Info
Background:
Photoacoustic imaging (PAI) uses laser-induced thermoelastic expansion of oxyhemoglobin and deoxyhemoglobin to generate ultrasonic waves, enabling sub-millimeter mapping of tumor vasculature and oxygen saturation. Because many tumors exhibit increased vascular density, PAI can help identify and delineate tumor regions of interest. Exogenous contrast agents can further improve tumor conspicuity, partly through preferential accumulation via enhanced permeability and retention mechanisms.¹ However, many optoacoustic contrast agents require high tissue concentrations to generate sufficient signal, which can necessitate relatively large injected doses. Molecularly targeted PAI agents are also often limited to biomarkers with high expression levels, restricting the number of practical imaging targets. Recent advances in in vivo tissue clearing using topically applied tartrazine have shown reduced optical scattering in superficial tissues and improved light penetration, effectively increasing apparent tissue transparency.²
Hypothesis/Objective:
We hypothesized that increasing superficial tissue transparency would improve the detectability of injected optoacoustic contrast agents. As a proof of concept, we applied topical tartrazine to surface tumors to determine whether tissue clearing increases contrast-agent–derived PAI signal and improves tumor visualization.
Methods:
To confirm that tartrazine would not interfere with near-infrared (NIR) PAI, 0.6 M tartrazine and 0.3 M tartrazine solutions, corresponding to 30 wt% and 15 wt%, respectively, were placed in cuvettes, and absorbance was measured from 370 to 1000 nm. To evaluate whether tartrazine introduced optoacoustic artifacts in the NIR range, optoacoustic spectra were acquired from 700 to 900 nm. To assess tissue-clearing feasibility in nude mice, nude mouse skin approximately 1–2 mm thick was treated with different tartrazine solutions for 60 minutes, and transparency was evaluated by visualizing a grid through the treated skin. For in vivo imaging, orthotopic 4T1 mammary fat pad tumors were established in ten 4–8-week-old athymic nude mice by injecting 10⁶ cells into the inguinal mammary fat pad. Ten days after tumor cell implantation, mice received an intravenous injection of 150 µL of 2.14 mM indocyanine green (ICG). Twenty-four hours later, mice were anesthetized with 2% isoflurane in 100% oxygen. Depilatory cream was applied for 6 minutes and then removed. A 0.6 M tartrazine solution or NaCl control was then topically applied for 6 minutes with gentle rubbing. PAI was performed using an iThera MSOT system. Images were acquired at 700, 730, 760, 800, 850, and 875 nm with 10 averages per frame and 5 repetitions per slice. Images were acquired at 1 mm step sizes across the tumor, typically spanning 9–12 mm in length, corresponding to 18–24 slices. Data was reconstructed and spectrally unmixed in ViewMSOT 4.0 to quantify unmixed ICG signal within tumor regions of interest. To evaluate the clearing effectiveness of topical tartrazine in thicker tissues, a 15 wt% tartrazine solution was applied to the surface of dark porcine skin and muscle samples and allowed to clear for 3 or 24 hours. PAI was performed using a clinical PAI instrument, the Seno Medical Imagio system, and signal-to-noise ratios were calculated.
Results:
Both tartrazine solutions exhibited minimal absorbance in the NIR range and generated negligible optoacoustic signal between 700 and 900 nm (Figure 1A). Topical tissue clearing was visually apparent, as gridlines became clearly visible through treated skin with strong contrast (Figure 1B). Light transmission increased over 60 minutes, reaching up to 20% transmission in the 15 wt% tartrazine group and up to 50% transmission in the 30 wt% tartrazine group.
In vivo PAI demonstrated a marked increase in spectrally unmixed ICG signal in tartrazine-treated tumors compared with controls. A representative reconstructed 3D volume was rotated by two 45° increments to highlight increased signal within the tumor ROI (Figure 1C). Quantification of tumor ROIs showed that tartrazine treatment significantly increased tumor ICG signal relative to controls 24 hours after IV ICG administration (Figure 1D). Importantly, tumor physiology and oxygenation remain unaffected by tartrazine treatment (Figure 1E). In dark-colored porcine skin, tartrazine treatment also improved PAI signal detection. Signal-to-noise ratio of a covered ICG sample placed under the skin increased from 1.1–1.9 at baseline to 2.1–3.0 after 3 hours of tartrazine treatment and further increased to 4.3 after 24 hours.
Conclusion:
Topical tartrazine-mediated tissue clearing substantially increased detectable PAI signal from IV-administered ICG and improved visual delineation of tumor regions relative to surrounding tissue. These findings support in vivo tissue clearing as a potential strategy to enhance contrast-agent–based PAI of superficial tumors. Tartrazine also improved PAI signal in thicker tissue samples, enabling delineation of regions of interest that were previously difficult or unobtainable. Future studies will evaluate whether this approach can improve the performance of molecularly targeted PAI contrast agents and enable detection at lower injected doses.
Image/Figure:
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Image/Figure Caption:
Figure 1: A) Absorbance and PAI spectrum highlighting minimal tartrazine signal in the NIR range. B) Confirming tartrazine clearing capabilities on athymic nude mouse skin via increased gridline visualization and light transmission. C) Representative 3D PA images of uncleared and cleared tumors D) Quantitative measurements of increased PAI signal in tartrazine-cleared tumors vs uncleared tumors, demonstrating improved ICG detection with D) no changes in tumor oxygenation. F) Topical tartrazine application increases the SNR of ICG through 1 cm of dark colored porcine skin and muscle. **** p < 0.0001. Error bars represent the standard deviation.
Author
Marty Pagel, PhD
University of Wisconsin Madison