40M; objective: 20x; acquisition time: 1 s. (d, e) Time-dependent fluorescence imaging of U87MG tumor-bearing mouse (tumors are indicated by white arrows and circles, and are 4 mm in diameter) injected with (d) RET1IR@cRGD or (e) RET1IR NPs (each at ~50 μg) after 5 min, 2 h, 24 h and 48 h. Autofluorescence is coded in green and the unmixed polymer nanoparticle signal in red. (f) Fluorescence spectra of tissue autofluorescence (green) and the unmixed nanoparticle signal (red) in a living mouse. (g) ROI analysis of fluorescence intensity of tumor over background of mice in (d) and (e). Using one-tailed paired Student's t-test (n = 3 mice injected with RET1IR@cRGD), p < 0.05 at 24 h. (h, i) NIR fluorescence imaging of urine samples (h) collected 48 h after injection from mouse in (d), and (i) from a mouse without any nanoparticle injection.

Liqin Xiong, et al. Nat Commun. ;3:1193-1193.
4.
Figure 2

Figure 2. From: Self-luminescing BRET-FRET near infrared dots for in vivo lymph node mapping and tumor imaging.

In vitro characterization of RET IR nanoparticles. (a) UV-Vis absorption and fluorescence emission spectra of RET2IR NPs in PBS buffer. (b) Bioluminescence emission spectrum of RET2IR NPs in PBS buffer. (c) Representative transmission electron microscopy (TEM) image of RET2IR NPs. Scale bar = 200 nm. (d) Dynamic light scattering (DLS) measurement of four indicated NP formulations in water by lognormal size distribution. (e) Gel electrophoresis (0.5% agarose) analysis of RET NPs in tris-borate-EDTA (TBE) buffer: RET1IR (lane 1), RET1IR@cRGD (lane 2), RET2IR (lane 3), RET2IR@cRGD (lane 4). (f) Bioluminescent and NIR fluorescent intensity of RET2IR NPs (1 μg) in mouse serum at 37°C from 0 h to 24 h. Data points represent mean±s.d. (n=3). (g) The NIR fluorescence signals of blood samples of mice injected with RET1IR NPs (~20 μg) from 5 min to 24 h. Data represent mean±s.d. (n=4). (h) Viability values (%) of U87MG cells estimated by MTT assay versus incubation concentrations of RET1IR NPs. Data represent mean+s.d. (n=3).

Liqin Xiong, et al. Nat Commun. ;3:1193-1193.
5.
Figure 5

Figure 5. From: Self-luminescing BRET-FRET near infrared dots for in vivo lymph node mapping and tumor imaging.

In vivo imaging of U87MG tumors in mice with RET2IR@cRGD. (a, b) Time-dependent (a) in vivo bioluminescence and (b) fluorescence imaging of U87MG tumor-bearing mouse (indicated by a red arrow and circle; tumor size was about 5 mm in diameter) injected with RET2IR@cRGD NPs (~50 μg). Acquisition time for images in (a) from left to right: 15 s (5 min), 15 s (2 h), 1 min (24 h), 1 min (48 h), and 3 min (48 h). (c) ROI analysis of the bioluminescence and fluorescence intensity between tumor and background of mice in (a) and (b). Using one-tailed paired Student's t-test (n = 3), p < 0.00002 at 5 min, and 2 h, p < 0.04 at 24 h, and p > 0.05 at 48 h. (d) In vivo bioluminescence imaging of a mouse with a small tumor of 2 mm in diameter, as indicated by a red arrow, 2 h after tail vein injection of RET2IR@cRGD. (e) NIR fluorescence imaging of urine samples collected 48 h after injection from mice in (a). Data points represent mean±s.d. (n=4) (f-i) Histological imaging of frozen U87MG tumor slices from mouse in (a): (f) bright field, (g) NIR fluorescence (excitation filter: 480/30 nm, dichroic beamsplitter: Q570LP, emission filter: D755/40M, acquisition: 1 s), (h) Alexa Fluor 488 anti-mouse CD31 (excitation: 480/30 nm, dichroic beamsplitter: 505DCLP, emission: D535/40 nm, acquisition time: 200 ms), and (i) overlay of images in (g) and (h). Scale bar: 20 μm, objective: 20x.

Liqin Xiong, et al. Nat Commun. ;3:1193-1193.

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