Multimodality imaging for preclinical assessment of nanoparticles and information provided by each modality.

<p>Multimodality imaging for preclinical assessment of nanoparticles and information provided by each modality.</p

TUNEL and H&E stained images of untreated, HerDox-treated, and Dox-treated tumors: (A) TUNEL images of untreated tumors (left), HerDox-treated tumors (middle) (injection drug dosage: 0.004 mg/kg×6 injections), Dox-treated tumors (right) (injection drug dosage: 0.04 mg/kg×6 injections) were acquired using confocal imaging (ex: 488 nm, em: 510–560 nm, and 10×) of tumor specimens.

<p>The arrows indicate the boundary between apoptotic and normal cells. Insets represent magnified images. (B) Average fluorescence of the TUNEL images of untreated, Dox-treated, HerDox-treated tumor sections (*: p<0.01) (C) H&E images of untreated (left), HerGa-treated tumors (middle), and Dox-treated tumors (right) were obtained using the Olympus microscope incorporating a CCD camera with a RGB filter (10×). The scale bar represents 200 µm. Arrows indicate nuclear shrinkage and fragmentation.</p

Fluorescence intensity image of harvested tissue <i>and in situ</i> confocal fluorescence of tumors: (A) Fluorescence image (left-panel; Ex: 532 nm; Em: 590±30 nm) of organs and tumors extracted from mice receiving HerDox (0.004 mg/kg/day; see Methods).

<p>Confocal fluorescence images of the tumors extracted from HerDox-treated (A, right-panel), Dox-treated (B, right), and untreated mice (B, left) were acquired at different z-depths (a total of thickness: 32 µm) with a step size of 2 µm. Maximum intensity z-projection of confocal fluorescence images of HerDox-treated and untreated tumors was performed. HerDox and Dox fluorescence in (A) and (B) are indicated by a color shift toward 255 on the scale bar in (A). The magnified image (A, right-panel) shows HerDox distribution in the tumor. Arrows indicate nuclei-localized fluorescence. (C) Mean fluorescence intensity of HerDox-treated, Dox-treated and untreated tumor. The error bar indicates standard deviation. *: p<0.01.</p

High-frequency ultrasound imaging of tumor regions in mice receiving (A) 0.004 mg/kg/day HerDox or (B) 0.04 mg/kg/day Dox, by i.v. delivery and in (C) untreated mice: Ultrasound images of tumor regions were obtained after the indicated drug injection (before drug injection, after the third daily drug injection, and after the sixth daily drug injection) while mice received daily i.v. injections of the indicated drugs for six sequential days.

<p>Graphs (right-panel) depict average echo intensities of tumor regions (0, before drug injection; 3, immediately after the third daily drug injection; 6, 24 h after the sixth daily drug injection). *: p<0.01, +: p = ∼0.6097, and − : p = ∼0.95. The error bars indicate standard deviations. The arrows indicate tumor regions.</p

Ratiometric spectral classification images of HerDox-treated tumors <i>in situ:</i> (A) Ratiometric spectral classification images (middle and right-panel) of HerDox-treated tumors.

<p>Ratiometric spectral classification was performed using our previously developed program. Green: autofluorescence (Autofl.), Red: 0.2 Dox+0.8 autofl., Cyan: 0.3 Dox+0.7 autofl., and Blue: 0.1 Dox+0.9 autofl.. The reference spectral signatures used to generate the spectrally classified image are shown in the left-panel. (B) Spectral unmixed image. Scale bar: 80 µm.</p

High-frequency ultrasound images of tumors <i>ex vivo</i> after extracting from mice treated with Dox and HerDox: Ultrasound images were obtained of tumors extracted from (A) an untreated mouse, (B) a mouse treated with 0.004 mg/kg/day HerDox, or (C) a mouse treated with 0.04 mg/kg/day Dox.

<p>(D) Echo signal intensities of A–C, respectively. The error bars reflect standard deviation. *: p<0.01.</p