Photoacoustic Microscopy.

Photoacoustic microscopy (PAM) is a hybrid in vivo imaging technique that acoustically detects optical contrast via the photoacoustic effect, a physical phenomenon that converts optical energy into acoustic energy. Unlike pure optical microscopic techniques, PAM takes advantage of the weak acoustic scattering in tissue and thus breaks through the optical diffusion limit (∼1 mm in soft tissue). With its excellent scalability, PAM can provide high-resolution images at desired maximum imaging depths up to a few millimeters. Compared with backscattering-based confocal microscopy and optical coherence tomography, PAM provides unique absorption contrast instead of scattering contrast. Furthermore, PAM can image more molecules, endogenous or exogenous, at their absorbing wavelengths than fluorescence-based methods, such as wide-field, confocal, and multiphoton microscopy. Most importantly, PAM can simultaneously image anatomical, functional, molecular, flow dynamic and metabolic contrasts in vivo. Focusing on state-of-the-art developments in PAM, this chapter discusses the key features of PAM implementations and their applications in biomedical studies. We introduce the fundamentals of PAM and highlight novel system designs. In particular, we compare the imaging speeds of different PAM systems and list several important areas where PAM has been increasingly applied in biomedical research.

Photoacoustic tomography has been developed for in vivo functional, metabolic, molecular, and histologic imaging by physically combining optical and ultrasonic waves. Broad applications include early-cancer detection and brain imaging. High-resolution optical imaging—such as confocal microscopy, two-photon microscopy, and optical coherence tomography—is limited to superficial imaging within the optical diffusion limit (~1 mm in the skin) of the surface of scattering tissue. By synergistically combining light and sound, photoacoustic tomography conquers the optical diffusion limit and provides deep penetration at high ultrasonic resolution and high optical contrast. Photoacoustic tomography has two major embodiments: photoacoustic computed tomography and photoacoustic microscopy. In photoacoustic computed tomography, a pulsed broad laser beam illuminates the biological tissue to generate a small but rapid temperature rise, which leads to emission of ultrasonic waves due to thermoelastic expansion. The unscattered pulsed ultrasonic waves are then detected by ultrasonic transducers. High-resolution tomographic images of optical contrast are then formed through image reconstruction. In photoacoustic microscopy, a pulsed laser beam is delivered into the biological tissue to generate ultrasonic waves, which are then detected with a focused ultrasonic transducer to form a depth resolved 1D image. Raster scanning yields 3D high-resolution tomographic images. Super-depths beyond the optical diffusion limit have been reached with high spatial resolution. The annual conference on PAT has grown exponentially since early 2000 and become the largest in SPIE’s 20,000-attendee Photonics West since 2010.

Photoacoustic tomography has been developed for in vivo functional, metabolic, molecular, and histologic imaging by physically combining optical and ultrasonic waves. Broad applications include early-cancer detection and brain imaging. High-resolution optical imaging—such as confocal microscopy, two-photon microscopy, and optical coherence tomography—is limited to superficial imaging within the optical diffusion limit (~1 mm in the skin) of the surface of scattering tissue. By synergistically combining light and sound, photoacoustic tomography in the form of either photoacoustic computed tomography or photoacoustic microscopy provides deep penetration at high ultrasonic resolution and high optical contrast. The annual conference on photoacoustic tomography has become the largest in SPIE’s 20,000-attendee Photonics West since 2010.

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.

Purpose: To demonstrate the value of the laser-scanning optical-resolution (LSOR)-photoacoustic (PA) microscopy (PAM) system and the conventional multimodal imaging techniques in the evaluation of laser-induced retinal injury and choroidal neovascularization (CNV) in rats. Methods: Different degrees of retinal injury were induced using laser photocoagulation. We compared the LSOR-PAM system with conventional imaging techniques in evaluating retinal injury with or without CNV. Six additional rats, treated with an anti-VEGF antibody or immunoglobulin G immediately after photocoagulation, were imaged 7 and 14 days after injection, and CNV lesion areas were compared. Results: In the retinal injury model, fundus autofluorescence showed well-defined hyperreflection, while the lesion displayed abundant PA signals demonstrating nonuniform melanin distribution in retinal pigment epithelium (RPE). RPE was detected with higher contrast in the PAM B-scan image than optical coherence tomography (OCT). Additionally, the CNV lesion was present with multiple PA signal intensities which distinctly characterized the location and area of CNV as found in fundus fluorescein angiography. Furthermore, the decreased PA signals extending from the CNV lesion were similar to those of the vascular bud in ex vivo imaging, which was invisible in other in vivo images. When treated with anti-VEGF agents, statistically significant differences can be demonstrated by PAM similar to other modalities. Conclusions: LSOR-PAM can detect the melanin distribution of RPE in laser-induced retinal injury and CNV in rats. PAM imaging provides a potential new tool to evaluate the vitality and functionality of RPE in vivo as well as to monitor the development and treatment of CNV.

In this work we derive a system model for a laser-scanning, optical-resolution photoacoustic microscopy system. We use the model to derive a simple image reconstruction algorithm and then analyze the depth resolution achievable by this algorithm. There has recently been development of high-frequency photoacoustic microscopy (PAM) systems with the ability to image biological tissue at a microscopic scale. The imaging depths achievable (a few mm) are shallower than in photoacoustic tomography (which has lower spatial resolution), but deeper than conventional optical microscopy. PAM usually employs a focused single-element high-frequency ultrasonic transducer and a spatially overlapped optical illumination. These existing PAMs require mechanical scanning of the ultrasonic-optical assembly, which is relatively slow and also not compatible with other optical microscopic modalities such as confocal microscopy, two-photon microscopy, and optical coherence tomography. Recently one of us (H.Z.) has developed a laser-scanning OR-PAM (LSOR-PAM) to demonstrate the feasibility of employing optical scanning in PAM. In LSOR-PAM, the ultrasonic detector is kept stationary and only the laser light is raster-scanned within the FOV during data acquisition. Further improvements in image quality and the development of image quality metrics will benefit from the system model derived in this work.

Photoacoustic tomography (PAT), combining optical and ultrasonic waves via the photoacoustic effect, provides in vivo multiscale non-ionizing functional and molecular imaging. Light offers rich tissue contrast but does not penetrate biological tissue in straight paths as x-rays do. Consequently, high-resolution pure optical imaging (e.g., confocal microscopy, two-photon microscopy, and optical coherence tomography) is limited to depths within the optical diffusion limit (~1 mm in the skin). In PAT, pulsed laser light penetrates the tissue and generates a small but rapid temperature rise, which induces emission of ultrasonic waves due to thermoelastic expansion. The ultrasonic waves, ~1000 times less scattering than optical waves in tissue, are then detected to form high-resolution images at depths up to 7 cm, breaking through the optical diffusion limit. PAT is the only modality capable of imaging across the length scales of organelles, cells, tissues, and organs with consistent contrast. Such a technology has the potential to enable multiscale systems biology and accelerate translation from microscopic laboratory discoveries to macroscopic clinical practice. PAT may also hold the key to the earliest detection of cancer by in vivo label-free quantification of hypermetabolism, the quintessential hallmark of cancer. The technology is commercialized by several companies.

Early identification of the margins and location of choroidal neovascularization (CNV) is critical for the precise diagnosis and treatment of numerous neovascular eye diseases, including age-related macular degeneration (AMD). Integration of multimodal photoacoustic microscopy (PAM) and optical coherence tomography (OCT) imaging has been developed to complement the strengths of each modality. A major challenge remains in selectively distinguishing CNV from native microvasculature due to the high optical absorption of hemoglobin. To overcome such limitations, RGD targeting peptides conjugated with gold nanorods (GNR-RGD) was used as multimodal contrast agents to increase the sensitivity of PAM and OCT, allowing for enhanced visualization of CNV due to RGD’s selective binding to integrins in neovascularization. The ability of GNR-RGD enhanced PAM and OCT imaging was evaluated in three New Zealand White rabbits with CNV models. The CNV model was created at day 28 post laser-induced retinal vein occlusion. In vivo color fundus photography, fluorescein angiography, PAM, and OCT imaging was acquired before and after intravenous injection of 400 μL GNR-RGD at concentration of 5 mg/mL at days 1, 3, 5, 7, and 14. Longitudinal studies show that GNR accumulated at CNV sites and led the PAM and OCT signal increased by 27.2-fold in PAM and 171.4 % in OCT peaking at 48 h post-injection and decreased at day 14. Histological analysis, TUNEL assay, and liver and kidney function tests show no systemic toxicity of GNR in the retina or vital organs. The above approaches can provide a potential multimodal molecular imaging tool for precise evaluation of CNV in AMD and other neovascular diseases.

Regenerative therapies such as stem cell therapies are an area of active investigation that will likely play an important role in the future to improve vision loss from retinal diseases including macular degeneration and retinitis pigmentosa. It is important to visualize these cells after administration to determine their fate and effect. In this study, an advanced non-invasive photoacoustic microscopy (PAM) and optical coherence tomography (OCT) imaging system was developed to monitor cells in vivo. To boost the sensitivity of PAM and OCT, novel ultrapure functionalized chain-like gold nanoparticle (CGNP) clusters were synthesized and cultured into a precursor human retinal pigment epithelial cell line with differentiated properties (ARPE-19) cells. The fabricated CGNP clusters have redshifted plasmonic peak absorption from 520nm to 650nm, resulting in reduced background signal from hemoglobin. The position of cells following subretinal injection into the rabbit retina having laser injury is selectively tracked longitudinally in vivo using integrated photoacoustic microscopy (PAM) and OCT over 3 months in 3 rabbits. PAM images obtained at two different optical wavelengths of 578 nm and 650 nm were overlaid on the same image plane and on the OCT image allowed to distinguish transplanted cells from the adjacent native choroidal vessels and track the migration of the cells over time. Quantification of PAM and OCT signals illustrated that the PAM signal increased by 30-fold, and OCT signal increased by 180 %. Histological analysis confirmed that ARPE-19 cells migrated to the injured sites and correlated with the location noted on the PAM/OCT imaging. This work provides a comprehensive imaging and nanoparticle system that could be used for labeling and tracking of cell-based regenerative therapies.

Optical coherence tomography (OCT) and Photoacoustic microscopy (PAM) are two noninvasive, high-resolution, three-dimensional, biomedical imaging modalities based on different contrast mechanisms. OCT detects the light backscattered from a biological sample either in the time or spectral domain using an interferometer to form an image. PAM is sensitive to optical absorption by detecting the light-induced acoustic waves to form an image. Due to their complementary contrast mechanisms, OCT and PAM are suitable for being combined to achieve multimodal imaging. In this dissertation, an optical coherence photoacoustic microscopy (OC-PAM) system was developed for in vivo multimodal retinal imaging with a pulsed broadband NIR light source. To test the capabilities of the system on multimodal ophthalmic imaging, the retina of pigmented rats was imaged. The OCT images showed the retinal structures with quality similar to conventional OCT, while the PAM images revealed the distribution of melanin in the retina since the NIR PAM signals are generated mainly from melanin in the posterior segment of the eye. By using the pulsed broadband light source, the OCT image quality highly depends on the pulse-to-pulse stability of the light source without averaging. In addition, laser safety is always a concern for in vivo applications, especially for eye imaging with a pulsed light source. Therefore, a continuous wave (CW) light source is desired for OC-PAM applications. An OC-PAM system using an intensity-modulated CW superluminescent diode was then developed. The system was tested for multimodal imaging the vasculature of a mouse ear in vivo by using Gold Nanorods (GNRs) as contrast agent for PAM, as well as excised porcine eyes ex vivo. Since the quantitative information of the optical properties extracted from the proposed NIR OC-PAM system is potentially able to provide a unique technique to evaluate the existence of melanin and lipofuscin specifically, a phantom study has been conducted and the relationship between image intensity of OCT and PAM was interpreted to represent the relationship between the optical scattering property and optical absorption property. It will be strong evidence for practical application of the proposed NIR OC-PAM system.

Photoacoustic microscopy (PAM) is a hybrid imaging technique based on the photoacoustic effect and which has begun to develop in recent years. Thanks to the system structure that senses the optical contrast acoustically, it is able to present deep imaging with high resolution beyond the optical diffusion limit. Signals recorded in imaging with the PAM system are exposed to noise by system components and environmental effectcs. In the first stage of the work, a synthetic noise is added at a certain rate on the acoustic signal generated by the solution of the acoustic wave equation. Noisy signals are filtered using discrete wavelet transforms using different main wavelets and noise metrics are calculated on the signals to evaluate the filtering performance. In the second step, the noise metrics are examined on the images by generating the images with the filtered wavelet signals, which are suitable for filtering the PA signals in the direction of the data obtained in the first stage.

Photoacoustic imaging (PAI) is an emerging hybrid imaging modality that can provide a strong optical absorption contrast using the photoacoustic (PA) effect, and breaks through the fundamental imaging depth limit of existing optical microscopy such as optical coherence tomography (OCT), confocal or two-photon microscopy. In PAI, a short-pulsed laser is illuminated to the tissue, and the PA waves are generated by thermoelastic expansion. Despite the high lateral resolution of optical-resolution photoacoustic microscopy (OR-PAM) thanks to the tight optical focus, the lateral resolution of OR-PAM is limited to the optical diffraction limit, which is approximately a half of the excitation wavelength. Here, we demonstrate a new super-resolution photoacoustic microscopy (SR-PAM) system by breaking the optical diffraction limit. The conventional microscopes with nanoscale resolutions such as a scanning electron microscope (SEM) and transmission electron microscope (TEM) are typically used to image the structures of nanomaterials, but these systems should work in a high vacuum environment and cannot provide the optical properties of the materials. Our newly developed SR-PAM system provides the optical properties with a nanoscale resolution in a normal atmosphere. We have photoacoustically imaged single gold nanoparticles with an average size of 80 nm in diameter and shown their PA expansion properties individually. The lateral resolution of this system was approximately 20 nm. Therefore, this tool will provide an unprecedented optical absorption property with an accurate nanoscale resolution and greatly impact on materials science and nanotechnology field.

Photoacoustic microscopy (PAM) is emerging as a powerful technique for imaging microvasculature at depths beyond the ~1 mm depth limit associated with confocal microscopy, two-photon microscopy and optical coherence tomography. PAM, however, is currently qualitative in nature and cannot quantitatively measure important functional parameters including oxyhemoglobin (HbO2), deoxyhemoglobin (HbR), oxygen saturation (sO2), blood flow (BF) and rate of oxygen metabolism (MRO2). Here we describe a new photoacoustic microscopic method, termed photoacoustic computed microscopy (PACM) that combines current PAM technique with a model-based inverse reconstruction algorithm. We evaluate the PACM approach using tissue-mimicking phantoms and demonstrate its in vivo imaging ability of quantifying HbO2, HbR, sO2, cerebral BF and cerebral MRO2 at the small vessel level in a rodent model. This new technique provides a unique tool for neuroscience research and for visualizing microvasculature dynamics involved in tumor angiogenesis and in inflammatory joint diseases.

The application of molecular and cellular imaging in ophthalmology has numerous benefits. It can enable the early detection and diagnosis of ocular diseases, facilitating timely intervention and improved patient outcomes. Molecular imaging techniques can help identify disease biomarkers, monitor disease progression, and evaluate treatment responses. Furthermore, these techniques allow researchers to gain insights into the pathogenesis of ocular diseases and develop novel therapeutic strategies. Molecular and cellular imaging can also allow basic research to elucidate the normal physiological processes occurring within the eye, such as cell signaling, tissue remodeling, and immune responses. By providing detailed visualization at the molecular and cellular level, these imaging techniques contribute to a comprehensive understanding of ocular biology. Current clinically available imaging often relies on confocal microscopy, multi-photon microscopy, PET (positron emission tomography) or SPECT (single-photon emission computed tomography) techniques, optical coherence tomography (OCT), and fluorescence imaging. Preclinical research focuses on the identification of novel molecular targets for various diseases. The aim is to discover specific biomarkers or molecular pathways associated with diseases, allowing for targeted imaging and precise disease characterization. In parallel, efforts are being made to develop sophisticated and multifunctional contrast agents that can selectively bind to these identified molecular targets. These contrast agents can enhance the imaging signal and improve the sensitivity and specificity of molecular imaging by carrying various imaging labels, including radionuclides for PET or SPECT, fluorescent dyes for optical imaging, or nanoparticles for multimodal imaging. Furthermore, advancements in technology and instrumentation are being pursued to enable multimodality molecular imaging. Integrating different imaging modalities, such as PET/MRI (magnetic resonance imaging) or PET/CT (computed tomography), allows for the complementary strengths of each modality to be combined, providing comprehensive molecular and anatomical information in a single examination. Recently, photoacoustic microscopy (PAM) has been explored as a novel imaging technology for visualization of different retinal diseases. PAM is a non-invasive, non-ionizing radiation, and hybrid imaging modality that combines the optical excitation of contrast agents with ultrasound detection. It offers a unique approach to imaging by providing both anatomical and functional information. Its ability to utilize molecularly targeted contrast agents holds great promise for molecular imaging applications in ophthalmology. In this review, we will summarize the application of multimodality molecular imaging for tracking chorioretinal angiogenesis along with the migration of stem cells after subretinal transplantation in vivo.

Determination of the precise location and degree of condition of the Choroidal neovascularization (CNV) lesion is essential for diagnosation Neovascular age-related macular degeneration (AMD) and evaluation the efficacy of treatment. Given the complimentary contrast mechanisms of Photoacoustic microscopy (PAM) and Optical coherence tomography (OCT), the combination of PAM and OCT imaging could potentially provide much sensitive and specific detection of CNV. In this paper, we validated the opportunity to evaluate the information of laser-induced CNV and presented the in vivo time-serial evaluation of the CNV by simultaneously using PAM and OCT techniques. In vivo PAM and OCT examination was performed after laser photocoagulation applied to the rat fundus at days 1, 3, 5, 7, 14. Time-serial results showed that CNV in rats increased to its maximum at day 7 and decreased at day 14. Evolution of CNV information was given in PAM images with a high contrast and details of high axial resolution OCT images were simultaneously given to show the hyperreflective reaction progress.