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.

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.

Laser diodes (LDs) have been considered as cost-effective and compact excitation sources to overcome the requirement of costly and bulky pulsed laser sources that are commonly used in photoacoustic microscopy (PAM). However, the spatial resolution and/or imaging speed of previously reported LD-based PAM systems have not been optimized simultaneously. In this paper, we developed a high-speed and high-resolution LD-based PAM system using a continuous wave LD, operating at a pulsed mode, with a repetition rate of 30 kHz, as an excitation source. A hybrid scanning mechanism that synchronizes a one-dimensional galvanometer mirror and a two-dimensional motorized stage is applied to achieve a fast imaging capability without signal averaging due to the high signal-to-noise ratio. By optimizing the optical system, a high lateral resolution of 4.8 μm has been achieved. In vivo microvasculature imaging of a mouse ear has been demonstrated to show the high performance of our LD-based PAM system.

Ultraviolet photoacoustic microscopy (UV-PAM) has recently been demonstrated as a potential imaging tool for surgical margin analysis (SMA). UV-PAM does not require staining or micrometer-thick slicing, which is inevitable in conventional histological imaging. To promote UV-PAM as a practical intraoperative diagnostic tool, the imaging speed should be improved while preserving the high-resolution imaging capability and simplistic system design. In this Letter, we developed a galvanometer mirror-based UV-PAM (GM-UV-PAM) system for high-speed histology-like imaging. By using a UV laser with a high repetition rate (55 kHz) and a one-dimensional galvanometer mirror, our GM-UV-PAM system can generate subcellular images in less than 15 min for a typical brain biopsy ( 5 m m × 5 m m ), with a lateral resolution of ∼ 1.0 µ m . The images of mouse brain slices obtained by our GM-UV-PAM system show that it can provide histological information for SMA.

Simultaneously monitoring cortex-wide microvascular dynamics, blood oxygenation metabolism, and neuronal activities with high spatiotemporal resolution is essential for understanding neurovascular coupling (NVC). However, it remains a challenge for existing neuroimaging tools. Here, we report a linear transducer array–based hybrid microscope (LiTA-HM) that integrates photoacoustic microscopy and confocal fluorescence microscopy to achieve simultaneous neurovascular imaging with a field of view (FOV) of 6 millimeters by 5 millimeters, a spatial resolution of ~6 micrometers, and a temporal resolution of 0.8 seconds. We design an array of eight miniature transducers to enlarge FOV and optimize a polygon mirror–based ultrafast scanner to achieve real-time imaging. Proof-of-concept imaging experiments were conducted on awake mice. Different couplings between thousands of neuron somas and vessel branches down to capillaries were observed in hypoxia and anesthesia experiments. Spatiotemporal correlations and functional connectivity in NVC were uncovered for spreading depolarization waves occurred in a drug-induced epilepsy experiment. We expect that LiTA-HM will enable broad applications of neuroimaging.

Optical-resolution photoacoustic microscopy (OR-PAM) has been widely used for imaging blood vessel and oxygen saturation of hemoglobin (sO₂), providing high-resolution functional images of living animals in vivo. However, most of them require one or multiple bulky and costly pulsed lasers, hindering their applicability in preclinical and clinical settings. In this paper, we demonstrate a reflection-mode low-cost high-resolution OR-PAM system by using two cost-effective and compact laser diodes (LDs), achieving microvasculature and sO₂ imaging with a high lateral resolution of ∼6 µm. The cost of the excitation sources has dramatically reduced by ∼20–40 times compared to that of the pulsed lasers used in state-of-the-art OR-PAM systems. A blood phantom study was performed to show a determination coefficient R² of 0.96 in linear regression analysis. Experimental results of in vivo mouse ear imaging show that the proposed dual-wavelength LD-based PAM system can provide high-resolution functional images at a low cost.