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The last decade has seen the development of a wide set of tools, such as wavefront shaping, computational or fundamental methods, that allow to understand and control light propagation in a complex medium, such as biological tissues or multimode fibers. A vibrant and diverse community is now working on this field, that has revolutionized the prospect of diffraction-limited imaging at depth in tissues. This roadmap highlights several key aspects of this fast developing field, and some of the challenges and opportunities ahead.

Recent advances in functional ultrasound imaging (fUS) and photoacoustic tomography (PAT) offer powerful tools for studying brain function. Complementing each other, fUS and PAT, respectively, measure the cerebral blood flow (CBF) and hemoglobin concentrations, allowing synergistic characterization of cerebral hemodynamics. Here, cross-ray ultrasound tomography (CRUST) and its combination with PAT are presented. CRUST employs a virtual point source from a spherically focused ultrasonic transducer (SFUST) to provide widefield excitation at a 4-kHz pulse repetition frequency. A full-ring-shaped ultrasonic transducer array whose imaging plane is orthogonal to the SFUST's acoustic axis receives scattered ultrasonic waves. Superior to conventional fUS, whose sensitivity to blood flow is angle-dependent and low for perpendicular flow, the crossed transmission and panoramic detection fields of CRUST provide omnidirectional sensitivity to CBF. Using CRUST-PAT, the CBF, oxygen saturation, and hemoglobin concentration changes of the mouse brain during sensory stimulation are measured, with a field of view of ≈7 mm in diameter, spatial resolution of ≈170 µm, and temporal resolution of 200 Hz. The results demonstrate CRUST-PAT as a unique tool for studying cerebral hemodynamics.

Long-duration in vivo simultaneous imaging of multiple anatomical structures is useful for understanding physiological aspects of diseases, informative for molecular optimization in preclinical models, and has potential applications in surgical settings to improve clinical outcomes. Previous studies involving simultaneous imaging of multiple anatomical structures, e.g., blood and lymphatic vessels as well as peripheral nerves and sebaceous glands, have used genetically engineered mice, which require expensive and time-consuming methods. Here, an IgG4 isotype control antibody is labeled with a near-infrared dye and injected into a mouse ear to enable simultaneous visualization of blood and lymphatic vessels, peripheral nerves, and sebaceous glands for up to 3 hours using photoacoustic microscopy. For multiple anatomical structure imaging, peripheral nerves and sebaceous glands are imaged inside the injected dye-labeled antibody mass while the lymphatic vessels are visualized outside the mass. The efficacy of the contrast agent to label and localize deep medial lymphatic vessels and lymph nodes using photoacoustic computed tomography is demonstrated. The capability of a single injectable contrast agent to image multiple structures for several hours will potentially improve preclinical therapeutic optimization, shorten discovery timelines, and enable clinical treatments.

This Conference Presentation, “Functional photoacoustic tomography of the human brain,” was recorded at SPIE Photonics West held in San Francisco, California, United States.

Obtaining frozen sections of bone tissue for intraoperative examination is challenging. To identify the bony edge of resection, orthopaedic oncologists therefore rely on pre-operative X-ray computed tomography or magnetic resonance imaging. However, these techniques do not allow for accurate diagnosis or for intraoperative confirmation of the tumour margins, and in bony sarcomas, they can lead to bone margins up to 10-fold wider (1,000-fold volumetrically) than necessary. Here, we show that real-time three-dimensional contour-scanning of tissue via ultraviolet photoacoustic microscopy in reflection mode can be used to intraoperatively evaluate undecalcified and decalcified thick bone specimens, without the need for tissue sectioning. We validate the technique with gold-standard haematoxylin-and-eosin histology images acquired via a traditional optical microscope, and also show that an unsupervised generative adversarial network can virtually stain the ultraviolet-photoacoustic-microscopy images, allowing pathologists to readily identify cancerous features. Label-free and slide-free histology via ultraviolet photoacoustic microscopy may allow for rapid diagnoses of bone-tissue pathologies and aid the intraoperative determination of tumour margins.

Photoacoustic tomography with genetically encoded near-infrared probes enables visualization of specific cell populations in vivo at high resolution in deep tissues. Here, we report a near-infrared Forster resonance energy transfer biosensor based on a pair of the near-infrared fluorescent proteins, miRFP670-iRFP720, which enables dynamic photoacoustic imaging of active biological processes. Specifically, we detected apoptosis in single cells at a resolution of ~3 µm in a mouse ear and mouse brain tumors (>3 mm in depth) at a spatial resolution of ~150 µm. These results open the way for high-resolution photoacoustic imaging of dynamic biological processes in deep tissues.

One of the primary challenges in breast cancer diagnosis and treatment is intratumor heterogeneity (ITH), i.e., the coexistence of different genetically and epigenetically distinct malignant cells within the same tumor. Identification of ITH is critical for designing better treatments and hence to increase patient survival rates. Here, we report a non-invasive approach that integrates photoacoustic computed tomography (PACT) with multitargeted and multiplexed patchy polymeric photoacoustic contrast agents (MTMPPPCAs). The target specificity of MTMPPPCAs to distinguish estrogen and progesterone receptor-positive in breast tumors was demonstrated through both fluorescence and photoacoustic measurements and validated by tissue pathology analysis.

Over 100 monoclonal antibodies have been approved by the FDA for clinical use; however, a paucity of knowledge exists regarding the injection site behavior of these formulated therapeutics, i.e., the effect of antibody and formulation on the tissue around the injection site and vice versa. In this report, we injected a near-infrared dye-labeled IgG4 isotope control antibody into the subcutaneous space in mouse ears to analyze the injection site dynamics, including quantifying molecular movement. Surprisingly, we discovered that the antibody reduces the local blood oxygen saturation levels in mice after prolonged anesthesia without affecting the total hemoglobin content and oxygen extraction fraction. The local oxygen saturation results open a new pathway to study the functional effects of monoclonal antibodies.