Photoacoustic Microscopy System Research Articles

High-speed high-resolution laser diode-based photoacoustic microscopy for in vivo microvasculature imaging

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.

Chain-like gold nanoparticle clusters for multimodal photoacoustic microscopy and optical coherence tomography enhanced molecular imaging

Colloidal gold nanoparticles (GNPs) serve as promising contrast agents in photoacoustic (PA) imaging, yet their utility is limited due to their absorption peak in the visible window overlapping with that of hemoglobin. To overcome such limitation, this report describes an ultrapure chain-like gold nanoparticle (CGNP) clusters with a redshift peak wavelength at 650 nm. The synthesized CGNP show an excellent biocompatibility and photostability. These nanoparticles are conjugated with arginine-glycine-aspartic acid (RGD) peptides (CGNP clusters-RGD) and validated in 12 living rabbits to perform multimodal photoacoustic microscopy (PAM) and optical coherence tomography (OCT) for visualization of newly developed blood vessels in the sub-retinal pigment epithelium (RPE) space of the retina, named choroidal neovascularization (CNV). The PAM system can achieve a 3D PAM image via a raster scan of 256 × 256 pixels within a time duration of 65 s. Intravenous injection of CGNP clusters-RGD bound to CNV and resulted in up to a 17-fold increase in PAM signal and 176% increase in OCT signal. Histology indicates that CGNP clusters could disassemble, which may facilitate its clearance from the body.

Compact long-working-distance laser-diode-based photoacoustic microscopy with a reflective objective

Photoacoustic microscopy (PAM) has quickly developed into a noninvasive biomedical imaging technique to achieve detection, diagnosis, and monitoring. Compared with Q-switched neodymium-doped yttrium aluminum garnet or optical parametric oscillator lasers, a low-cost and small-size laser diode (LD) used as an alternative light source is conducive to achieving the miniaturization and integration for preclinical transformation. However, the LD’s low peak output power needs the high numerical aperture objective to attain tight focus, which limits the working distance (WD) of the system in only 2–3 mm, resulting in not achieving the backward coaxial confocal mode. Here, we present a compact visible LD-based PAM system with a reflective objective to achieve a 22 mm long WD and a 10 µm lateral resolution. Different depth subcutaneous microvascular networks in label-free mouse ears have successfully reappeared in vivo with a signal-to-noise ratio up to 14 dB by a confocal alignment. It will be a promising tool to develop into a handy tool for subcutaneous blood vessel imaging.

Beamforming for large-area scan and improved SNR in array-based photoacoustic microscopy

Beamforming enhances the performance of array-based photoacoustic microscopy (PAM) systems for large-area scan. In this study, we quantify the imaging performance of a large field-of-view optical-resolution photoacoustic-microscopy system using an phased-array detector. The system combines a low-cost pulsed-laser diode with a 128-element linear ultrasound probe. Signal-to-noise ratio (SNR) and generalized contrast-to-noise ratio (gCNR) are quantified using the phased-array detector and applying three beamforming strategies: a no-beamforming method equivalent to a single-element flat transducer, a fixed focus beamforming method that mimics a single-element focused transducer, and a dynamic focus beamforming using a delay-and-sum (DAS) algorithm. The imaging capabilities of the system are demonstrated generating high-resolution images of tissue-mimicking phantoms containing sub-millimetre ink tubes and an ex vivo rabbit’s ear. The results show that dynamic focus DAS beamforming increases and homogenizes SNR along 1-cm2 images, reaching values up to 15 dB compared to an unfocused detector and up to 30 dB compared to out-of-focus regions of the fixed focus configuration. Moreover, the obtained values of gCNR using the DAS beamformer indicate an excellent target visibility, both on phantoms and ex vivo. This strategy makes it possible to scan larger surfaces compared to standard configurations using single-element detectors, paving the way for advanced array-based PAM systems.

Retinal safety evaluation of photoacoustic microscopy

Photoacoustic microscopy (PAM) has significant potential as a promising diagnostic method for eye diseases and can provide anatomic and functional information of the retinal and choroidal vasculature. However, there are no FDA-approved PAM systems for ophthalmic imaging. In this study, a comprehensive safety evaluation was performed to evaluate the safety of PAM retinal imaging and whether PAM causes damage to retinal structure or function in rabbit eyes. 12 Dutch-Belted pigmented rabbits received photoacoustic imaging to 57% of the retinal surface area with a laser energy of 5% of the ANSI safety limit for five consecutive days and followed before imaging and 3 days, 1, 2, 3, and 4 weeks post imaging. Retinal morphologic analyses using slit lamp examination, fundus photography, red free, FA, FAF, ICGA, and OCT showed no retinal hemorrhage, edema, detachment, vascular abnormalities, or pigmentary abnormalities in the retina or choroid after PAM imaging. Full-field ERG analysis showed no significant difference in scotopic or photopic a- and b-wave amplitudes or implicit times between the control and experimental eyes over time (n = 6, P values > 0.05). Retinal ultrastructural evaluation using TEM showed normal structure of organelles and nuclei, and no significant loss of cells after PAM. TUNEL assay showed no evidence of cells apoptosis in retina. Retinal histopathology indicated that the architecture and thickness of the retinal layers was well preserved in all experimental eyes. A positive control at 500% of the ANSI limit demonstrated significant damage. The comprehensive retinal safety evaluation demonstrated no damage to retinal structure or function for 4 weeks after PAM imaging in rabbits.

Label-free imaging of lipid-rich biological tissues by mid-infrared photoacoustic microscopy.

.Significance: Mid-infrared (IR) imaging based on the vibrational transition of biomolecules provides good chemical-specific contrast in label-free imaging of biology tissues, making it a popular tool in both biomedical studies and clinical applications. However, the current technology typically requires thin and dried or extremely flat samples, whose complicated processing limits this technology’s broader translation.Aim: To address this issue, we report mid-IR photoacoustic microscopy (PAM), which can readily work with fresh and thick tissue samples, even when they have rough surfaces.Approach: We developed a transmission-mode mid-IR PAM system employing an optical parametric oscillation laser operating in the wavelength range from 2.5 to . Due to its high sensitivity to optical absorption and the low ultrasonic attenuation of tissue, our PAM achieved greater probing depth than Fourier transform IR spectroscopy, thus enabling imaging fresh and thick tissue samples with rough surfaces.Results: In our spectroscopy study, the symmetric stretching at (3508 nm) was found to be an excellent source of endogenous contrast for lipids. At this wavenumber, we demonstrated label-free imaging of the lipid composition in fresh, manually cut, and unprocessed tissue sections of up to 3-mm thickness.Conclusions: Our technology requires no time-consuming sample preparation procedure and has great potential in both fast clinical histological analysis and fundamental biological studies.

Multiscale high-speed photoacoustic microscopy based on free-space light transmission and a MEMS scanning mirror.

The conventional photoacoustic microscopy (PAM) system allows trade-offs between lateral resolution and imaging depth, limiting its applications in biological imaging in vivo. Here we present an integrated optical-resolution (OR) and acoustic-resolution (AR) multiscale PAM based on free-space light transmission and fast microelectromechanical systems (MEMS) scanning. The lateral resolution for OR is 4.9 µm, and the lateral resolution for AR is 114.5 µm. The maximum imaging depth for OR is 0.7 mm, and the maximum imaging depth for AR is 4.1 mm. The imaging speed can reach 50 k Alines per second. The high signal-to-noise ratios and wavelength throughput are achieved by delivering light via free-space, and the high speed is achieved by a MEMS scanning mirror. The blood vasculature from superficial skin to the deep tissue of a mouse leg was imaged in vivo using two different resolutions to demonstrate the multiscale imaging capability.

In vivo volumetric monitoring of revascularization of traumatized skin using extended depth-of-field photoacoustic microscopy.

Faster and better wound healing is a critical medical issue. Because the repair process of wounds is closely related to revascularization, accurate early assessment and postoperative monitoring are very important for establishing an optimal treatment plan. Herein, we present an extended depth-of-field photoacoustic microscopy system (E-DOF-PAM) that can achieve a constant spatial resolution and relatively uniform excitation efficiency over a long axial range. The superior performance of the system was verified by phantom and in vivo experiments. Furthermore, the system was applied to the imaging of normal and trauma sites of volunteers, and the experimental results accurately revealed the morphological differences between the normal and traumatized skin of the epidermis and dermis. These results demonstrated that the E-DOF-PAM is a powerful tool for observing and understanding the pathophysiology of cutaneous wound healing.

Ultralow energy photoacoustic microscopy for ocular imaging in vivo.

.Significance: The development of ultralow energy photoacoustic microscopy (PAM) on the clinically relevant pigmented rabbit eye model paves a road toward translation of the emerging PAM technology in ophthalmology clinics.Aim: Since the eye is particularly vulnerable to laser damage, we aim to develop an ultralow energy PAM system to significantly improve the laser safety of PAM by increasing the sensitivity of the system and reducing the incident laser energy for imaging.Approach: A multichannel data acquisition circuit with two-stage signal amplification was specially designed, which, in combination with the application of 3 by 3 median filter in the spatial domain, significantly improved the signal-to-noise ratio of the PAM system. The safety of this system was validated by histopathology, fluorescein angiography, and fundus photography.Results: Experiments performed on pigmented rabbits demonstrated that, when using this ultralow energy PAM system, satisfactory image quality can be achieved in the eye with an incident laser fluence that is only 1% of the American National Standards Institute safety limit. Fundus photography, fluorescein angiography, and histopathology were performed after the imaging procedure, and no retinal or ocular damage was observed.Conclusions: The proposed ultralow energy PAM system has excellent safety and holds potential to be developed into a clinical tool for ocular imaging.

Resolution-matched reflection mode photoacoustic microscopy and optical coherence tomography dual modality system

Photoacoustic microscopy (PAM) and optical coherence tomography (OCT) are sensitive to optical absorption and scattering characteristics, respectively. As such, the integration of these two modalities in order to combine important complementary information has garnered much attention. Due to the relatively low axial resolution of PAM, PAM and OCT dual modality systems generally have a large resolution gap, especially for reflection mode systems. In this study, based on a wide-band transparent pure-optical ultrasonic detector, we developed a dual modality system (PAM-OCT system) in which PAM has a similar spatial resolution (i.e. several micrometers in both the lateral and axial directions) to OCT. In addition, due to the optical transparency advantage, the integrated system works in reflection mode, which is ideal for in vivo biomedical imaging. We successfully imaged the skin of a mouse hindlimb, which cannot be done by a transmission mode dual modality system. Our work demonstrates this dual modality system has potential in biomedical studies with complementary imaging contrasts.

High-speed wide-field multi-parametric photoacoustic microscopy.

Capable of imaging blood perfusion, oxygenation, and flow simultaneously at the microscopic level, multi-parametric photoacoustic microscopy (PAM) has quickly emerged as a powerful tool for studying hemodynamic and metabolic changes due to physiological stimulations or pathological processes. However, the low scanning speed poised by the correlation-based blood flow measurement impedes its application in studying rapid microvascular responses. To address this challenge, we have developed a new, to the best of our knowledge, multi-parametric PAM system. By extending the optical scanning range with a cylindrically focused ultrasonic transducer (focal zone, 76µm×4.5mm) for simultaneous acquisition of 500 B-scans, the new system is 112 times faster than our previous multi-parametric system that uses a spherically focused transducer (focal diameter, 40 µm) and enables high-resolution imaging of blood perfusion, oxygenation, and flow over an area of 4.5×1mm2 at a frame rate of 1 Hz. We have demonstrated the feasibility of this system in the living mouse ear. Further development of this system into reflection mode will enable real-time cortex-wide imaging of hemodynamics and metabolism in the mouse brain.

Development of optical resolution photoacoustic microscopy with sub-micron lateral resolution for visualization of cells and their structures

Photoacoustic (PA) imaging is a hybrid imaging technique with light-dependent contrast and ultrasound-dependent depth. In previous studies, the interest of PA imaging was mainly clinical research. However, in recent years, PA imaging that targets cells also has attracted attention. Cells are the smallest unit in our body, and cell abnormalities cause various diseases. Therefore, PA imaging of cells is important for elucidating the relationship between diseases and PA properties and linking the results to the clinical application of PA imaging. For PA imaging of cells and its internal structures, sub-micron lateral resolution is required. In this study, we have developed an optical resolution photoacoustic microscopy (OR-PAM) system using a high NA objective lens and a single-mode fiber to achieve sub-micron lateral resolution. With the OR-PAM, it was confirmed that the lateral resolution was better than 700 nm, and the characteristic shapes of red blood cells and melanoma cells could be visualized.

Improved Depth-of-Field Photoacoustic Microscopy with a Multifocal Point Transducer for Biomedical Imaging.

In this study, a photoacoustic microscopy (PAM) system based on a multifocal point (MFP) transducer was fabricated to produce a large depth-of-field tissue image. The customized MFP transducer has seven focal points, distributed along with the transducer’s axis, fabricated by separate spherically-focused surfaces. These surfaces generate distinct focal zones that are overlapped to extend the depth-of-field. This design allows extending the focal zone of 10 mm for the 11 MHz MFP transducer, which is a great improvement over the 0.48 mm focal zone of the 11 MHz single focal point (SFP) transducer. The PAM image penetration depths of a chicken-hemoglobin phantom using SFP and MFP transducers were measured as 5 mm and 8 mm, respectively. The significant increase in the PAM image-based penetration depth of the chicken-hemoglobin phantom was a result of using the customized MFP transducer.

Functional optical coherence tomography and photoacoustic microscopy imaging for zebrafish larvae.

We present a dual modality functional optical coherence tomography and photoacoustic microscopy (OCT-PAM) system. The photoacoustic modality employs an akinetic optical sensor with a large imaging window. This imaging window enables direct reflection mode operation, and a seamless integration of optical coherence tomography (OCT) as a second imaging modality. Functional extensions to the OCT-PAM system include Doppler OCT (DOCT) and spectroscopic PAM (sPAM). This functional and non-invasive imaging system is applied to image zebrafish larvae, demonstrating its capability to extract both morphological and hemodynamic parameters in vivo in small animals, which are essential and critical in preclinical imaging for physiological, pathophysiological and drug response studies.

Wide-field monitoring and real-time local recording of microvascular networks on small animals with a dual-raster-scanned photoacoustic microscope.

Photoacoustic microscopy (PAM) provides a new method for the imaging of small-animals with high-contrast and deep-penetration. However, the established PAM systems have suffered from a limited field-of-view or imaging speed, which are difficult to both monitor wide-field activity of organ and record real-time change of local tissue. Here, we reported a dual-raster-scanned photoacoustic microscope (DRS-PAM) that integrates a two-dimensional motorized translation stage for large field-of-view imaging and a two-axis fast galvanometer scanner for real-time imaging. The DRS-PAM provides a flexible transition from wide-field monitoring the vasculature of organs to real-time imaging of local dynamics. To test the performance of DRS-PAM, clear characterization of angiogenesis and functional detail was illustrated, hemodynamic activities of vasculature in cerebral cortex of a mouse were investigated. Furthermore, response of tumor to treatment were successfully monitored during treatment. The experimental results demonstrate the DRS-PAM holds the great potential for biomedical research of basic biology.

A Focused Optically Transparent PVDF Transducer for Photoacoustic Microscopy

This papers reports the development of a novel focused optically-transparent ultrasound transducer with a wide bandwidth for photoacoustic microscopy (PAM). It consists of a 9- $\mu \text{m}$ -thick polyvinylidene fluoride (PVDF) film coated with Indium-tin oxide (ITO) and metal electrodes, which is laminated onto a concave glass lens. A new fabrication process was developed to ensure the formation of continuous ITO coatings on the curved transducer surface without fracturing. Both optical and acoustic experiments were conducted to characterize the performance of the transducer. The testing results show that the transducer has an optical transmittance of 60% (@ 532 nm), an acoustic center frequency of 24 MHz, and an acoustic bandwidth of 26 MHz. With an acoustic numerical aperture (NA) of 0.23, it provides a 0.13-mm acoustic focal spot and a 1.6-mm focal depth. For demonstration, a PAM setup was built for conducting PA imaging on different targets. In contrast to existing PAM systems, the use of the optically-transparent focused transducer allows a simpler and more compact configuration and easier alignment of the optical excitation and acoustic focal points. Such features are especially useful for the development of miniaturized PAM systems for handheld, wearable and even endoscopic applications.

Towards virtual biopsies of gastrointestinal tissues using photoacoustic remote sensing microscopy.

Gastrointestinal (GI) tissue biopsies provide critical diagnostic information for a wide variety of conditions such as neoplastic diseases (colorectal, small bowel and stomach cancers) and non-neoplastic diseases (inflammatory disorders, infection, celiac disease). Endoscopic biopsies collect small tissue samples that require resource intensive processing to permit histopathological analysis. Unfortunately, the sparsely collected biopsy samples may fail to capture the pathologic condition because selection of biopsy sites relies on macroscopic superficial tissue features and clinician judgement. Here, we present the first all-optical non-contact label-free non-interferometric photoacoustic microscopy system capable of performing "virtual biopsies". A modular photoacoustic remote sensing (PARS™) architecture is used facilitating imaging of unprocessed tissues providing information similar to conventional histopathological staining techniques. Prospectively this would allow gastroenterologists to assess subcellular tissue morphology in situ when selecting biopsy location. Tested on preserved unstained human and freshly resected murine tissues, the presented PARS microscope rapidly retrieves images of similar area to current biopsies, while maintaining comparable quality to the current standard for histopathological analysis. Additionally, results show the first label free assessment of subsurface cellular morphology in FFPE GI tissue blocks. Clinically relevant features are recovered including cellular details such as lamina propria within colon tissue and cell nuclear structure in resected smooth muscle. Constructed with a modular architecture, this system facilitates the future development of compact imaging heads. The modular PARS system overcomes many of the challenges with imaging unstained thick tissue in situ, representing a significant milestone in the development of a clinical microscope providing virtual biopsy capabilities.

A compact scanning probe for photoacoustic microscopy using ultrasonic actuator stage

Recently, photoacoustic microscopy (PAM) has attracted attention as a novel modality for medical imaging, because PAM can visualize a-few-mm deep structures with a several-tens-micrometer spatial resolution in highly-scattering living tissues. For future use of PAM in clinics, downsizing of the scanning probe is essential. In this work, we constructed a PAM system with a miniature ultrasonic actuator that allowed us to significantly downsize the scanning probe (61 mm × 50 mm × 47 mm). We demonstrated lateral spatial resolution of ∼4.4 μm and imaging speed of 16 s/B-mode image (80 A-scan lines) in the proposed PAM system, being comparable to those of the conventional PAM system using stepping motor stage.

Fully waterproof two-axis galvanometer scanner for enhanced wide-field optical-resolution photoacoustic microscopy.

A large field-of-view and fast scanning of photoacoustic microscopy (PAM) relatively have been difficult to obtain due to the water-drowned structure of the system for the transmission of ultrasonic signals. Researchers have widely studied the achievement of a waterproof scanner for dynamic biological applications with a high-resolution and high signal-to-noise ratio. This Letter reports a novel, to the best of our knowledge, waterproof galvanometer scanner-based PAM system with a successfully attainable ${9.0}\;{\rm mm} \times {14.5}\;{\rm mm}$9.0mm×14.5mm scan region, amplitude scan rate of 40 kHz, and spatial resolution of 4.9 µm. The in vivo characterization of a mouse brain in intact-skull microvascular visualization demonstrated its capability in biomedical imaging and is anticipated to be an effective technique for various preclinical and clinical studies.

Design of photoacoustic microscope system based on labVIEW platform

A photoacoustic microscope system based on virtual instrument development environment is presented, including ultrasonic sensor, digital oscilloscope, laser diode, personal calculation and other hardware platforms. and developed supporting software and image reconstruction algorithms. In the subcutaneous angiography experiment, the distribution characteristics of the ear blood vessels in mouse were completely reproduced perfectly, and the spatial resolution of the system can reach 14um. The system and method can potentially to develop into a non-invasive biological tissue structure and functional imaging technique.