Functional photoacoustic imaging: from nano- and micro- to macro-scale

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TL;DR

This comprehensive review describes functional photoacoustic imaging (fPAI) across nanoscale, microscale, and macroscale, highlighting its ability to provide scalable resolution and depth while quantifying physiological parameters such as oxygen saturation, blood flow, and metabolic rate. Key advances include super-resolution nanoscopy achieving 88 nm lateral resolution for single organelles, and clinical applications such as differentiating benign from malignant thyroid nodules with 83% sensitivity and 93% specificity. The clinical significance lies in fPAI's potential for noninvasive diagnosis and monitoring of cancers, stroke, liver steatosis, and other diseases.

Full summary

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**Background** Functional photoacoustic imaging (fPAI) is a hybrid biomedical imaging technique that combines optical excitation with acoustic detection, enabling scalable resolution and imaging depth from the nanoscale to the macroscale. Unlike pure optical methods limited to superficial depths (<1 mm), PAI uses the photoacoustic effect—where pulsed laser light induces thermoelastic expansion and generates acoustic waves—to image deeper tissues. fPAI can quantify functional biomarkers such as total hemoglobin concentration (HbT), oxygen saturation (SO₂), blood flow, vessel morphology, and metabolic rate of oxygen (MRO₂). This review summarizes recent advances in fPAI across scales, from super-resolution nanoscopy to clinical applications.

**Methods** The review covers three scales: (1) Nanoscale: Techniques include photoactivated atomic force microscopy (pAFM) with ~8 nm resolution, dual-pulse pAFM for organic semiconductor crack detection, near-field scanning photoacoustic microscopy (NSPM), and far-field super-resolution methods such as photoimprint photoacoustic microscopy (PI-PAM) and nonlinear PA nanoscopy. PI-PAM improved lateral resolution by a factor of √(1+b) (b = photobleaching rate) and imaged live rose petal epidermal cells at 570 nm. Nonlinear PA nanoscopy achieved 88 nm lateral resolution for single mitochondria. (2) Microscale: Sub-wavelength photoacoustic microscopy (SW-PAM) with 220 nm resolution imaged single melanoma cells and red blood cells. Bessel-beam SW-PAM achieved 300 nm resolution with a seven-fold improvement in depth of field. Ultrafast wide-field OR-PAM (UFF-PAM) achieved 2 Hz volumetric imaging rate, 10 µm lateral resolution, and 11 mm field-of-view for whole-brain hemodynamics in stroke models. Functional OR-PAM with five wavelengths (532, 545, 558, 620, 640 nm) quantified HbT, SO₂, and blood flow speed in tumor angiogenesis. A quadruple imaging system combining OR-PAM, ultrasound, OCT, and fluorescence imaging was applied to corneal neovascularization. (3) Macroscale: Raster-scan optoacoustic mesoscopy (RSOM) with a through-hole broadband transducer achieved 4×2 mm² field-of-view in ~15 s, enabling statistical classification of cutaneous melanomas (n=10) vs. benign nevi (n=10) using vessel morphology biomarkers. Photoacoustic computed tomography (PACT) with a 1024-element spherical array and deep-learning-assisted reconstruction monitored brain hemodynamics in rats under alternating oxygen/nitrogen mixtures. Hybrid systems (CRUST-PAT, FLOT, MROT) combined PACT with ultrasound Doppler, fluorescence, or fMRI to study neurovascular coupling. Multispectral optoacoustic tomography (MSOT) at 680–960 nm detected hepatic steatosis in 5 patients vs. 5 healthy controls, with significant PA intensity increases at 930 nm (p<0.001). 3D PACT with four arc-shaped arrays quantified angiographic irregularity and speed of sound in fatty liver rats.

**Key Results** - Nanoscale: pAFM achieved ~8 nm resolution; nonlinear PA nanoscopy resolved two 5 nm targets 90 nm apart and imaged mitochondria at 88 nm lateral resolution. - Microscale: SW-PAM resolved single red blood cells at 220 nm. UFF-PAM measured spreading depolarization wave speed and vasoconstriction in stroke mice. Functional OR-PAM showed SO₂ restoration after SPK2 injection in hypoxic conditions. In thyroid nodules (52 patients), fPAI achieved 83% sensitivity and 93% specificity for malignancy detection. - Macroscale: RSOM differentiated melanoma from nevi using total blood volume, vessel length, tortuosity, and fractal number. PACT with deep learning enabled real-time SO₂ mapping in rat brain under 10% O₂/90% N₂ vs. 90% O₂/10% N₂. MSOT showed significantly higher PA intensity at 930 nm in steatotic livers (p<0.001). 3D PACT in fatty liver rats revealed statistically clear distinctions in vessel volume occupancy, angiographic irregularity, and speed of sound between lean and obese groups.

**Clinical Implications** fPAI offers noninvasive, label-free functional imaging with scalable resolution, making it suitable for diagnosing cancers (thyroid, breast, melanoma, prostate), monitoring stroke and neurovascular coupling, assessing liver steatosis, and evaluating dermatologic diseases. The technique's ability to quantify oxygen metabolism and vessel morphology provides biomarkers for early disease detection and treatment monitoring. Clinical systems (e.g., MSOT, PACT) have received FDA approval or are in global use for Crohn's disease, psoriasis, and Duchenne muscular dystrophy. Future advances in contrast agents, multimodal integration, and real-time imaging will further enhance clinical translation.