Quantitative photoacoustic evaluation of graded ischemic stroke and the therapeutic efficacy of low-intensity transcranial ultrasound stimulation

This study demonstrates that chlorophosphonazo III (CPZ III) can be used as a contrast agent for photoacoustic calcium imaging. CPZ III can pass across the plasma membrane for labeling intracellular Ca2+ without cytotoxicity. In optical-resolution photoacoustic microscopy (OR-PAM), the photoacoustic (PA) signal intensity was strongly correlated with the presence of CPZ III and Ca2+ at various concentrations. The sensitivity of PA signal reception was enhanced by using an 8 MHz single-element focused ultrasound detector due to their matched frequency characteristics. Differences in the PA signal intensity were successfully found between the core and margin areas of tumorspheres in three-dimensional cell cultures. These findings indicate that CPZ III can serve as a novel PA contrast agent for functional Ca2+ imaging using OR-PAM.

Low-intensity transcranial ultrasound stimulation (LITUS) is an emerging non-invasive neuromodulation technique for pain treatment, with the unique ability to modulate deep brain nuclei associated with pain. The aim of this study is to systematically review and summarize the evidence for the efficacy of LITUS in pain management and to elucidate the potential mechanisms underlying its analgesic effects. A systematic search was conducted across five databases up to Mar 31st, 2025. Controlled studies in both human and animal subjects were included. Two independent reviewers completed the screening and risk of bias assessment process following predefined inclusion and exclusion criteria. A total of thirteen studies were included in the review. These studies demonstrated LITUS's potential in managing various types of pain among different populations and animal models, particularly targeting the anterior cingulate cortex, thalamus, insular cortex, primary sensorimotor cortex, and periaqueductal gray. Most included studies showed positive effects and verified the safety of LITUS on pain, reporting few adverse effects. LITUS is an effective and non-invasive tool for pain regulation in animals and humans, enabling precise modulation of deep brain circuits. Analgesic effects may be affected by pain-related risk factors, insufficient dosage, suboptimal protocols, and target selection. Initial evidence has highlighted the direct link between LITUS parameters, brain region responses, and pain behavior. Modulation of brain excitatory, nociceptive circuit, electrophysiological response, autonomic response, biochemistry, neuroinflammation, and psychology are proposed as the potential mechanisms underlying the efficacy of LITUS. More high-quality research is urgently needed to advance clinical LITUS use and reveal its mechanisms.

Imaging intracellular calcium dynamics to evaluate the cellular activity is crucial for an understanding of the related cell behavior and signal transduction. Identifying spatial-temporal activity patterns in three-dimensional (3D) tumor cell culture can provide novel insights into calcium-associated tumorigenesis and lead to optimizing the tumor-killing strategies. However, the information cannot be obtained without adequate penetration in 3D cell culture. Herein, we develop optical-resolution photoacoustic microscopy (OR-PAM) by tuning the laser wavelength to 627 nm to investigate calcium waves in 3D tumor cell culture using a novel photoacoustic contrast agent, Chlorophosphonazo-III (CPZ). CPZ has a peak optical absorbance at 650 nm. Not like another Bisazo derivative that can also be used for PA calcium imaging, Arsenazo-III, non-toxic and the high survival rate is observed even when cells are treated with 150 μM of CPZ (<97%). Moreover, CPZ can differentiate calcium changes using single wavelength like some conventional single wavelength calcium indicators used in optical imaging. Phantom results show that the photoacoustic (PA) signal intensity is highly correlated with calcium concentration using 100 μM or 150 μM CPZ, where R2 values are 0.94 and 0.97, respectively. To investigate the feasibility of live-cell PA calcium imaging in 3D cell culture, we evoke the intracellular calcium puffs of tumorspheres using thapsigargin and high concentration of extracellular calcium. A 2-fold enhancement of PA calcium intensity is observed after stimulating tumorspheres with thapsigargin or extracellular calcium. It demonstrates that the functional calcium imaging in 3D tumor cell culture can be detected by our OR-PAM system and CPZ can serve as a functional PA contrast agent.

Efficacy of Low-Intensity Transcranial Ultrasound Stimulation in Enhancing Motor Recovery via Modulation of Microglial Polarization, Angiogenesis and Neurogenesis in MCAO Rats.

In medical imaging, a number of imaging modalities have been used to visualize the structural information of different internal organs in the human body and some modalities can even visualize structures at a cellular level for diagnostic and treatment purposes. Optical resolution photoacoustic microscopy (OR-PAM) is one of the emerging imaging modalities to analyze the anatomy and functionality of tissues non-invasive. It is a hybrid imaging technology that combines photoacoustic (PA) contrast and acoustic resolution to reconstruct images of tissues in humans and animals. However, in OR-PAM the received ultrasonic signal by the thermal expansion of tissues has a low signal-to-noise ratio because most of the signal power is lost in the conversion process from light to acoustic waves which makes it difficult to visualize the structural information in the PA images. Traditional denoising methods such as wiener filter, bandpass filter, wavelet-based denoising, noise reduction by SVD and dictionary-based denoising methods can denoise PA images to some extent but it is still a difficult task to preserve structural information in the images by such methods. In this research, a convolutional autoencoder (CAE) based model is proposed to denoise and learn the structural patterns of blood vessels in PA images. To achieve this task, a CAE model is first trained between noisy and Gabor filtered sub-images, those contain the patterns of different vascular structures. Then, the trained model is used to approximate the denoise version of the input noisy sub-images of blood vessels. The proposed model is trained and tested on PA images of blood vessels of a mouse ear, acquired by the OR-PAM imaging system and the results show that our proposed method can effectively approximate and reconstruct the noisy vascular structures than traditional images denoising filtering methods.

A cross-correlation-based method is proposed to quantitatively measure transverse flow velocity using optical resolution photoacoustic (PA) microscopy enhanced with a digital micromirror device (DMD). The DMD is used to alternately deliver two spatially separated laser beams to the target. Through cross-correlation between the slow-time PA profiles measured from the two beams, the speed and direction of transverse flow are simultaneously derived from the magnitude and sign of the time shift, respectively. Transverse flows in the range of 0.50 to 6.84 mm/s are accurately measured using an aqueous suspension of 10-μm-diameter microspheres, and the root-mean-squared measurement accuracy is quantified to be 0.22 mm/s. The flow measurements are independent of the particle size for flows in the velocity range of 0.55 to 6.49 mm/s, which was demonstrated experimentally using three different sizes of microspheres (diameters: 3, 6, and 10 μm). The measured flow velocity follows an expected parabolic distribution along the depth direction perpendicular to the flow. Both maximum and minimum measurable velocities are investigated for varied distances between the two beams and varied total time for one measurement. This technique shows an accuracy of 0.35 mm/s at 0.3-mm depth in scattering chicken breast, making it promising for measuring flow in biological tissue.

Detection of weak optical absorption by optical-resolution photoacoustic microscopy

We demonstrate the feasibility of simultaneous multi-probe detection for an optical-resolution photoacoustic microscopy (OR-PAM) system. OR-PAM has elicited the attention of biomedical imaging researchers because of its optical absorption contrast and high spatial resolution with great imaging depth. OR-PAM allows label-free and noninvasive imaging by maximizing the optical absorption of endogenous biomolecules. However, given the inadequate absorption of some biomolecules, detection sensitivity at the same incident intensity requires improvement. In this study, a modulated continuous wave with power density less than 3 mW/cm2 (1/4 of the ANSI safety limit) excited the weak photoacoustic (PA) signals of biological cells. A microcavity transducer is developed based on the bulk modulus of gas five orders of magnitude lower than that of solid; air pressure variation is inversely proportional to cavity volume at the same temperature increase. Considering that a PA wave expands in various directions, detecting PA signals from different positions and adding them together can increase detection sensitivity and signal-to-noise ratio. Therefore, we employ four detectors to acquire tiny PA signals simultaneously. Experimental results show that the developed OR-PAM system allows the label-free imaging of cells with weak optical absorption.

Photoacoustic (PA) imaging (PAI), or optoacoustic imaging, is a hybrid imaging modality that combines optical absorption contrast and ultrasound image formation. In PAI, the target is excited by a short laser pulse and subsequently absorbs the photon energy, leading to a transient local temperature rise. The temperature rise induces a local pressure rise that propagates as acoustic waves. As acoustic waves generally undergo less scattering and attenuation in tissue compared with light, PAI can provide high-resolution images in both the optical (quasi)ballistic and (quasi)diffusive regimes (1,2). Based on the image formation methods, PAI can be classified into two categories: photoacoustic microscopy (PAM) and photoacoustic computed tomography (PACT). PAM uses a focused excitation light beam and/or a focused single-element ultrasonic transducer for direct image formation through position scanning (1,2). PAM has a maximum imaging depth ranging from a few hundred micrometers to a few millimeters with spatial resolution ranging from sub-micrometer to sub-millimeter (2,3). PAM can be further classified into optical-resolution PAM (OR-PAM) and acoustic-resolution PAM (AR-PAM). For both OR-PAM and AR-PAM, the axial resolution is determined by the bandwidth of the ultrasonic transducer (4). OR-PAM works in the optical (quasi)ballistic regime, whereas the light is tightly focused that it can penetrate about one optical transport mean free path (~1 mm in soft tissue). Therefore, the lateral resolution of OR-PAM is mainly determined by the optical focal spot size (4-6). The optical focusing is diffraction-limited as λ/2NA, where λ is the light wavelength, and NA is the numerical aperture of objective lens. On the contrary, in AR-PAM, the laser is loosely focused to fulfill the entire acoustic focal spot, thereby penetrating a few optical transport mean free paths, i.e., in the quasi-diffusive regime. The lateral resolution of AR-PAM is thus determined by the size of acoustic focus (4,7,8), limited by acoustic diffraction. In PACT, the object is illuminated with a wide-field laser beam in the diffusive regime, and the generated acoustic waves are detected at multiple locations or by using a multi-element transducer array. The image formed by PACT is reconstructed by an inverse algorithm. The spatial resolution of PACT is fundamentally limited by acoustic diffraction, and additionally affected by the directionality and spacing of the detector elements (9). Recently, several studies have shown that sub-diffraction imaging of biological samples can be achieved through PAI by breaking optical-diffraction limit in the (quasi)ballistic regime or acoustic-diffraction limit in the (quasi)diffusive regime, which have opened new possibilities for fundamental biological studies. Yao et al. developed a photoimprint PAM using the intensity-dependent photobleaching effect and acquired a melanoma cell PA image with a lateral resolution of 90 nm (10). Danielli et al. reported a label-free PA nanoscopy based on the optical-absorption saturation effect and acquired a mitochondria PA image with a lateral resolution of 88 nm (11). Chaigne et al. exploited the sample-dynamics-induced inherent temporal fluctuation in the PA signals and achieved a resolution enhancement of about 1.4 over conventional PACT (12). Murray et al. broke the acoustic diffraction limit by implementing a blind speckle illumination and block-FISTA reconstruction algorithm and achieved a resolution close to the acoustic speckle size (13). Dean-Ben et al. also overcame the acoustic diffraction limit by incorporating rapid sequential acquisition of 3D PA images of flowing absorbing particles and further enhanced the visibility of structures under limited-view tomographic conditions (14). Conkey et al. optimized wavefront shaping with photoacoustic feedback and achieved up to ten times improvement in signal-to-noise ratio and five to six times sub-acoustic-diffraction resolution (15). In this concise review, we summarize and analyze the recent development in super-resolution (SR) PAI (SR-PAI) in both the optical (quasi)ballistic and (quasi)diffusive regime, as well as their representative applications. We also discuss the current challenges in SR-PAI and envision the potential breakthroughs.

Photoacoustic (PA) imaging is rapidly progressing imaging modality in which very short pulsed laser causes thermal expansion to generate PA signal. In previous PA microscope systems, relatively long time was required for image acquisition because they required mechanical scan of the PA transducer. The objective of the present study is to develop fast laser scanning optical resolution photoacoustic microscopy (OR-PAM) with fixed PA signal detector to realize very high frame rate PA imaging. Q-switched Nd:YAG laser with the wavelength of 532 nm, pulse width of 5.5 ns and pulse repetition rate of up to 50 kHz was equipped in the system. Low frequency PA detector was comprised of a glass prism configuring acoustic focusing and a polymethyl methacrylate (PMMA) prism with 5 MHz PZT (lead zirconate titanate) transducer. High frequency PA detector was comprised of the same glass prism and a glass prism with ZnO (zinc oxide) thin film transducer. Galvano scanner operating in the air was controlled by a microcomputer to scan laser beam. PA signal was detected with the fixed PA detector thus realized the frame rate of 5 fps for C-mode equivalent to 500 fps for B-mode. The lateral resolution of the low frequency system was found to be 11.6 μm and the blood vessel on the surface of cod roe was clearly visualized. The system may be applicable for imaging blood flow and vascular dynamics of micro vessel.

Optical resolution photoacoustic microscopy (OR-PAM) has been shown as a promising tool for label-free microvascular and single-cell imaging in clinical and bioscientific applications. However, most OR-PAM systems are realized by using a bulky laser for photoacoustic excitation. The large volume and high price of the laser may restrain the popularity of OR-PAM. In this study, we attempt to develop a compact, portable, and low cost OR-PAM based on a consumer Blu-ray (405 nm) DVD pickup head for label-free micro-vascular imaging and red-blood-cell related blood examination. According to the high optical absorption of the hemoglobin at 405 nm, the proposed OR-PAM has potential to be an alternative for the conventional optical microscopy in the examinations of hematological morphology for blood routine. We showed that the Blu-ray DVD pickup head owns the required laser energy and focusing optics for OR-PAM. The firmware of a Blu-ray DVD drive was modified to allow its pickup head to generate nano-second laser pulses with a tunable pulse repetition rate of &gt;30 kHz and a tunable pulse width ranging from 10 to 30 ns. The laser beam was focused onto the target after passing through a transparent cover slide, and then aligned to be confocal with a 50-MHz focused ultrasonic transducer in forward mode. To keep the target on focus, a scan involving auto-tracking procedure was performed. The measured maximum achievable lateral resolution was 1 &mu;m which was mainly limited by the minimum step size of the used motorized stage. A blood smear was imaged without any staining. The red blood cells were well resolved and the biconcave structure could be clearly visualized. In addition, to verify the in vivo imaging capability of the proposed OR-PAM, the micro-vasculature of a mouse ear was imaged without any contrast agent. The results showed that it performed better than a 200x digital optical microscope in terms of image contrast and vascular morphology. In summaries, the proposed OR-PAM has been demonstrated as a promising tool for label-free blood imaging in both small animal studies and blood examinations, and potentially can be a compact and low-cost OR-PAM platform.

Optical resolution photoacoustic microscopy (ORPAM) has demonstrated both high resolution and rich contrast imaging of optical chromophores in biologic tissues. To date, sensitivity remains a major challenge for ORPAM, which limits the capability of resolving biologic microvascular networks. In this study, we propose and evaluate a new ORPAM modality termed as optical resolution photoacoustic computed microscopy (ORPACM), through the combination of a two-dimensional laser-scanning system with a medical ultrasonographic platform. Apart from conventional ORPAMs, we record multiple photoacoustic (PA) signals using a 128-element ultrasonic transducer array for each pulse excitation. Then, we apply a reconstruction algorithm to recover one depth-resolved PA signal referred to as an A-line, which reveals more detailed information compared with conventional single-element transducer-based ORPAMs. In addition, we carried out both in vitro and in vivo experiments as well as quantitative analyses to show the advanced features of ORPACM.

Optical resolution photoacoustic microscopy (OR-PAM) provides high-resolution, label-free and non-invasive functional imaging for broad biomedical applications. Dual-polarized fiber laser sensors have high sensitivity, low noise, a miniature size, and excellent stability; thus, they have been used in acoustic detection in OR-PAM. Here, we review recent progress in fiber-laser-based ultrasound sensors for photoacoustic microscopy, especially the dual-polarized fiber laser sensor with high sensitivity. The principle, characterization and sensitivity optimization of this type of sensor are presented. In vivo experiments demonstrate its excellent performance in the detection of photoacoustic (PA) signals in OR-PAM. This review summarizes representative applications of fiber laser sensors in OR-PAM and discusses their further improvements.

Optical resolution photoacoustic microscopy (OR-PAM) can achieve an optical-dependent high lateral resolution (<; 1 μm) and visualize photoacoustic (PA) property at single-cell level. The spatial resolvability of the PA imaging is expected to precisely characterize biological activities of cells such as their interactions with drugs and contrast agents by comparing the acquired PA characteristics with the cell's morphology. However, since the conventional OR-PAM systems only visualize objects that generate PA signals, it was difficult to acquire a PA image and an optical morphological image simultaneously. Here, we report a novel system of the hybrid optical / PA microscopy that can acquire morphological and PA characteristics of cells at the same time. The performance of the developed system was validated by measuring a USAF1951 target and bovine red blood cells.

Photoacoustic (PA) imaging is one of the fastest growing imaging technologies nowadays in both research and clinical applications, especially due to its unique capability to visualize blood vessels. The PA microscopy (PAM) is classified into two types: optical-resolution PAM (OR-PAM) and acoustic-resolution PAM (AR-PAM). OR-PAM image has a point spread function (PSF) much smaller than AR-PAM because it uses a tightly focused optical beam and the PSF is determined by the optical focus. In contrast, AR-PAM uses an unfocused optical illumination to excite a relatively large area and detects the PA signal from a small area determined by its acoustic focus. Because ultrasound is less scattered than light in biological tissue, AR-PAM can achieve deeper imaging depth than OR-PAM at the expense of image resolution. Due to the limited resolution and imaging depth scale of each PAM type, it is challenging to image vessels in various area of small animals. In this study, we demonstrated in vivo OR-/AR-PAM imaging of blood vessels in various areas such as eye, ear, and hind limb by using a single commercial PAM system. Additionally, we quantified micro-vessel density (MVD) of the mouse eye and ear images, and applied a synthetic aperture focusing technique (SAFT) to correct the distorted PA signal at the out-of-focus in AR-PAM image. As a result, we have demonstrated multiscale PAM imaging of small animal vasculature in various areas with vessel quantification and resolution enhancement, so we believe that this multiscale PAM imaging technique would be helpful in biology research such as ischemia and neovascularization.