We present fast functional photoacoustic microscopy (PAM), which is capable of three-dimensional high-resolution high-speed imaging of the mouse brain, complementary to other imaging modalities. A single-wavelength pulse-width-based method was implemented to image blood oxygenation with capillary-level resolution and a one-dimensional imaging rate of 100 kHz. We applied PAM to image the vascular morphology, blood oxygenation, blood flow, and oxygen metabolism in the brain in both resting and stimulated states.

A photoacoustic microscope system provides label-free multilayered histology-like imaging of unprocessed human breast specimens.

Three-dimensional (3D) optical imaging of whole biological organs with microscopic resolution has remained a challenge. Most versions of such imaging techniques require special preparation of the tissue specimen. Here we demonstrate microtomy-assisted photoacoustic microscopy (mPAM) of mouse brains and other organs, which automatically acquires serial distortion-free and registration-free images with endogenous absorption contrasts. Without tissue staining or clearing, mPAM generates micrometer-resolution 3D images of paraffin- or agarose-embedded whole organs with high fidelity, achieved by label-free simultaneous sensing of DNA/RNA, hemoglobins, and lipids. mPAM provides histology-like imaging of cell nuclei, blood vessels, axons, and other anatomical structures, enabling the application of histopathological interpretation at the organelle level to analyze a whole organ. Its deep tissue imaging capability leads to less sectioning, resulting in negligible sectioning artifact. mPAM offers a new way to better understand complex biological organs.

Optical-based sensing approaches have long been an indispensable way to detect molecules in biological tissues for various biomedical research and applications. The advancement in optical microscopy is one of the main drivers for discoveries and innovations in both life science and biomedical imaging. However, the shallow imaging depth due to the use of ballistic photons fundamentally limits optical imaging approaches’ translational potential to a clinical setting. Photoacoustic (PA) tomography (PAT) is a rapidly growing hybrid imaging modality that is capable of acoustically detecting optical contrast. PAT uniquely enjoys high-resolution deep-tissue imaging owing to the utilization of diffused photons. The exploration of endogenous contrast agents and the development of exogenous contrast agents further improve the molecular specificity for PAT. PAT’s versatile design and non-invasive nature have proven its great potential as a biomedical imaging tool for a multitude of biomedical applications. In this review, representative endogenous and exogenous PA contrast agents will be introduced alongside common PAT system configurations, including the latest advances of all-optical acoustic sensing techniques.

Accelerating imaging speed in optical microscopy is often realized at the expense of image contrast, image resolution, and detection sensitivity – a common predicament for advancing high-speed and high-throughput cellular imaging. We here demonstrate a new imaging approach, called asymmetric-detection time-stretch optical microscopy (ATOM), which can deliver ultrafast label-free high-contrast flow imaging with well delineated cellular morphological resolution and in-line optical image amplification to overcome the compromised imaging sensitivity at high speed. We show that ATOM can separately reveal the enhanced phase-gradient and absorption contrast in microfluidic live-cell imaging at a flow speed as high as ~10 m/s, corresponding to an imaging throughput of ~100,000 cells/sec. ATOM could thus be the enabling platform to meet the pressing need for intercalating optical microscopy in cellular assay, e.g. imaging flow cytometry – permitting high-throughput access to the morphological information of the individual cells simultaneously with a multitude of parameters obtained in the standard assay.

We demonstrate optical time-stretch confocal microscopy in the 1 μm spectral window for high-speed and high-resolution cellular imaging. In contrast to the prior demonstrations of time-stretch imaging, which all operated in the telecommunication band, the present work extends the utility of this imaging modality to a wavelength regime (~1 μm), which is well known to be the optimal diagnostic window in biophotonics. This imaging technique enables us to image the nasopharyngeal epithelial cells with cellular resolution (<2 μm), at a line scan rate of 10 MHz, and with a field of view as wide as ~0.44 mm × 0.1 mm. We also theoretically and experimentally characterized the system performance. As the low-loss dispersive fibers for the time-stretch process as well as other essential optical components for enhancing the imaging sensitivity are commonly available at 1 μm, time-stretch confocal microscopy in this wavelength range could usher in realizing high-speed cell imaging with an unprecedented throughput.

Histological images can reveal rich cellular information of tissue sections, which are widely used by pathologists in disease diagnosis. However, the gold standard for histopathological examination is based on thin sections on slides, which involves inevitable time-consuming and labor-intensive tissue processing steps, hindering the possibility of intraoperative pathological assessment of the precious patient specimens. Here, by incorporating ultraviolet photoacoustic microscopy (UV-PAM) with deep learning, we show a rapid and label-free histological imaging method that can generate virtually stained histological images (termed Deep-PAM) for both thin sections and thick fresh tissue specimens. With the tissue non-destructive nature of UV-PAM, the imaged intact specimens can be reused for other ancillary tests. We demonstrated Deep-PAM on various tissue preparation protocols, including formalin-fixation and paraffin-embedding sections (7-µm thick) and frozen sections (7-µm thick) in traditional histology, and rapid assessment of intact fresh tissue (~ 2-mm thick, within 15 min for a tissue with a surface area of 5 mm × 5 mm). Deep-PAM potentially serves as a comprehensive histological imaging method that can be simultaneously applied in preoperative, intraoperative, and postoperative disease diagnosis.

Photoacoustic computed tomography (PACT), a fast-developing modality for deep tissue imaging, images the spatial distribution of optical absorption. PACT usually treats the absorption coefficient as a scalar. However, the absorption coefficients of many biological tissues exhibit an anisotropic property, known as dichroism or diattenuation, which depends on molecular conformation and structural alignment. Here, we present a novel imaging method called dichroism-sensitive PACT (DS-PACT), which measures both the amplitude of tissue's dichroism and the orientation of the optic axis of uniaxial dichroic tissue. By modulating the polarization of linearly polarized light and measuring the alternating signals through lock-in detection, DS-PACT can boost dichroic signals from biological tissues. To validate the proposed approach, we experimentally demonstrated the performance of DS-PACT by imaging plastic polarizers and ex vivo bovine tendons deep inside scattering media. We successfully detected the orientation of the optic axis of uniaxial dichroic materials, even at a depth of 4.5 transport mean free paths. We anticipate that the proposed method will extend the capability of PACT to imaging tissue absorption anisotropy.