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Topical corticosteroids manage inflammatory skin conditions via their action on the immune system. An effect of application of corticosteroids to the skin is skin blanching caused by peripheral vasoconstriction. This has been used to characterize, in some cases relative potency and also as a way to compare skin penetration. Chromameters have been used to assess skin blanching—the outcome of vasoconstriction caused by topical corticosteroids—but do not directly measure vasoconstriction. Here, we demonstrate quantitative volumetric photoacoustic microscopy (PAM) as a tool for directly assessing the vasoconstriction followed by topical corticosteroid application, noninvasively visualizing skin vasculature without any exogeneous contrast agent. We photoacoustically differentiated the vasoconstrictive ability of four topical corticosteroids in small animals through multiparametric analyses, offering detailed 3D insights into vasoconstrictive mechanisms across different skin depths. Our findings highlight the potential of PAM as a noninvasive tool for measurement of comparative vasoconstriction with potential for clinical, pharmaceutical, and bioequivalence applications.

A fast scanner of optical-resolution photoacoustic microscopy is inherently vulnerable to perturbation, leading to severe image distortion and significant misalignment among multiple 2D or 3D images. Restoration and registration of these images is critical for accurately quantifying dynamic information in long-term imaging. However, traditional registration algorithms face a great challenge in computational throughput. Here, we develop an unsupervised deep learning based registration network to achieve real-time image restoration and registration. This method can correct artifacts from B-scan distortion and remove misalignment among adjacent and repetitive images in real time. Compared with conventional intensity based registration algorithms, the throughput of the developed algorithm is improved by 50 times. After training, the new deep learning method performs better than conventional feature based image registration algorithms. The results show that the proposed method can accurately restore and register the images of fast-scanning photoacoustic microscopy in real time, offering a powerful tool to extract dynamic vascular structural and functional information.

Photoacoustic microscopy (PAM) has gained increasing popularity in biomedical imaging, providing new opportunities for tissue monitoring and characterization. With the development of deep learning techniques, convolutional neural networks have been used for PAM image resolution enhancement and denoising. However, there exist several inherent challenges for this approach. This work presents a Unified PhotoAcoustic Microscopy image reconstruction Network (UPAMNet) for both PAM image super-resolution and denoising. The proposed method takes advantage of deep image priors by incorporating three effective attention-based modules and a mixed training constraint at both pixel and perception levels. The generalization ability of the model is evaluated in details and experimental results on different PAM datasets demonstrate the superior performance of the method. Experimental results show improvements of 0.59 dB and 1.37 dB, respectively, for 1/4 and 1/16 sparse image reconstruction, and 3.9 dB for image denoising in peak signal-to-noise ratio.

Photoacoustic microscopy is advancing with research on utilizing ultraviolet and visible light. Dual-wavelength approaches are sought for observing DNA/RNA- and vascular-related disorders. However, the availability of high numerical aperture lenses covering both ultraviolet and visible wavelengths is severely limited due to challenges such as chromatic aberration in the optics. Herein, we present a groundbreaking proposal as a pioneering simulation study for incorporating multilayer metalenses into ultraviolet-visible photoacoustic microscopy. The proposed metalens has a thickness of 1.4 µm and high numerical aperture of 0.8. By arranging cylindrical hafnium oxide nanopillars, we design an achromatic transmissive lens for 266 and 532 nm wavelengths. The metalens achieves a diffraction-limited focal spot, surpassing commercially available objective lenses. Through three-dimensional photoacoustic simulation, we demonstrate high-resolution imaging with superior endogenous contrast of targets with ultraviolet and visible optical absorption bands. This metalens will open new possibilities for downsized multispectral photoacoustic microscopy in clinical and preclinical applications.

Pathology interpretations of tissue rely on the gold standard of histology imaging, potentially hampering timely access to critical information for diagnosis and management of neoplasms because of tedious sample preparations. Slide-free capture of cell nuclei in unprocessed specimens without staining is preferable; however, inevitable irregular surfaces in fresh tissues results in limitations. An ultraviolet metasurface with the ability to generate an ultraviolet optical focus maintaining

In addition to offering morphological visualizations via capture of the spatial distributions of optical absorption, photoacoustic imaging technology can reveal abundant physical information about biological particles, including their orientation, density, and viscoelasticity, through analysis of the pressure transients in the spectral domain. However, the low-amplitude wideband photoacoustic signals of intrinsic microscopic optically-absorbing objects under the action of confined photoacoustic excitation power continue to hinder simultaneous photoacoustic structural imaging and spectroscopic analysis of the nonfluorescent chromophores in living biological tissues because of the inadequate responses to photoacoustic impulses observed in most photoacoustic imaging setups that include piezoelectric transducers. Building upon a recently-developed optical evanescent wave sensor that can respond to ultrasound with high sensitivity over a broad frequency range, we propose in vivo spatial-spectral photoacoustic microscopy for recovery of structural imaging in three dimensions and characterization of anatomical features in the acoustic frequency domain. Label-free photoacoustic images of a living zebrafish are acquired in which spectroscopically-resolved differentiation of the microarchitecture is accessed, along with isometric micrometer-scale volumetric visualizations. The proposed imaging technology could potentially provide more comprehensive evaluations of the physiopathological status of living small animals.

Neurovascular imaging is essential for investigating neurodegenerative diseases. However, the existing neurovascular imaging technology suffers from a trade-off between a field of view (FOV) and resolution in the whole brain, resulting in an inhomogeneous resolution and lack of information. Here, homogeneous-resolution arched-scanning photoacoustic microscopy (AS-PAM), which has an ultrawide FOV to cover the entire mouse cerebral cortex, was developed. Imaging of the neurovasculature was performed with a homogenous resolution of 6.9 µm from the superior sagittal sinus to the middle cerebral artery and caudal rhinal vein in an FOV of 12 × 12 mm2. Moreover, using AS-PAM, vascular features of the meninges and cortex were quantified in early Alzheimer’s disease (AD) and wild-type (WT) mice. The results demonstrated high sensitivity to the pathological progression of AD on tortuosity and branch index. The high-fidelity imaging capability in large FOV enables AS-PAM to be a promising tool for precise brain neurovascular visualization and quantification.

Photoacoustic imaging (PAI) has emerged as a molecular-selective imaging technology based on optical absorption contrast. Dichroism-sensitive photoacoustic (DS-PA) imaging has been reported, where the absorption coefficient has a vector characteristic, featuring dimensions of contrast in polarization and wavelength. Herein, we present a DS-PA microscopy (DS-PAM) system that implements optical anisotropy contrast and molecular selectivity. Moreover, we propose mathematical solutions to fully derive dichroic properties. A wavelength for the PAI of collagenous tissue was used, and the proposed algorithms were validated using linear dichroic materials. We successfully mapped dichroic information in fibrous tissue imaging based on the degree of anisotropy and axis orientation, and also deduced mechanical assessment from the tissue arrangement. The proposed DS-PAM system and algorithms have great potential in various diagnostic fields using polarimetry, such as musculoskeletal and cardiovascular systems.

In this paper, we propose an efficient label-free in vivo photoacoustic (PA) imaging of melanoma using a condensed near infrared-I (NIR-I) supercontinuum light source. Although NIR-II spectral window is advantageous such as longer penetration depth compared to the NIR-I region, supercontinuum light sources emitting both NIR-I and NIR-II region could lower the efficiency to target melanoma because of low optical power density in the melanoma’s absorption spectra. To exploit efficient in vivo PA imaging of melanoma, we demonstrated the light source emitting from visible (532–600 nm) to NIR-I (600–1000 nm) by optimizing stimulated Raman scattering induced supercontinuum generation. The melanoma’s structure is successfully differentiated from blood vessels at a high pulse energy of 2.5 µJ and a flexible pulse repetition rate (PRR) of 5–50 kHz. The proposed light source with the microjoules energies and tens of kHz of PRR can potentially accelerate clinical trials such as early diagnosis of melanoma.

Spatial sampling density and data size are important determinants of the imaging speed of photoacoustic microscopy (PAM). Therefore, undersampling methods that reduce the number of scanning points are typically adopted to enhance the imaging speed of PAM by increasing the scanning step size. Since undersampling methods sacrifice spatial sampling density, by considering the number of data points, data size, and the characteristics of PAM that provides three-dimensional (3D) volume data, in this study, we newly reported deep learning-based fully reconstructing the undersampled 3D PAM data. The results of quantitative analyses demonstrate that the proposed method exhibits robustness and outperforms interpolation-based reconstruction methods at various undersampling ratios, enhancing the PAM system performance with 80-times faster-imaging speed and 800-times lower data size. The proposed method is demonstrated to be the closest model that can be used under experimental conditions, effectively shortening the imaging time with significantly reduced data size for processing.

Optical-resolution photoacoustic microscopy offers high-resolution, label-free hemodynamic and functional imaging to many biomedical applications. However, long-standing technical barriers, such as limited field of view, bulky scanning probes, and slow imaging speed, have limited the application of optical-resolution photoacoustic microscopy. Here, we present freehand scanning photoacoustic microscopy (FS-PAM) that can flexibly image various anatomical sites. We develop a compact handheld photoacoustic probe to acquire 3D images with high speed, and great flexibility. The high scanning speed not only enables video camera mode imaging but also allows for the first implementation of simultaneous localization and mapping (SLAM) in photoacoustic microscopy. We demonstrate fast in vivo imaging of some mouse organs, and human oral mucosa. The high imaging speed greatly reduces motion artifacts and distortions from tissue moving, breathing, and unintended handshaking. We demonstrate small-lesion localization in a large region of the brain. FS-PAM offers a flexible high-speed imaging tool with an extendable field of view, enabling more biomedical imaging applications.

The ischemic stroke animal model evaluates the efficacy of reperfusion and neuroprotective strategies for ischemic injuries. Various conventional methods have been reported to induce the ischemic models; however, controlling specific neurological deficits, mortality rates, and the extent of the infarction is difficult as the size of the affected region is not precisely controlled. In this paper, we report a single laser-based localized target ischemic stroke model development method by simultaneous vessel monitoring and photothrombosis induction using photoacoustic microscopy (PAM), which has minimized the infarct size at precise location with high reproducibility. The proposed method has significantly reduced the infarcted region by illuminating the precise localization. The reproducibility and validity of suggested method have been demonstrated through repeated experiments and histological analyses. These results demonstrate that our method can provide the ischemic stroke model closest to the clinical pathology for brain ischemia research from inducement, occurrence mechanisms to the recovery process.

In most multispectral optical-resolution photoacoustic microscopy (OR-PAM), spatial scanning is repeated for each excitation wavelength, which decreases throughput and causes motion artifacts during spectral processing. This study proposes a new spectroscopic OR-PAM technique to acquire information on the photoacoustic signal intensity and excitation wavelength from single spatial scans. The technique involves irradiating an imaging target with two broadband optical pulses with and without wavelength-dependent time delays. The excitation wavelength of the sample is then calculated by measuring the time delay between the photoacoustic signals generated by the two optical pulses. This technique is validated by measuring the excitation wavelengths of dyes in tubes. Furthermore, we demonstrate the three-dimensional spectroscopic OR-PAM of cells stained with suitable dyes. Although the tradeoff between excitation efficiency and excitation bandwidth must be adjusted based on the application, combining the proposed technique with fast spatial scanning methods can significantly contribute to recent OR-PAM applications, such as monitoring quick biological events and microscale tracking of moving materials.

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-resolution photoacoustic microscopy (OR-PAM) enjoys superior spatial resolution and has received intense attention in recent years. The application, however, has been limited to shallow depths because of strong scattering of light in biological tissues. In this work, we propose to achieve deep-penetrating OR-PAM performance by using deep learning enabled image transformation on blurry living mouse vascular images that were acquired with an acoustic-resolution photoacoustic microscopy (AR-PAM) setup. A generative adversarial network (GAN) was trained in this study and improved the imaging lateral resolution of AR-PAM from 54.0 µm to 5.1 µm, comparable to that of a typical OR-PAM (4.7 µm). The feasibility of the network was evaluated with living mouse ear data, producing superior microvasculature images that outperforms blind deconvolution. The generalization of the network was validated with in vivo mouse brain data. Moreover, it was shown experimentally that the deep-learning method can retain high resolution at tissue depths beyond one optical transport mean free path. Whilst it can be further improved, the proposed method provides new horizons to expand the scope of OR-PAM towards deep-tissue imaging and wide applications in biomedicine.

Near-infrared photoacoustics receives increasing interest as an intravital modality to sense key biomolecules. One of the most central types of biomolecules of interest are lipids as they constitute essential bio-hallmarks of cardiovascular and metabolic diseases and their in-vivo detection holds insightful information about disease progression and treatment monitoring. However, the full potential of near-infrared photoacoustic for high-resolution and high-sensitivity biomedical studies of lipids has so far not been exploited due a lack of appropriate excitation sources delivering short-pulses at high-repetition-rate, high-pulse-energy, and wavelength around 1200 nm. Here, we demonstrate a custom-built SRS fiber amplifier that provides optical excitations at 1192.8 nm, repetition rates of 200 kHz, pulse durations below 2 ns, and pulse energies beyond 5 μJ. We capitalize on the performance of our excitation source and show near-infrared photoacoustics resolving intrinsic lipid contrast in biomedically relevant specimens ranging from single cells to lipid-rich tissue with subcellular resolution.

Ultraviolet photoacoustic microscopy (UV-PAM) has been investigated to provide label-free and registration-free volumetric histological images for whole organs, offering new insights into complex biological organs. However, because of the high UV absorption of lipids and pigments in tissue, UV-PAM suffers from low image contrast and shallow image depth, hindering its capability for revealing various microstructures in organs. To improve the UV-PAM imaging contrast and imaging depth, here we propose to implement a state-of-the-art optical clearing technique, CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis), to wash out the lipids and pigments from tissues. Our results show that the UV-PAM imaging contrast and quality can be significantly improved after tissue clearing. With the cleared tissue, multilayers of cell nuclei can also be extracted from time-resolved PA signals. Tissue clearing-enhanced UV-PAM can provide fine details for organ imaging.

The investigation of neuronal activity in non-human primate models is of critical importance due to their genetic similarity to human brains. In this study, we tested the feasibility of using photoacoustic imaging for the detection of cortical and subcortical responses due to peripheral electrical stimulation in a squirrel monkey model. Photoacoustic computed tomography and photoacoustic microscopy were applied on squirrel monkeys for real-time deep subcortical imaging and optical-resolution cortical imaging, respectively. The electrically evoked hemodynamic changes in primary somatosensory cortex, premotor cortices, primary motor cortex, and underlying subcortical areas were measured. Hemodynamic responses were observed in both cortical and subcortical brain areas at the cortices during external stimulation, demonstrating the feasibility of photoacoustic technique for functional imaging of non-human primate brain.

In recent years, many methods have been investigated to improve imaging speed in photoacoustic microscopy (PAM). These methods mainly focused upon three critical factors contributing to fast PAM: laser pulse repetition rate, scanning speed, and computing power of the microprocessors. A high laser repetition rate is fundamentally the most crucial factor to increase the PAM speed. In this paper, we review methods adopted for fast PAM systems in detail, specifically with respect to light sources. To the best of our knowledge, ours is the first review article analyzing the fundamental requirements for developing high-speed PAM and their limitations from the perspective of light sources.

High-speed optical-resolution photoacoustic microscopy (OR-PAM), integrating the merits of high spatial resolution and fast imaging acquisition, can observe dynamic processes of the optical absorption-based molecular specificities. However, it remains challenging for the evaluation to morphological and physiological parameters that are closely associated with photoacoustic spectrum due to the inadequate ultrasonic frequency response of the routinely-employed piezoelectric transducer. By utilizing the galvanometer for fast optical scanning and our previously-developed surface plasmon resonance sensor as an unfocused broadband ultrasonic detector, high-speed spectroscopic photoacoustic imaging was accessed in the OR-PAM system, achieving an acoustic bandwidth of ∼125 MHz and B-scan rate at ∼200 Hz over a scanning range of ∼0.5 mm. Our system demonstrated the dynamic imaging of the moving phantoms’ structures and the simultaneous characterization of their photoacoustic spectra over time. Further, fast volumetric imaging and spectroscopic analysis of microanatomic features of a zebrafish eye ex vivo was obtained label-freely.

Optical-resolution photoacoustic microscopy (OR-PAM) can provide functional, anatomical, and molecular images at micrometer level resolution with an imaging depth of less than 1 mm in tissue. However, the imaging speed of traditional OR-PAM is often low due to the point-by-point mechanical scanning and cannot capture time-sensitive dynamic information. In this work, we demonstrate a recent effort in improving the imaging speed of OR-PAM, using a newly developed water-immersible two-axis scanner. Driven by water-compatible electromagnetic actuation force, the new scanning mirror employs a novel torsion-bending mechanism to achieve fast 2D scanning. The torsion scanning along the fast-axis works in the resonant model, and the bending scanning along the slow-axis operate at the quasi-static mode. The scanning speed and scanning range along the two axes can be independently adjusted. Steered by the two-axis torsion-bending scanning mirror immersed in water, the focused excitation light and the generated acoustic wave can be confocally aligned over the entire imaging area. Thus, a high imaging speed can be achieved without sacrificing the detection sensitivity. Equipped with the torsion-bending scanner, the high-speed OR-PAM system has achieved a cross-sectional frame rate of 400 Hz, and a volumetric imaging speed of 1 Hz over a field of view of 1.5 × 2.5 mm2. We have also demonstrated high-speed OR-PAM of the hemodynamic changes in response to pharmaceutical and physiological challenges in small animal models in vivo. We expect the torsion-bending scanner based OR-PAM will find matched biomedical studies of tissue dynamics.

Photoacoustic (PA) microscopy (PAM) has achieved remarkable progress in biomedicine in the past decade. It is a fast-rising imaging modality with diverse applications, such as hemodynamics, oncology, metabolism, and neuroimaging. Combining optical excitation and acoustic detection, the hybrid nature of PAM provides advantages of rich contrast and deep penetration. In recent years, high-speed PAM has flourished and enabled high‐speed wide‐field imaging of functional activity. In this review, we summarize the most recent advances in high-speed PAM technologies, including high-repetition-rate multi-wavelength laser development, fast scanning techniques, and novel PA signal acquisition strategies.

Functional imaging of microvascular dynamics in extremities delivers intuitive information for early detection, diagnosis, and prognosis of vascular diseases. High-resolution and high-speed photoacoustic microscopy (PAM) visualizes and measures multiparametric information of microvessel networks in vivo such as morphology, flow, oxygen saturation, and metabolic rate. Here, we demonstrate high-resolution photoacoustic monitoring of vascular dynamics in human fingers. We photoacoustically monitored the position displacement of blood vessels associated with arterial pulsation in human fingers. Then, during and after arterial occlusion, we photoacoustically quantified oxygen consumption and blood perfusion in the fingertips. The results demonstrate that high-resolution functional PAM could be a vital tool in peripheral vascular examination for measuring heart rate, oxygen consumption, and/or blood perfusion.

The point-by-point scanning mechanism of photoacoustic microscopy (PAM) results in low-speed imaging, limiting the application of PAM. In this work, we propose a method to improve the quality of sparse PAM images using convolutional neural networks (CNNs), thereby speeding up image acquisition while maintaining good image quality. The CNN model utilizes attention modules, residual blocks, and perceptual losses to reconstruct the sparse PAM image, which is a mapping from a 1/4 or 1/16 low-sampling sparse PAM image to a latent fully-sampled one. The model is trained and validated mainly on PAM images of leaf veins, showing effective improvements quantitatively and qualitatively. Our model is also tested using in vivo PAM images of blood vessels of mouse ears and eyes. The results suggest that the model can enhance the quality of the sparse PAM image of blood vessels in several aspects, which facilitates fast PAM and its clinical applications.

Photoacoustic microscopy (PAM) is an emerging imaging method combining light and sound. However, limited by the laser’s repetition rate, state-of-the-art high-speed PAM technology often sacrifices spatial sampling density (i.e., undersampling) for increased imaging speed over a large field-of-view. Deep learning (DL) methods have recently been used to improve sparsely sampled PAM images; however, these methods often require time-consuming pre-training and large training dataset with ground truth. Here, we propose the use of deep image prior (DIP) to improve the image quality of undersampled PAM images. Unlike other DL approaches, DIP requires neither pre-training nor fully-sampled ground truth, enabling its flexible and fast implementation on various imaging targets. Our results have demonstrated substantial improvement in PAM images with as few as 1.4 % of the fully sampled pixels on high-speed PAM. Our approach outperforms interpolation, is competitive with pre-trained supervised DL method, and is readily translated to other high-speed, undersampling imaging modalities.

Photoacoustic imaging (PAI), featuring rich contrast, high spatial/temporal resolution and deep penetration, is one of the fastest-growing biomedical imaging technology over the last decade. To date, numbers of handheld and semi-handheld photoacoustic imaging devices have been reported with corresponding potential clinical applications. Here, we summarize emerged handheld and semi-handheld systems in terms of photoacoustic computed tomography (PACT), optoacoustic mesoscopy (OAMes), and photoacoustic microscopy (PAM). We will discuss each modality in three aspects: laser delivery, scanning protocol, and acoustic detection. Besides new technical developments, we also review the associated clinical studies, and the advantages/disadvantages of these new techniques. In the end, we propose the challenges and perspectives of miniaturized PAI in the future.

Background: Photoacoustic microscopy is expected to have clinical applications as a noninvasive and three-dimensional (3D) method of observing intradermal structures., Objective: Investigate the applicability of a photoacoustic microscope equipped with two types of pulsed lasers that can simultaneously recognize hemoglobin and melanin., Methods: 16 skin lesions including erythema, pigmented lesions, vitiligo and purpura, were analyzed to visualize 3D structure of melanin granule distribution and dermal blood vessels. 13 cases of livedo racemosa in cutaneous polyarteritis nodosa (cPN) were further analyzed to visualize the 3D structure of dermal blood vessels in detail. Vascular structure was also analyzed in the biopsy specimens obtained from tender indurated erythema of cPN by CD34 immunostaining., Results: Hemoglobin-recognition signal clearly visualized the 3D structure of dermal blood vessels and melanin-recognition signal was consistently reduced in vitiligo. In livedo racemosa, the hemoglobin-recognition signal revealed a relatively thick and large reticular structure in the deeper layers that became denser and finer toward the upper layers. The numerical analysis revealed that the number of dermal blood vessels was 1.29-fold higher (p<0.05) in the deeper region of the lesion than that of normal skin. The CD34 immunohistochemical analysis in tender indurated erythema revealed an increased number of dermal vessels compared with normal skin in 88.9% (8/9) of the cases, suggesting that vascular network remodeling had occurred in cPN., Conclusion: The photoacoustic system has an advantage in noninvasively detecting dermal blood vessel structures that are difficult to recognize by two-dimensional histopathology specimen examination and is worth evaluating in various skin diseases., Competing Interests: Conflicts of Interest None declared., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The goal of this study is to further develop a multi-modality eye imaging system and evaluate its feasibility of acquiring images of different modalities simultaneously. An integrated multimodality imaging system combining spectral-domain optical coherence tomography (SD-OCT), photoacoustic microscopy (PAM), and fluorescence microscopy (FM) was developed, and its performance for eye imaging was validated on multiple clinically-relevant retinal disease models in vivo in rabbits. OCT imaging allows for visualization of the different anatomic retinal layers with high axial resolution. PAM can be used to image vasculature, angiogenesis, and hemorrhages. The leakage of neovascularization can be verified with FM and fluorescein dye. Simultaneous imaging with OCT, PAM, and FM ensures co-registration of the three modalities without being affected by motion artifacts caused by breathing, body or eye movements, and heartbeat. This simultaneous multi-modality eye imaging system could be a new tool for applications both in ophthalmology and other fields.

We report wide-field polygon-scanning functional OR-PAM that for the first time achieves 1-MHz A-line rate of oxygen saturation in vivo. We address two technical challenges. The first is a 1-MHz dual-wavelength pulsed laser that has sufficient pulse energy and ultrafast wavelength switching. The second is a polygon-scanning imaging probe that has a fast scanning speed, a large field of view, and great sensitivity. The OR-PAM system offers a B-scan rate of 477.5 Hz in a 12-mm range and a volumetric imaging rate of ∼1 Hz over a 12 × 5 mm2 scanning area. We image microvasculature and blood oxygen saturation in a 12 × 12 mm2 scanning area in 5 s. Dynamic imaging of oxygen saturation in the mouse ear is demonstrated to monitor fast response to epinephrine injection. The new wide-field fast functional imaging ability broadens the biomedical application of OR-PAM.

Photoacoustic imaging (PAI) takes advantage of both optical and ultrasound imaging properties to visualize optical absorption with high resolution and contrast. Photoacoustic microscopy (PAM) is usually categorized with all-optical microscopy techniques such as optical coherence tomography or confocal microscopes. Despite offering high sensitivity, novel imaging contrast, and high resolution, PAM is not generally an all-optical imaging method unlike the other microscopy techniques. One of the significant limitations of photoacoustic microscopes arises from their need to be in physical contact with the sample through a coupling media. This physical contact, coupling, or immersion of the sample is undesirable or impractical for many clinical and pre-clinical applications. This also limits the flexibility of photoacoustic techniques to be integrated with other all-optical imaging microscopes for providing complementary imaging contrast. To overcome these limitations, several non-contact photoacoustic signal detection approaches have been proposed. This paper presents a brief overview of current non-contact photoacoustic detection techniques with an emphasis on all-optical detection methods and their associated physical mechanisms.

Multispectral photoacoustic microscopy uses a wavelength-dependent absorption difference as a contrast mechanism to image the target molecule. In this paper, we present a novel multispectral pulsed fiber laser source, which selectively alternates the excitation wavelengths between green and red colors based on the stimulated Raman scattering (SRS) effect for imaging. This laser has a high pulse repetition rate (PRR) of 300 kHz and high pulse energy of more than 200 nJ meeting the real-time requirements of optical-resolution photoacoustic microscopy imaging. By switching the polarization state of the pump light and optical paths of the pump light, the operating wavelengths of the light source can be selectively alternated at the same fast PRR for any two SRS peak wavelengths between 545 and 655 nm. At 545 nm excitation wavelength, molecular photoacoustic signals from both blood vessels and gold nanorods were obtained simultaneously. However, at 655 nm, the photoacoustic signals of gold nanorods were dominant because the absorption of light by the blood vessels decreased drastically in the spectral region over 600 nm. Thus the multispectral photoacoustic system designed using the novel laser source implemented here could simultaneously monitor the time-dependent fast movement of two molecules independently, having different wavelength-dependent absorption properties at a high repetition rate of 0.49 frames per second (fps).

Bone microvasculature plays a paramount role in bone marrow maintenance, development, and hematopoiesis. Studies of calvarian vascular patterns within living mammalian skull with the available intravital microscopy techniques are limited to small scale observations. We developed an optical-resolution optoacoustic microscopy method combined with ultrasound biomicroscopy in order to reveal and discern the intricate networks of calvarian and cerebral vasculature over large fields of view covering majority of the murine calvaria. The vasculature segmentation method is based on an angle-corrected homogeneous model of the rodent skull, generated using simultaneously acquired three-dimensional pulse-echo ultrasound images. The hybrid microscopy design along with the appropriate skull segmentation method enable high throughput studies of a living bone while facilitating correct anatomical interpretation of the vasculature images acquired with optical resolution optoacoustic microscopy.

Among the numerous endogenous biological molecules, information on lipids is highly coveted for understanding both aspects of developmental biology and research in fatal chronic diseases. Due to the pronounced absorption features of lipids in the extended near-infrared region (1650−1850 nm), visualisation and identification of lipids become possible using multi-spectral photoacoustic (optoacoustic) microscopy. However, the spectroscopic studies in this spectral region require lasers that can produce high pulse energies over a broad spectral bandwidth to efficiently excite strong photoacoustic signals. The most well-known laser sources capable of satisfying the multi-spectral photoacoustic microscopy requirements (tunability and pulse energy) are tunable nanosecond optical parametric oscillators. However, these lasers have an inherently large footprint, thus preventing their use in compact microscopy systems. Besides, they exhibit low-repetition rates. Here, we demonstrate a compact all-fibre, high pulse energy supercontinuum laser that covers a spectral range from 1440 to 1870 nm with a 7 ns pulse duration and total energy of 18.3 μJ at a repetition rate of 100 kHz. Using the developed high-pulse energy source, we perform multi-spectral photoacoustic microscopy imaging of lipids, both ex vivo on adipose tissue and in vivo to study the development of Xenopus laevis tadpoles, using six different excitation bands over the first overtone transition of C–H vibration bonds (1650−1850 nm).

Carotid artery atherosclerosis is a main cause of stroke. Understanding atherosclerosis biology is critical in the development of targeted prevention and treatment strategies. Consequently, there is demand for advanced tools investigating atheroma pathology. We consider hybrid optoacoustic and multiphoton microscopy for the integrated and complementary interrogation of plaque tissue constituents and their mutual interactions. Herein, we visualize human carotid plaque using a hybrid multimodal imaging system that combines optical resolution optoacoustic (photoacoustic) microscopy, second and third harmonic generation microscopy, and two-photon excitation fluorescence microscopy. Our data suggest more comprehensive insights in the pathophysiology of atheroma formation and destabilization, by enabling congruent visualization of structural and biological features critical for the atherosclerotic process and its acute complications, such as red blood cells and collagen.

We demonstrate optical resolution photoacoustic microscopy (OR-PAM) of lipid-rich tissue using a multi-wavelength pulsed laser based on nonlinear fiber optics. 1047 nm laser pulses are converted to 1098, 1153, 1215, and 1270 nm pulses via stimulated Raman scattering in a graded-index multimode fiber. Multispectral PAM of a lipid phantom is demonstrated with our low-cost and simple technique.

High resolution ultrasound and photoacoustic images of stained neutrophils, lymphocytes and monocytes from a blood smear were acquired using a combined acoustic/photoacoustic microscope. Photoacoustic images were created using a pulsed 532 nm laser that was coupled to a single mode fiber to produce output wavelengths from 532 nm to 620 nm via stimulated Raman scattering. The excitation wavelength was selected using optical filters and focused onto the sample using a 20× objective. A 1000 MHz transducer was co-aligned with the laser spot and used for ultrasound and photoacoustic images, enabling micrometer resolution with both modalities. The different cell types could be easily identified due to variations in contrast within the acoustic and photoacoustic images. This technique provides a new way of probing leukocyte structure with potential applications towards detecting cellular abnormalities and diseased cells at the single cell level.

The quantized vibration of chemical bonds provides a way of detecting specific molecules in a complex tissue environment. Unlike pure optical methods, for which imaging depth is limited to a few hundred micrometers by significant optical scattering, photoacoustic detection of vibrational absorption breaks through the optical diffusion limit by taking advantage of diffused photons and weak acoustic scattering. Key features of this method include both high scalability of imaging depth from a few millimeters to a few centimeters and chemical bond selectivity as a novel contrast mechanism for photoacoustic imaging. Its biomedical applications spans detection of white matter loss and regeneration, assessment of breast tumor margins, and diagnosis of vulnerable atherosclerotic plaques. This review provides an overview of the recent advances made in vibration-based photoacoustic imaging and various biomedical applications enabled by this new technology.

Three-dimensional photoacoustic microscopy (PAM) has gained considerable attention within the biomedical imaging community during the past decade. Detecting laser-induced photoacoustic waves by optical sensing techniques facilitates the idea of all-optical PAM (AOPAM), which is of particular interest as it provides unique advantages for achieving high spatial resolution using miniaturized embodiments of the imaging system. The review presents the technology aspects of optical-sensing techniques for ultrasound detection, such as those based on optical resonators, as well as system developments of all-optical photoacoustic systems including PAM, photoacoustic endoscopy, and multi-modality microscopy. The progress of different AOPAM systems and their representative applications are summarized.

We have developed reflection-mode multispectral photoacoustic microscopy (PAM) based on a novel optical–acoustic objective that integrates a customized ultrasonic transducer and a commercial reflective microscope objective into one solid piece. This technical innovation provides zero chromatic aberration and convenient confocal alignment of the optical excitation and acoustic detection. With a wavelength-tunable optical-parametric-oscillator laser, we have demonstrated multispectral PAM over an ultrabroad spectral range of 270–1300 nm. A near-constant lateral resolution of ∼2.8 μm is achieved experimentally. Capitalizing on the consistent performance over the ultraviolet, visible, and near-infrared range, multispectral PAM enables label-free concurrent imaging of cell nucleus (DNA/RNA contrast at 270 nm), blood vessel (hemoglobin contrast at 532 nm), and sebaceous gland (lipid contrast at 1260 nm) at the same spatial scale in a living mouse ear.