3.1.

PLD-based PAM

In 2012, the first attempt to visualize the blood vessel phantom in 3D was performed using a PLD at 905 nm with light focused by collimating and focusing lenses.³⁰ They generated preliminary 3D volumetric renderings of the knotted and helical blood vessel phantoms, which were accurately represented compared to photographs. However, the lateral resolution of their PLD-based PAM system was $500\mu \mathrm{m}$, which was not ideal for real human blood vessel imaging because some blood vessels, such as arterioles, venules, and capillaries, are extremely small, ranging from 0.5 to $30\mu \mathrm{m}$ in diameter.³¹ The next year, Zeng et al.^32,33 developed a 905 nm PLD-based OR-PAM system with a 0.8 kHz repetition rate, 100 ns pulse width, and $4.9\mu \mathrm{J}$ pulse energy, shown in Fig. 1(a). Compared with previous studies, they improved the lateral resolution from hundreds of microns to $1.5\mu \mathrm{m}$, and the whole system was assembled in portable equipment. A dead ant (a length of $\sim 3.5\mathrm{mm}$ and a diameter of 0.3 to 1 mm) was imaged to demonstrate its feasibility for imaging small animals. 2D PAM images of the dead ant are shown in Fig. 1(b), which shows the ant’s size and shape. Another PLD-based PAM system, with a 1 kHz repetition rate, 124 ns pulse width, and $3\mu \mathrm{J}$ pulse energy, imaged the phantoms made of polyethylene tubes filled with blood and a mouse ear to demonstrate the feasibility of imaging biological tissues.³⁷ Each A-line signal was averaged 128 times to achieve $\sim 13\mathrm{dB}$ of SNR, and more averages are needed to further improve the SNR.

Fig. 1

PLD-based PAM. (a) A schematic of a 905 nm PLD-based OR-PAM system. ADC, analog-to-digital converter. (b) Anatomic schematic of an ant, 2D PA MAP image of the ant, and 2D PA B-scan image along the white dashed line, respectively. NPA, normalized photoacoustic amplitude. (c) A schematic of PLD-based PAM using aspheric lens and CL for light beam collimating and focusing. DAQ, data acquisition. (d) PAM images and photographs of a mouse ear and porcine ovary, respectively. (e) A schematic of transmission-mode PLD-based PAM system. (f) A schematic of reflection-mode PLD-based PAM system. (g) Reflection-mode PLD-based PAM images of a phantom and ex vivo mouse skin. (h) A schematic of the VIS PLD-based PAM system using a reflective objective. (i) Photograph and VIS PLD-based PAM image of the microvascular structure in a mouse ear in vivo. Reproduced with permission from Refs. 3233.34.35.–36.

Erfanzadeh et al.³⁴ came up with an optical scheme combining an aspheric lens and a cylindrical lens (CL) to efficiently collimate and focus the excitation light beam of a 905 nm PLD. As shown in Fig. 1(c), the light was first collimated by an aspheric lens and then by two perpendicularly arranged CLs in perpendicular directions. After collimation, the rectangular light beam ($\sim 15\mathrm{mm}\times 10\mathrm{mm}$) was focused on the sample by an aspheric lens with a numerical aperture (NA) of 0.71. A 3.5 MHz UT was used to detect the PA signal with 128 times averaging. Benefiting from the optical design, the SNR of phantom and mouse ear images was improved by at least 12 dB in comparison with the work in Ref. 37 with a similar PLD. Images of the vasculature on ex vivo mouse ear and porcine ovarian tissue [Fig. 1(d)] with 22 dB and 25 dB SNR, respectively, demonstrate the feasibility of this low-cost OR-PAM system for imaging biological samples and potentially characterizing ovarian cancer. However, it is hard to visualize microvasculature due to the limited resolution, showing some discontinuous or mixed blood vessels in PAM images.

Another group presented PLD-based OR-PAM systems in both transmission and reflection modes.³⁵ According to the schematic of the two systems [Figs. 1(e) and 1(f)], the only difference is that the transducer was held at a 180 deg angle with the incident light in transmission mode and at a 40 deg angle in reflection mode. PAM images of tissue-mimicking phantoms and ex vivo mouse skin vasculature are shown in Fig. 1(g). They demonstrate the feasibility of the OR-PAM system with a very low-energy PLD (with an output peak power of $\sim 6\mathrm{W}$) for imaging biological tissues. However, due to the low excitation energy, averaging numerous PA signals at each position was still performed to increase the SNR. Otherwise, complex signal processing algorithms are required to reduce the number of averaging.³⁸

Motor scanning was used in the aforementioned systems, and multiple averaging was required in data acquisition (DAQ). Erfanzadeh et al.³⁹ demonstrated a laser scanning LD-PAM system without signal averaging during DAQ. The 905 nm PLD beam, with a 1 kHz repetition rate and 50 ns pulse width, was well collimated and focused into a small enough beam spot for 2D galvanometer scanning. A 3.5 MHz UT was used to detect PA signals without averaging, achieving $\sim 370$ A-lines per second. Images of the vasculature on ex vivo mouse ear and porcine ovarian tissue with $\sim 12$ and $\sim 18\mathrm{dB}$ SNR, respectively, demonstrate the feasibility of this low-cost and fast laser scanning OR-PAM system for imaging biological samples and characterization of ovarian cancer.

Very recently, a new OR-PAM system was developed with the PLD beam homogenized and shaped by a square-core multimode optical fiber (MMF).⁴⁰ In the PLD beam transport optics, a set of plano-convex aspheric lenses were first used for fiber coupling, maximizing the beam power to the MMF. A $20\mu \mathrm{m}$ circular pinhole was attached to the fiber end, followed by another set of aspheric lenses for light delivery to the samples. A 128-element linear phased array was used for the detection of PA signals. The relatively high-quality PA images of ex vivo rabbit ears, obtained with this fiber-coupled LD reflection-mode OR-PAM system, have shown the system’s great potential for the characterization of blood vessels and hair follicles.

The absorption coefficient of hemoglobin is much lower in the near-infrared (NIR) range than in the visible range, resulting in low image quality for blood capillary imaging when using NIR PLDs-based PAM systems. Zeng et al.⁴¹ developed a visible PLD OR-PAM (VIS PLD-OR-PAM) system, achieving a lateral resolution of $\sim 0.95\mu \mathrm{m}$. The PLD was operated at a 405 nm wavelength with a 1 kHz repetition rate, 174 ns pulse width, and $\sim 52\mathrm{nJ}$ pulse energy ($\sim 3$ orders of magnitude less than that of NIR PLDs). Subcutaneous microvasculature on a mouse back was clearly visualized, validating the label-free imaging feasibility of the VIS PLD-OR-PAM in superficial tissues. To ensure sufficient energy density on the samples and generate effective PA signals, a PLD with low peak power necessitates a high NA objective to focus the laser beam tightly while limiting the working distance. To overcome this dilemma, Deng et al.³⁶ developed a VIS PLD-OR-PAM system with a reflective objective ($\mathrm{NA}=0.3$) to maximize the working distance up to 22 mm [Fig. 1(h)]. In vivo imaging of blood vessels and capillaries in mouse ears using this system demonstrate its potential for preclinical applications, shown in Fig. 1(i).

It is worth noting that, with these PLDs, PAM systems could be low-cost and compact due to their economical price and small volume. However, most of the commercially available PLDs are at NIR wavelengths and provide low pulse energy of several $\mu \mathrm{J}$, resulting in a low SNR for biological imaging. To solve this issue, Manwar et al.⁴² developed an adaptive denoising filtering method to enhance the SNR of PA signals obtained using low-energy PLDs.

3.2.

CWLD-based PAM

By contrast, CWLDs are available in a wide range of wavelengths (visible and NIR). Visible CWLD-based PAM systems can obtain better SNR images even when the pulse energy is low due to the high absorption of blood in the visible range. Taking advantage of CWLDs, including the low cost and wide range of available wavelengths, they have been heavily explored to be used as an excitation source in PAM.

CWLDs are commonly operated in CW mode across various applications. CWLDs with a specific light intensity modulation scheme can be employed to generate PA signals in the frequency domain. Langer et al.⁴³ incorporated a CWLD with a wavelength of 405 nm and an output power of 120 mW in a PAM system. The CWLD was modulated with a frequency of 10 MHz to facilitate PA imaging. Figure 2(a) shows the PAM image in which the distinct donut shape of red blood cells is readily discernible. In addition, another fluorescence imaging modality was also combined in their system, providing luminescence images [Fig. 2(b)] with complementary information.

Fig. 2

CWLD-based PAM. (a) A schematic of a 405 nm CWLD-based OR-PAM system. APD, avalanche photodiode; LIA, lock-in amplifiers; f-gen, function generator. (b) PAM image of human red blood cells. (c) Corresponding luminescence image. (d) A schematic of the high-speed high-resolution LD-based PAM system. AL, aspheric lens; CL, cylindrical lens; GM, galvanometer mirror; OL, objective lens; S, sample; WT, water tank; UT, ultrasonic transducer. (e) PAM image of a leaf phantom dyed with black ink. (f) In vivo PAM images of a mouse ear. (g) A schematic of the dual-wavelength LD-based PAM system. (h) In vivo PAM image of a mouse ear with blue LD excitation. (i) ${\mathrm{sO}}_2$ image of the mouse ear. Reproduced with permission from Refs. 4344.–45.

Recently, the feasibility of using CWLDs in pulsed mode has been demonstrated by overdriving the diode while avoiding catastrophic optical damage and thermal damage. This approach represents a complementary solution to PLDs. Stylogiannis et al.⁴⁶ overdrove CWLDs at 6 different wavelengths, ranging from 445 to 830 nm with a peak current up to 45 times higher than the manufacturer-specified maximum current, resulting in short pulses of about 10 ns with a pulse energy of about 200 nJ and a repetition rate of higher than 600 kHz. The diode characterization experiment shows that CWLDs, operated in pulsed mode, can provide higher optical power than those operated in CW mode, with a stable performance at different repetition rates. Combined with a raster-scanning system, the pulse-driven 450 nm CWLD was used for PA measurement to characterize their imaging performance in a reflection-mode PAM system. The lateral and axial resolutions were 110 and $33\mu \mathrm{m}$, respectively. The vasculature of the mouse ear and human forearm at the epidermal and dermal layers was imaged by the CWLD-based PAM system with 500 times averaging. The SNR was observed to be 15 to 20 dB for the initial $100\mu \mathrm{m}$ depth and declined sharply with increasing depth, indicating the high absorption and scattering of blue light by the tissue. Another implementation of PAM, utilizing a compact fingertip 450 nm CWLD, has demonstrated the capability of both pulsed modulation and continuous modulation for PAM.⁴⁷ However, it also suffered from suboptimal resolution and imaging speed, necessitating 256 times averaging to achieve acceptable results. To generate high-resolution PAM images with high imaging speed simultaneously, Li et al.⁴⁴ developed a high-speed high-resolution CWLD-based PAM system for in vivo microvasculature imaging [Fig. 2(d)]. Considering the emitter size of LDs, they optimized the optical system by reshaping the laser beams from LDs horizontally and vertically. A pair of CLs combined with an iris was used to control the laser beam to an ideal circular shape, achieving a high lateral resolution of $4.8\mu \mathrm{m}$. The maximum SNR can reach up to $\sim 42\mathrm{dB}$ with a pulse energy of $\sim 30\mathrm{nJ}$ for leaf phantom imaging [Fig. 2(e)] and $\sim 31\mathrm{dB}$ with a pulse energy of $\sim 60\mathrm{nJ}$ for in vivo microvasculature imaging of a mouse ear [Fig. 2(f)]. The exceptional SNR of the CWLD-based PAM system renders signal averaging unnecessary. In addition, the incorporation of a 1D galvanometer mirror scanning system enables high-speed imaging capabilities. However, the limited working distance of the high NA objective lens restricts the implementation of this system solely to transmission-mode imaging, making it unsuitable for visualizing microvasculature in thick tissues. In addition, using a single wavelength LD may preclude the estimation of crucial diagnostic parameters [such as oxygen saturation (${\mathrm{sO}}_2$)], which limits clinical applications of the PAM system. Therefore, they developed a reflection-mode high-resolution OR-PAM system for in vivo ${\mathrm{sO}}_2$ imaging using 2 LDs with different wavelengths, achieving a high lateral resolution of $\sim 6\mu \mathrm{m}$, shown in Fig. 2(g).⁴⁵ High-quality microvasculature imaging [Fig. 2(h)] and accurate ${\mathrm{sO}}_2$ calculation [Fig. 2(i)] can be achieved with this low-cost PAM system, which facilitates the widespread adoption of PAM in both preclinical and clinical applications.

As such, the use of CWLDs in pulsed mode has the potential to expand the range of applications for LD-based imaging techniques, particularly in the field of medical diagnostics.

With the full utilization of the Gruneisen relaxation effect, it is possible to induce the nonlinear PA effect. In this situation, the amplitude of PA signals exhibits variability depending on the baseline temperature of the tissue. Therefore, Zhong et al.^48,49 introduced an LD-based PAM system that combines a single-wavelength PLD with several CWLDs with different wavelengths, achieving multi-wavelength PAM. The CWLDs were utilized to heat the sample, and the PA signals were generated by the single-wavelength PLD. By controlling the laser irradiation sequence (pulse-CW-pulse), PAM images before and after thermal heating can be obtained. Proper differentiation operations can be applied to reveal images with different wavelengths and known light absorption. Ex vivo imaging and clear identification of three tubes filled with different color ink demonstrated its capability for multi-wavelength PA imaging.

4.1.

PAM Systems with GVS

In conventional PAM, 1D depth-resolved PA signals are first detected by the UT, and then two-axis motorized scanning stages are needed to obtain cross-sectional or volumetric images by linear or raster scanning. To increase the scanning speed and reduce the size of the PAM probe, GVS based on a raster scanning mechanism has been developed as a representative imaging scanner for miniaturized and handheld PAM. Hajireza et al.⁵⁰ demonstrated a handheld real-time OR-PAM system, which unveiled a new generation of PAM in various clinical applications. A 2D GVS mirror system was used to pass and scan the 532 nm laser beam with high and low scanning rates of 400 and 1 Hz in 2 directions, respectively. Then, the laser beam passed through the image guide fiber into the handheld probe with only a $40\mathrm{mm}\times 60\mathrm{mm}$ footprint and $<500\mathrm{g}$ in weight. Furthermore, this group attempted to further miniaturize the PAM probe by incorporating a graded-index lens, enabling precise focusing in a compact form.⁵¹ Zhang et al.⁵² also employed a field programmable gate array-driven fast 2D GVS to a compact PAM probe to facilitate high-resolution imaging of subcutaneous microvessels with deep penetration depth. It was able to capture a maximum amplitude projection (MAP) image of $400\times 400\text{pixels}$ within a $2\mathrm{mm}\times 2\mathrm{mm}$ imaging area, with an acquisition time of 16 s. In vivo animal experiments and human skin imaging demonstrated the capabilities of the miniaturized PAM for microvasculature visualization as well as quantitative analysis of the diameters and depths of the blood vessels. Instead of optical scanning only, Seong et al.⁵³ developed a waterproof, two-axis GVS-based handheld PAM (WF-GVS-HH-PAM) system that scans both optical and acoustic beams, providing an extended field of view (FOV) of $14.5\mathrm{mm}\times 9\mathrm{mm}$, as shown in Fig. 3(a). By positioning the GVS at the distal end of the probe, the proposed system successfully addressed the FOV limitation encountered in previous GVS-based handheld PAM systems. The microvasculature of the mouse ear, iris, and brain were imaged by the WF-GVS-HH-PAM system with lateral and axial resolutions of 11.5 and $31.3\mu \mathrm{m}$, respectively. The in vivo experiment results [Fig. 3(b)] demonstrate the ability of the WF-GVS-HH-PAM to visualize arteries, veins, single capillaries, and some distinctive blood vascular features (e.g., coronal suture and sagittal sinus). Furthermore, it provides the 3D volumetric and quantitative information in the depth-encoded PAM images. In 2022, Chen et al.⁵⁴ developed a freehand scanning handheld PAM (FS-PAM) probe to achieve fast speed, great flexibility, and a large FOV in one system. Dual-wavelength (532 and 558 nm) pulsed laser beams were connected to the FS-PAM probe, providing 3D images of vasculatures with not only anatomical but also functional information (e.g., ${\mathrm{sO}}_2$). The FS-PAM system incorporated a hybrid resonant-GVS, enabling video camera mode imaging and facilitating simultaneous localization and mapping in PAM.

Fig. 3

GVS-based PAM. (a) Schematic representation and photographs of the WF-GVS-HH-PAM system. AMP, amplifier; C, collimator; MMF, multi-mode fiber; OUC, opto-acoustic combiner; TM, transparent membrane; M, mirror; AL, acoustic lens; CL, correction lens; L, lens. (b) In vivo PAM MAP (top row) and depth-encoded (bottom row) images of microvasculature in mouse ear, iris, and brain, respectively. Reproduced with permission from Ref. 53.

Instead of raster scanning, PAM based on a rotatory scanning mechanism has also been developed recently. A cylindrically focused transducer is used to form an acoustic focal line, which is adjusted to be confocal with the optical imaging plane. The 2D GVS scans the focused laser spot in line from the left edge to the right edge of the acoustic focal line to generate a B-scan image. Following each optical line scan, both the optical scan line and acoustic focal line rotate simultaneously by a predetermined angle. Multiple scans and rotations with a total scanning angle of 180 deg are executed to encompass the entire imaging area. Based on the rotatory scanning mechanism, Jin et al.^55,56 reported a new portable OR-PAM (p-ORPAM). A 532 nm pulsed laser beam was coupled into a single mode fiber (SMF) and delivered into the imaging part. A 2D GVS was combined with a motorized rotator to achieve large FOV imaging. The performance of the system was evaluated in mouse ears and brains, rabbit ears and eyes, and human oral vasculature with a large FOV of up to 8.4 mm in diameter and a depth of field of 1.5 mm. Representative results demonstrate that the p-ORPAM is widely applicable to multiscale organisms. Subsequently, Qin et al.⁵⁷ conducted a study on the structural and functional cerebral vasculature of rhesus monkeys using the improved p-ORPAM system. This research holds great significance in the field of neuroscience due to the high resemblance of rhesus monkeys’ brains to that of humans. Furthermore, Qin et al.⁵⁸ developed a dual-modality system combining OR-PAM and spectral-domain optical coherence tomography for oral inspection, providing microvasculature and microstructure information of oral tissue simultaneously.

4.2.

PAM Systems with MEMS Scanner

In addition to GVS, the MEMS scanner has emerged as another prominent imaging scanner for miniaturized or handheld PAM due to its metrics: (1) the MEMS scanner is compact and lightweight, making it ideal to be integrated into various devices for portable applications; (2) the MEMS scanner can be designed to various scanning modes (e.g., linear, raster, and specific patterns), showing great flexibility in imaging applications; (3) the MEMS scanner provides a high scanning speed, which holds great significance in real-time and dynamic imaging of biological structures while reducing minimal motion artifacts; and (4) the MEMS scanner requires relatively low driven voltage and consumes lower power compared with other scanning devices, which enhances the overall energy efficiency, making it suitable for handheld PAM systems. Therefore, the MEMS scanner has been widely applied in the development of miniaturized and handheld PAM systems.

Chen et al.⁵⁹ developed a miniaturized probe head of a PAM system using a MEMS mirror. The MEMS mirror was placed after the fiber-based pulsed laser to perform 2D scanning of the laser beam with a maximum scanning area of $\sim 2.3\mathrm{mm}\times 3.5\mathrm{mm}$. A microring detector was used to detect the PA signals in a transmission mode with a lateral resolution of $17.5\mu \mathrm{m}$ and an axial resolution of $20\mu \mathrm{m}$. In addition, they further incorporated the confocal fluorescence microscopy imaging modality into the miniaturized system without adding bulk to the probe, demonstrating the capability of the dual-modality miniaturized system for both microvasculature and individual cell imaging.⁶⁰ However, the transmission-mode design limits its potential in in vivo applications. Qi et al.⁶¹ utilized a 2D MEMS scanning mirror to develop a miniaturized reflection-mode PAM system, shown in Fig. 4(a). They integrated optical components, a MEMS scanning mirror, and a flat UT into a cubic probe with a volume of $60\mathrm{mm}\times 30\mathrm{mm}\times 20\mathrm{mm}$ and a weight of 40 g. Figure 4(b) shows PAM imaging of carbon fibers of the in vivo microvasculature in mouse ear, brain, and human lip, respectively, with a lateral resolution of $10.4\mu \mathrm{m}$ and an active imaging area of $0.9\mathrm{mm}\times 0.9\mathrm{mm}$. Subsequently, Chen et al.⁶³ made further advancements by enhancing the resolution to $3.8\mu \mathrm{m}$ and expanding the effective imaging area to $2\mathrm{mm}\times 2\mathrm{mm}$. They achieved these improvements while simultaneously reducing the probe size to merely $22\mathrm{mm}\times 30\mathrm{mm}\times 13\mathrm{mm}$ and reducing its weight to 20 g. In vivo experiments conducted on the internal organs of a rat abdominal cavity and entire oral cavities of volunteers demonstrate the remarkable optimization of both the performance and physical characteristics of the system.

Fig. 4

MEMS-based PAM. (a) A schematic of a miniaturized 2D MEMS-based reflection-mode PAM system. (b) PAM images of carbon fibers, in vivo microvasculature in mouse ear, brain, and human lip, respectively. (c) A schematic of a MEMS-based handheld PAM probe system. (d) PA MAP images, depth map images, and 3D PA volumetric images of a mouse ear, iris, and brain, respectively. Reproduced with permission from Refs. 61 and 62.

In the aforementioned MEMS-based PAM systems, the scanning of only optical beams by the MEMS scanner limits the imaging FOV due to the efficient aperture of the UT. Lin et al.⁶⁴ presented a handheld PAM probe, by which both optical and acoustic beams were scanned confocally through a 2D water-immersible MEMS scanner with an imaging rate of 2 Hz over a $2.5\mathrm{mm}\times 2.0\mathrm{mm}\times 0.5\mathrm{mm}$ volume. Longitudinal experiments were also conducted to verify the system’s capability for monitoring the hemoglobin concentration in blood vessels. Nevertheless, there is still room for further optimization in reducing the probe size, which currently measures at $80\mathrm{mm}\times 115\mathrm{mm}\times 150\mathrm{mm}$. Figure 4(c) shows another MEMS-based handheld PAM probe with a diameter of 17 mm and a weight of 162 g.⁶² An opto-ultrasound combiner and an aluminum mirror on the MEMS scanner were used to facilitate confocal and co-axial alignment of both light and ultrasound, achieving a high SNR ($\sim 40\mathrm{dB}$ for the $6\mu \mathrm{m}$ diameter carbon fiber) and a high lateral resolution ($\sim 12\mu \mathrm{m}$). Due to the enhanced imaging performance, this system can screen microvasculature in a live animal without the need for data averaging. The mouse ear, iris, and brain were imaged by the handheld PAM system for in vivo demonstration. Figure 4(d) shows the MAP images, depth map images, and 3D PA volumetric images of a mouse ear, iris, and brain, respectively, providing distinct clarity in visualizing artery and vein pairs, capillaries, and brain cortical vessels. The imaging FOV of $2\mathrm{mm}\times 2\mathrm{mm}$ is sufficient to cover the entire iris in a single scan for the visualization of the microvascular morphology. For clinical studies, the handheld PAM system was used to image a mole on a human’s finger, providing valuable diagnostic information on the depth and boundary of moles. Subsequently, Qin et al.⁶⁵ developed a handheld dual-modality imaging microscope, which integrates MEMS-based OR-PAM and OCT, to provide complementary imaging contrasts. It has a remarkably lightweight design of 35.4 g and a compact volume of $65\mathrm{mm}\times 30\mathrm{mm}\times 18\mathrm{mm}$, providing an effective imaging area of $2\mathrm{mm}\times 2\mathrm{mm}$. The dual-modality microscope achieves lateral resolutions of 3.7 (PAM) and $5.6\mu \mathrm{m}$ (OCT), along with axial resolutions of 120 (PAM) and $7.3\mu \mathrm{m}$ (OCT). The animal and human oral imaging experiments have successfully demonstrated its clinical feasibility.