Detection limits for photoacoustic signals from target spheres in the presence of optically absorbing (A) or scattering (B) background PVCP.

<p>Phantoms of the ranges P2 (A) and P3 (B) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075533#pone-0075533-t001" target="_blank">Table 1</a>) were used to perform the measurements. Data in (A) were fit to a monoexponential decay with a plateau of 1. For clarity, data is shown only from the 0.128% BPC sphere at 750 nm; the same trend was observed for all BPC and TiO₂ concentrations, and wavelengths tested. Abbreviation VS = VisualSonics.</p

Photoacoustic imaging performance assessed using a PVCP quality control phantom in both small animal imaging instruments.

<p>(A) Absorption spectrum of the black plastic color (BPC) dye at three concentrations (0.032%, 0.064% and 0.128%) recorded on a spectrophotometer (B) Spectrum of laser energy as a function of wavelength recorded by an external power meter. (C) PA signal recorded from the target sphere without energy compensation. (D) PA signal recorded from target sphere once normalized for recorded energy per view (Endra) or per image (VisualSonics). (E) Linearity of BPC absorption as a function of concentration at five different wavelengths. (F) Linearity of PA signal recorded as a function of increasing BPC concentration at two different wavelengths.</p

Schematics of experimental systems used.

<p>(A) The VisualSonics Vevo LAZR uses intersecting planar laser beams (B) for excitation and a linear transducer array for 2D imaging of the resulting ultrasound. (C) The Endra Nexus 128 delivers diffuse laser light and detects the ultrasound using 128 transducers in a helical arrangement (D) for 3D reconstruction.</p

Placement of the quality control phantoms into the imaging systems and example images.

<p>Phantoms were positioned in VisualSonics Vevo LAZR (A) and Endra Nexus 128 (B) using water for ultrasound coupling. A simple acrylic box was used to position the phantom from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075533#pone-0075533-g001" target="_blank">Figure 1B</a> in the VisualSonics system, while the hemispherical mold that was used to make <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075533#pone-0075533-g001" target="_blank">Figure 1A</a> was placed directly into the Endra system. (C) Example of a cross section of the quality control phantom on the VisualSonics system. (D) Example of a tomographic reconstruction of the same phantom from the Endra system.</p

Verification of changing system settings and their effect on photoacoustic imaging signals.

<p>Data is shown for each instrument derived from imaging the quality control phantom as a function of wavelength. The effects of changing VisualSonics Vevo LAZR gain and persistence (signal averaging) on the photoacoustic signals from the target sphere are illustrated in A and B. Increasing the number of angular views (C) and pulses (D) increases the signal-to-noise ratio (SNR) logarithmically on the Endra system. The combined effect is illustrated in (E), while the resulting increase in scan time is illustrated in (F). All Endra SNR data is normalized to the SNR measured in the image volume acquired with 250 pulses and 360 views. Abbreviation VS = VisualSonics.</p

Detection limits as a function of depth in optically absorbing and scattering background PVCP (0.004% BPC and 0.5 mg/ml TiO₂; 0.256% BPC target).

<p>Data in A and B are images from phantom P4 acquired by VisualSonics and Endra respectively and were fit to a monoexponential decay function with a plateau of 1. As the Endra signal had yet to plateau, the second phantom P5 was used to push the limits of detection and this was imaged at multiple wavelengths as shown in (C). Abbreviation VS = VisualSonics.</p

Reproducibility of photoacoustic imaging measurements over time using the quality control phantom.

<p>Stability of VisualSonics (A) and Endra (B) over time assessed by repeated imaging of phantom P1 at 750 nm. First 30 days shown for clarity, but coefficient of variation values derived out to 4 months shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075533#pone-0075533-t002" target="_blank">Table 2</a>. Abbreviation VS = VisualSonics.</p

Characterization of tri-fusion transgenic mouse.

<p>(A) <i>In vivo</i> bioluminescence, micro-PET and fluorescence imaging of adult tri-fusion transgenic mice and non-transgenic littermate. Mice (N=15) with different expression levels of the tri-fusion reporter were scanned, and multimodality imaging results show that the expression level of all three genes are correlated with each other. No bioluminescence signal was found in non-transgenic littermate. Background micro-PET signal was found in heart and gastrointestinal system of non-transgenic littermate, and autofluorescence was also found in non-transgenic littermate. Transgenic positive mice show different signal levels in multimodality imaging due to the different gene expression levels. (B–C) <i>In vivo</i> bioluminescence and fluorescence imaging of strongly positive newborn and adult tri-fusion transgenic mice and non-transgenic littermates. Strong bioluminescence and fluorescence signals were found in tri-fusion transgenic mice, but not in non-transgenic littermates. p/s/cm² /sr=photons per second per cm² per steradian; % ID/g = percentage of injection dose per gram.</p

Schematic showing integrated computational/experimental modeling strategy involving both cell- and tumor-scale measurements.

<p>(<b>A</b>) Functional relationships involving cell-scale parameters such as proliferation (Ki-67), apoptosis (Caspase-3), and hypoxia (HIF-1α) are defined based on experimental observations, e.g., from immunohistochemistry the density of viable tissue as a function of vascularization is shown in the third panel (red: highest density; yellow: lowest; blue: vessels). These functional relationships as well as parameter values measured experimentally are then used as input to the model to create simulations of lymphoma growth. A sample simulated tumor cross-section showing vascularized viable tissue (highest density in red, lowest in yellow, with vessel cross-sections as small blue dots) is shown at the far right. (<b>B</b>) Lymphoma observations regarding size, morphology, and vasculature from macroscopic imaging of an inguinal lymph node in live mice provide part of the tumor-scale information to validate the model simulations. Note the pre-existing vasculature in the lymph node (in the center of each frame) from which oxygen and nutrients are supplied to the tissue. For comparison, a control group of lymph nodes in animals without tumors is also shown.</p

Characterization of MSCs.

<p>(A) FACS analysis of the immunophenotype profile of MSCs. MSCs are uniformly positive for extracellular matrix protein CD90, endothelial cells antigen CD105, and low-level hematopoietic progenitor cell marker CD34 and leukocyte common antigen, but are negative for macrophages monocyte antigen CD11b. (B) MSCs can differentiate into adipocytes, chondrocytes and osteocytes <i>in vitro</i> as assessed by oil Sudan III, alcian blue or alizarin S staining. Both adipocytes and chondrocytes showed fluorescent signal after differentiation.</p