Optical-resolution photoacoustic microscopy (OR-PAM) has recently gained significant attention from the biomedical-imaging community as it provides labelfree optical contrast from physiologically relevant tissue chromophores that are located a few millimeters deep, with subcellular spatial resolution [1
]. In OR-PAM, a tightly focused laser pulse illuminates the tissue and generates wideband acoustic waves from light-absorbing chromophores that are then detected by an acoustic transducer. Time-resolved photoacoustic waves, in combination with the two-dimensional raster scanning along the x
plane (lateral dimension), generate three-dimensional data from which maximum amplitude projection (MAP) and volume-rendered photoacoustic images can be created.
Conventional OR-PAM setups use complex imaging geometries to coaxially align optical illumination and acoustic detection paths. In early OR-PAM setups [6
], coaxial alignment was achieved using an acoustic-optic prism combiner consisting of one right-angle prism and one rhomboid prism pressed tightly to a thin layer of silicone oil. The laser light was focused by a system of optical lenses and then passed through the prism combiner before irradiating the tissue. A correction lens was attached to the prism combiner to refocus the light that was defocused through the combiner. Tissue-generated photoacoustic waves propagated through the rhomboid prism and were reflected by the silicone oil layer into the ultrasound detector attached to the prism. Since the entire imaging head, consisting of the above acoustic-optic prism combiner, the transducer, and the focused light, was moved to scan the subject, these systems exhibited slow acquisition speed, limited field of view (FOV), and significant acoustic loss.
The current generation of OR-PAMs reflect the light, instead of the acoustic waves, by sandwiching an aluminium foil in the acoustic-optic combiner. This allows dual axis optic only scanning using a two dimensional galvo mirror to improve the image acquisition speed and generate a wide FOV [7
]. The entire imaging head, including the galvo mirror, is submerged in a large volume (70 × 40 × 20 mm3
) of a nonconducting liquid coupling medium that rests above the imaged subject. Such a bulky imaging head limits high throughput and wearable imaging applications because it constrains animal imaging performed under anesthesia and causes discomfort to living subjects. Moreover, acoustic loss here is still significant because acoustic waves travel through the large coupling medium and the prism combiner before being detected by the transducer.
Alternatively, some OR-PAMs include a ring-shaped single-element ultrasound transducer to eliminate the off-axis alignment problems of optical illumination and acoustic detection. The focused light is directly delivered through a hole at the center of the transducer, or coupled using a single-mode fiber integrated with a gradient-index (GRIN) lens. The imaging head is then two-dimensionally raster-scanned using mechanical stages to generate volumetric images [8
]. Although the imaging head is miniaturized in these OR-PAM systems, the FOV, numerical aperture, and imaging speed (due to physical scanning of the imaging head) are still limited. Besides, they still require a-few-milimeter thick water coupling medium above the imaged subject due to long working distances.
The above drawbacks of conventional OR-PAM systems can be addressed if ultrasound detectors are transparent to light. To achieve this, all-optical ultrasound detection technologies, such as Fabry–Pérot etalons [13
], microring resonators [14
], and other photonic integrated circuits [15
] were studied for PAM. Although these are transparent technologies offering high photoacoustic sensitivity, they require complex fiber integration with an additional laser source and other optical-detection instruments. More importantly, they lack ultrasound excitation capabilities for applications that require combined ultrasound sensing and ultrasound tissue stimulation [16
]. Recently, transparent capacitive micromachined ultrasonic transducers (CMUTs) were developed [18
]. However, CMUTs need specialized front-end application-specific integrated circuits (ASICs), and involve a complicated fabrication process inside a cleanroom.
In order to address all the above limitations, we recently reported a photoacoustic-imaging technique using an optically transparent bulk piezoelectric lithium niobate (LiNbO3
) ultrasound transducer [20
offers several advantages over other piezoelectric materials:
On the basis of these advantages, our previous work [20
] introduced two transparent photoacoustic-imaging schematics, one that directly integrated a multimode optical fiber with the transparent LiNbO3
transducer, and the other that involved the optical-only scanning of the laser spot over a 10 × 10 mm2
Extending the window-transducer approach, here we present a novel OR-PAM that allows the optical-only scanning of a tightly focused light beam through a 10 × 10 mm2 single-element LiNbO3 transparent-ultrasound-transducer (TUT) window and demonstrates its applicability to image biological samples. The spatial resolution and signal-to-noise ratio (SNR) of the OR-PAM system were characterized using imaging experiments on resolution test targets and carbon-fiber phantoms. The biological imaging capabilities of the OR-PAM were studied using ex ovo chick-embryo chorioallantoic-membrane (CAM) vasculature and imaging melanoma phantoms through a piece of mouse skin.
The proposed TUT-based OR-PAM approach simplifies the coaxial alignment of optic and acoustic paths without the need for additional optical components (such as acoustic-optic prism combiners and correction lenses) and a large acoustic-coupling medium. Our approach provides other advantages:
The TUT can be fixed onto the imaging object (such as the skull of a mouse) to facilitate wearable imaging without a thick coupling medium. In the future, this will likely help in imaging the brains of freely behaving or awake mice in combination with ultrasound stimulation;
Depending on the size of the TUT, it enables the high-speed scanning of large areas with single-channel data acquisition.
In the future, our TUT approach can also be integrated into conventional optical microscopes to realize a multimodal microscopy platform with ultrasound-stimulation capabilities.
The rest of this paper is organized as follows. Section 2
describes the process of TUT fabrication and a schematic representation of the OR-PAM setup. TUT characterization and validation studies, including carbon-fiber imaging, CAM vasculature, and melanoma depth profiling, are presented in Section 3
. The advantages of the proposed TUT-based OR-PAM system, the limitations, and its future directions are discussed in Section 4
A novel OR-PAM system based on a transparent LiNbO3 ultrasound transducer was developed, characterized, and validated using both inanimate and biological subjects. The transducer was ITO-coated with 80% optical transmission in the visible and near-infrared optical-wavelength regions, and had a center frequency of 13 MHz with a fractional photoacoustic bandwidth of 60%. The resultant transparency of the LiNbO3 transducer facilitated a shared pathway for both light and acoustic-wave propagation.
Our approach also removed the need for additional optical components (such as acoustic-optic prisms) and large-coupling media used in conventional OR-PAM systems. Instead, the OR-PAM presented in this work has a much smaller, lighter imaging head—the TUT itself—with minimal acoustic coupling. Imaging experiments demonstrated an SNR of 38 dB, and a lateral and axial resolution of 8.5 and 150 µm, respectively.
The OR-PAM’s feasibility of vascular imaging was demonstrated using Day 4 CAM and melanoma depth profiling using melanoma-tissue phantoms. In the future, the proposed TUT could be integrated in conventional optical-microscopy techniques to allow multimodal microscopy that is capable of both ultrasound stimulation and sensing. Eventually, the TUT technology could be further scaled to develop miniaturized photoacoustic endomicroscopy and microendoscopy devices for space-constrained point-of-care clinical applications, e.g., prostate and pancreas needle biopsies [35