Tunable Gold Nanorods Enable Four-Channel Multiplex Photoacoustic Imaging

Introduction: : Deciphering complex biological systems requires the simultaneous visualization of several biochemical targets, for example, tracking multiple therapeutic cell cohorts, mapping tumor-microenvironment heterogeneity, or monitoring combination nanomedicines. Photoacoustic (PA) imaging is uniquely suited for in vivo multiplexing applications: it couples molecular imaging capabilities with centimeter-scale depth penetration, delivering information in real-time without ionizing radiation. Effective PA multiplexing, however, hinges on a palette of exogenous probes whose absorbance peaks are narrow and widely separated within the NIR-I window (650–950 nm), where tissue attenuation is minimal. Most current probes fall short. Organic dyes offer sharp spectra yet bleach or aggregate under nanosecond pulses; metal nanostructures can be tuned optically but often share broad, overlapping spectral bands and are susceptible to photothermal reshaping. As a consequence, in vivo PA studies rarely resolve more than two colors. We address this bottleneck by using a three-step synthesis to create a quartet of miniaturized gold nanorods (AuNRs) whose longitudinal surface-plasmon resonances are precision-spaced across NIR-I in ~70 nm increments. Aspect ratio is incrementally increased while core volume is held constant; a mesoporous-silica shell and PEG layer impart identical size profiles and surface chemistries, isolating optical signature as the sole differentiator. Integrated with a robust non-negative least-squares unmixing algorithm, this chemistry-matched yet optically orthogonal nanoparticle collection advances PA multiplexing from dual to quad channels without compromising quantitation accuracy, photostability or biocompatibility, thereby possibly broadening the biological questions addressable with spectral PA multiplexing.

Materials and

Methods: : Miniature tunable AuNRs were synthesized via a three-step seed-mediated protocol. First, 1-2 nm gold seeds were synthesized in CTAB/n-decanol micelles. Then, seeds were elongated to miniature anisotropic AuNR precursors (λmax ~ 730 nm). Finally, anisotropic AuNR precursors were tuned to discrete aspect ratios (λmax ~ 680, 770, 860, 950 nm) by adjusting the pH (HCl, 1M, 50-600 µL) while holding Ag⁺ (200 µL, 10 mM), Au(III), and ascorbate constant yielding four optically distinct AuNRs. AuNRs were coated with mesoporous silica and PEGylated (MW = 2000) resulting in PS-AuNRs.

Optical extinction (400–1100 nm) and transmission-electron microscopy (TEM; n > 100 rods per group) verified peak placement, aspect ratio, and shell uniformity. Spectroscopic PA imaging employed a Vevo LAZR system (LZ250 probe, 21 MHz, 680–970 nm, 2 nm step sizes) in water-submerged polyethylene tube phantoms. Photostability (6000 pulses, ~20 mJ/cm2) and linearity (0.125 OD – 1 OD) were characterized. Fifteen formulations encompassing central logical combinations were prepared: four single-agent samples; six binary mixtures at 50: 50 ratios; four ternary mixtures at 33% each; and one quaternary mixture containing 25 % each. A non-negative least-squares algorithm, pre-trained on individual spectra, unmixed the multi-wavelength PA stacks into individual contributions.

For in vivo validation, female Nu/J mice (8–12 weeks) received four subcutaneous injections of PS-AuNR1–4 (20 µL, 2 OD) on the dorsal surface. 3D and spectroscopic US/PA scans (LZ550 probe; 680–970 nm, 2 nm steps) were acquired immediately post-injection.

Results, Conclusions, and Discussions:: The three-step AuNR synthesis protocol was used to manufacture a quartet of optically orthogonal, yet chemically matched, AuNRs (Figure 1A). Transmission electron microscopy (TEM) confirmed discrete and incrementally increasing aspect ratios of AuNRs 1-4 with increasing lengths, proportionally contracting widths, maintaining core Au volume (Figure 1B). Subsequent mesoporous silica-coating and PEGylation gave PEGylated silica-coated AuNRs (PS-AuNRs) with similar sizes across PS-AuNRs 1-4; silica shell thickness compensated for core geometry, equalizing size and surface chemistry across the series (Figure 1C).

UV–vis–NIR spectra displayed four narrow, non-overlapping plasmon peaks at 690, 770, 870 and 940 nm, spaced by ~80 nm, minimizing spectral crosstalk (Figure 1D). 3D and spectral PA imaging showed wavelength-specific PA response. AuNR1 dominated at 690 nm, AuNR2 at 770 nm, AuNR3 at 870nm, and AuNR4 at 940 nm, while the PA spectra remained highly distinct across all AuNRs (Figure 1E). No photobleaching was observed after 6,000 laser pulse irradiation (20 mJ/cm2). Spectroscopic scans (680–970 nm) confirmed that PA profiles mirrored optical extinction (Figure 1F). Dose-response linearity testing spanning 12.5% to 100% concentration gradients showed linear correlation between particle concentration and PA spectra for each probe, supporting quantitative unmixing (Figure 1G).

Fifteen mixtures comprising single-, binary-, ternary- and quaternary compositions were created, and PA imaging was performed across multiple replicates. Resulting PA spectra were multiplexed with a non-negative least-squares algorithm and multiplexed to estimate contribution percentages of each AuNR within each mixture. The resulting heat-map yielded a mean absolute error of 3.84% (Figure 1H). Mixtures with maximally separated spectra (e.g., 50/50 AuNR1+AuNR4) registered < 1% error, whereas mixtures with significantly overlapping spectra (e.g., 50/50 AuNR3+AuNR4) accounted for the largest residuals ( < 8%). Subcutaneous implantation of all four probes in murine dorsum supported in vivo specificity: wavelength-gated PA images (690, 770, 870, 940 nm) selectively illuminated the corresponding depot, and spectral unmixing localized each injection site within 0.4 mm (Figure 1I).

Collectively, this aspect-ratio engineering strategy yields a photostable, four-color PA palette that remains linear, deconvolves with < 5 % error, and functions robustly in vivo, advancing PA multiplexing from dual to quad channel.

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