Portable optical‐resolution photoacoustic microscopy for volumetric imaging of multiscale organisms

Photoacoustic microscopy (PAM) provides a fundamentally new tool for a broad range of studies of biological structures and functions. However, the use of PAM has been largely limited to small vertebrates due to the large size/weight and the inconvenience of the equipment. Here, we describe a portable optical‐resolution photoacoustic microscopy (pORPAM) system for 3‐dimensional (3D) imaging of small‐to‐large rodents and humans with a high spatiotemporal resolution and a large field of view. We show extensive applications of pORPAM to multiscale animals including mice and rabbits. In addition, we image the 3D vascular networks of human lips, and demonstrate the feasibility of pORPAM to observe the recovery process of oral ulcer and cancer‐associated capillary loops in human oral cavities. This technology is promising for broad biomedical studies from fundamental biology to clinical diseases.      

Optical‐resolution photoacoustic microscopy with ultrafast dual‐wavelength excitation

Fast functional and molecular photoacoustic microscopy requires pulsed laser excitations at multiple wavelengths with enough pulse energy and short wavelength‐switching time. Recent development of stimulated Raman scattering in optical fiber offers a low‐cost laser source for multiwavelength photoacoustic imaging. In this approach, long fibers temporally separate different wavelengths via optical delay. The time delay between adjacent wavelengths may eventually limits the highest A‐line rate. In addition, a long‐time delay in fiber may limit the highest pulse energy, leading to poor image quality. In order to achieve high pulse energy and ultrafast dual‐wavelength excitation, we present optical‐resolution photoacoustic microscopy with ultrafast dual‐wavelength excitation and a signal separation method. The signal separation method is validated in numerical simulation and phantom experiments. We show that when two photoacoustic signals are partially overlapped with a 50‐ns delay, they can be recovered with 98% accuracy. We apply this ultrafast dual‐wavelength excitation technique to in vivo OR‐PAM. Results demonstrate that A‐lines at two wavelengths can be successfully separated, and sO₂ values can be reliably computed from the separated data. The ultrafast dual‐wavelength excitation enables fast functional photoacoustic microscopy with negligible misalignment among different wavelengths and high pulse energy, which is important for in vivo imaging of microvascular dynamics.     

Reflection‐mode switchable subwavelength Bessel‐beam and Gaussian‐beam photoacoustic microscopy in vivo

We have developed a reflection‐mode switchable subwavelength Bessel‐beam (BB) and Gaussian‐beam (GB) photoacoustic microscopy (PAM) system. To achieve both reflection‐mode and high resolution, we tightly attached a very small ultrasound transducer to an optical objective lens with numerical aperture of 1.0 and working distance of 2.5 mm. We used axicon and an achromatic doublet in our system to obtain the extended depth of field (DOF) of the BB. To compare the DOF performance achieved with our BB‐PAM system against GB‐PAM system, we designed our system so that the GB can be easily generated by simply removing the lenses. Using a 532 nm pulse laser, we achieved the lateral resolutions of 300 and 270 nm for BB‐PAM and GB‐PAM, respectively. The measured DOF of BB‐PAM was approximately 229 μm, which was about 7× better than that of GB‐PAM. We imaged the vasculature of a mouse ear using BB‐PAM and GB‐PAM and confirmed that the DOF of BB‐PAM is much better than the DOF of GB‐PAM. Thus, we believe that the high resolution achieved at the extended DOF by our system is very practical for wide range of biomedical research including red blood cell (RBC) migration in blood vessels at various depths and observation of cell migration or cell culture.      

Enhanced contrast acoustic‐resolution photoacoustic microscopy using double‐stage delay‐multiply‐and‐sum beamformer for vasculature imaging

In acoustic‐resolution photoacoustic microscopy (AR‐PAM) systems, the lateral resolution in the focal zone of the ultrasound (US) transducer is determined by the numerical aperture (NA) of the transducer. To have a high lateral resolution, a large NA is used. However, the larger the NA, the smaller the depth of focus [DOF]. As a result, the lateral resolution is deteriorated at depths out of the focal region. The synthetic aperture focusing technique (SAFT) along with a beamformer can be used to improve the resolution outside the focal region. In this work, for image formation in AR‐PAM, we propose the double‐stage delay‐multiply‐and‐sum (DS_DMAS) algorithm to be combined with SAFT. The proposed method is evaluated experimentally using hair targets and in vivo vasculature imaging. It is shown that DS_DMAS provides a higher resolution and contrast compared to other methods. For the B‐mode images obtained using the hair phantom, the proposed method reduces the average noise level for all the depths by about 134%, 57% and 23%, compared to the original low‐ resolution, SAFT+DAS and SAFT+DMAS methods, respectively. All the results indicate that the proposed method can be an appropriate algorithm for image formation in AR‐PAM systems.      

Volumetric imaging of erythrocytes using label‐free multiphoton photoacoustic microscopy

Photoacoustic microscopy (PAM) is an imaging modality well suited to mapping vasculature and other strong absorbers in tissue. However, one of the primary drawbacks to PAM when used for high‐resolution imaging is the relatively poor axial resolution due to the inverse dependence on the transducer bandwidth. While submicron lateral resolution PAM can be achieved by tightly focusing the excitation light, the axial resolution is fundamentally limited to 10s of microns for typical transducer frequencies. Here we present a multiphoton PAM technique called transient absorption ultrasonic microscopy (TAUM), which results in a completely optically resolved voxel with an experimentally measured axial resolution of 1.5 microns. This technique is demonstrated by imaging individual red blood cells in three dimensions in blood smear and ex vivo tissues. To the best of our knowledge, this is the first demonstration of fully resolved, volumetric photoacoustic imaging of erythrocytes. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Quantitative analysis on in vivo tumor‐microvascular images from optical‐resolution photoacoustic microscopy

Optical‐resolution photoacoustic microscopy (OR‐PAM) has been shown to be an excellent imaging modality for monitoring and study of tumor microvasculature. However, previous studies focused mainly on the normal tissues and did not quantify the tumor microvasculature. In this study, we present an in vivo OR‐PAM imaging of the melanomas and hepatoma implanted in the mouse ear. We quantify the vessel growth by extracting the skeletons of both dense and thin branches of the tumor microvasculature obtained by Hessian matrix enhancement followed by improved two‐step multistencils fast marching method. Compared with the previous methods of using OR‐PAM for normal tissues, our method was more effective in extracting the binary vascular network in the tumor images and in obtaining the complete and continuous microvascular skeleton maps. Our demonstration of using OR‐PAM in improving microvasculature of tumors and quantification of tumor growth would push deep this technology for the early diagnosis and treatment of cancers.      

Real‐time functional optical‐resolution photoacoustic microscopy using high‐speed alternating illumination at 532 and 1064 nm

Optical‐resolution photoacoustic microscopy (OR‐PAM), which has been widely used and studied as a noninvasive and in vivo imaging technique, can yield high‐resolution and absorption contrast images. Recently, metallic nanoparticles and dyes, such as gold nanoparticles, methylene blue, and indocyanine green, have been used as contrast agents of OR‐PAM. This study demonstrates real‐time functional OR‐PAM images with high‐speed alternating illumination at 2 wavelengths. To generate 2 wavelengths, second harmonic generation at 532 nm with an LBO crystal and a pump wavelength of 1064 nm is applied at a pulse repetition rate of 300 kHz. For alternating illumination, an electro‐optical modulator is used as an optical switch. Therefore, the A‐line rate for the functional image is 150 kHz, which is half of the laser repetition rate. To enable fast signal processing and real‐time displays, parallel signal processing using a graphics processing unit (GPU) is performed. OR‐PAM images of the distribution of blood vessels and gold nanorods in a BALB/c‐nude mouse's ear can be simultaneously obtained with 500 × 500 pixels and real‐time display at 0.49 fps.      

1064 nm acoustic resolution photoacoustic microscopy

Photoacoustic imaging is a noninvasive imaging technique having the advantages of high‐optical contrast and good acoustic resolution at improved imaging depths. Light transport in biological tissues is mainly characterized by strong optical scattering and absorption. Photoacoustic microscopy is capable of achieving high‐resolution images at greater depth compared to conventional optical microscopy methods. In this work, we have developed a high‐resolution, acoustic resolution photoacoustic microscopy (AR‐PAM) system in the near infra‐red (NIR) window II (NIR‐II, eg, 1064 nm) for deep tissue imaging. Higher imaging depth is achieved as the tissue scattering at 1064 nm is lesser compared to visible or near infrared window‐I (NIR‐I). Our developed system can provide a lateral resolution of 130 μm, axial resolution of 57 μm, and image up to 11 mm deep in biological tissues. This 1064‐AR‐PAM system was used for imaging sentinel lymph node and the lymph vessel in rat. Urinary bladder of rat filled with black ink was also imaged to validate the feasibility of the developed system to study deeply seated organs.      

Correcting the limited view in optical‐resolution photoacoustic microscopy

Optical‐resolution photoacoustic microscopy (OR‐PAM) has proven useful for anatomical and functional imaging with high spatial resolutions. However, the coherent signal generation and the desired reflection‐mode detection in OR‐PAM can result in a limited detectability of features aligned with the acoustic axis (ie, vertical structures). Here, we investigated the limited‐view phenomenon in OR‐PAM by simulating the generation and propagation of the acoustic pressure waves and determined the key optical parameters affecting the visibility of vertical structures. Proof‐of‐concept numerical experiments were performed with different illumination angles, optical foci and numerical apertures (NA) of the objective lens. The results collectively show that an NA of 0.3 can readily improve the visibility of vertical structures in a typical reflection‐mode OR‐PAM system. This conclusion was confirmed by numerical simulations on the cortical blood vessels in a mouse brain and by experiments in a suture‐cross phantom and in a mouse brain in vivo.      

Impacts of the murine skull on high‐frequency transcranial photoacoustic brain imaging

Non‐invasive photoacoustic tomography (PAT) of mouse brains with intact skulls has been a challenge due to the skull's strong acoustic attenuation, aberration, and reverberation, especially in the high‐frequency range (>15 MHz). In this paper, we systematically investigated the impacts of the murine skull on the photoacoustic wave propagation and on the PAT image reconstruction. We studied the photoacoustic acoustic wave aberration due to the acoustic impedance mismatch at the skull boundaries and the mode conversion between the longitudinal wave and shear wave. The wave's reverberation within the skull was investigated for both longitudinal and shear modes. In the inverse process, we reconstructed the transcranial photoacoustic computed tomography (PACT) and photoacoustic microscopy (PAM) images of a point target enclosed by the mouse skull, showing the skull's different impacts on both modalities. Finally, we experimentally validated the simulations by imaging an in vitro mouse skull phantom using representative transcranial PAM and PACT systems. The experimental results agreed well with the simulations and confirmed the accuracy of our forward and inverse models. We expect that our results will provide better understanding of the impacts of the murine skull on transcranial photoacoustic brain imaging and pave the ways for future technical improvements.      

High-resolution volumetric information fusion for depth of field enhancement in photoacoustic microscopy

Optical-resolution photoacoustic microscopy suffers from limited depth of field due to the strongly focused laser beam. Here, a novel volumetric information fusion is proposed to achieve large volumetric and high-resolution imaging. First, three-dimensional stationary wavelet transform was performed on the multi-focus data to obtain eight wavelet coefficients. Differential evolution based on joint weighted evaluation was then employed to optimize the block size of division for each wavelet coefficient. The proposed fusion rule using standard deviation for focus detection was used to fuse the corresponding sub-coefficients. Finally, photoacoustic imaging with large depth of field can be achieved by the inverse stationary wavelet transform. Performance test shows that the depth of field of photoacoustic imaging can be doubled without sacrificing lateral resolution. The proposed volumetric information fusion can further promote the capability of volumetric imaging of optical-resolution photoacoustic microscopy and will be helpful in the acquisition of physiological and pathological process.     

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.     

In vivo label‐free functional photoacoustic monitoring of ischemic reperfusion

Pressure ulcer formation is a common problem among patients confined to bed or restricted to wheelchairs. The ulcer forms when the affected skin and underlying tissues go through repeated cycles of ischemia and reperfusion, leading to inflammation. This theory is evident by intravital imaging studies performed in immune cell–specific, fluorescent reporter mouse skin with induced ischemia‐reperfusion (I‐R) injuries. However, traditional confocal or multiphoton microscopy cannot accurately monitor the progression of vascular reperfusion by contrast agents, which leaks into the interstitium under inflammatory conditions. Here, we develop a dual‐wavelength micro electro mechanical system (MEMS) scanning–based optical resolution photoacoustic microscopy (OR‐PAM) system for continuous label‐free functional imaging of vascular reperfusion in an IR mouse model. This MEMS‐OR‐PAM system provides fast scanning speed for concurrent dual‐wavelength imaging, which enables continuous monitoring of the reperfusion process. During reperfusion, the revascularization of blood vessels and the oxygen saturation (sO₂) changes in both arteries and veins are recorded, from which the local oxygen extraction ratios of the ischemic tissue and the unaffected tissue can be quantified. Our MEMS‐OR‐PAM system provides novel perspectives to understand the I‐R injuries. It solves the problem of dynamic label‐free functional monitoring of the vascular reperfusion at high spatial resolution.      

Photoacoustic microscopy for evaluating a lipopolysaccharide‐induced inflammation model in mice

Photoacoustic microscopy (PAM) is a noninvasive imaging technique and is excellent to study structural and functional changes in the microcirculation. In this work, a lipopolysaccharide (LPS)‐induced inflammation model in mice is noninvasively evaluated by PAM. PAM is used to image the microvascular structural changes in mice for 8 hours after the LPS with different concentrations is applied. Quantitative analysis of five vessel parameters is conducted, which shows that the rate of reduction in microvasculature is highly dependent on the applied LPS concentrations. For low‐concentration LPS, changes in the microvasculature are not obvious over the observation period, whereas for high‐concentration LPS, quick and marked reduction in the microvasculature is observed. In addition, changes in capillaries are more significant than those in relatively large vessels. The results show that PAM is able to evaluate the inflammation mouse model by studying structural (and potentially functional) changes in the microcirculation. Furthermore, PAM may have potential for early intervention and treatment plan optimization of sepsis by monitoring the microcirculation and inflammatory response.      

Acoustic resolution photoacoustic microscopy based on microelectromechanical systems scanner

Photoacoustic microscopy (PAM) can be classified as optical resolution (OR)‐PAM and acoustic resolution (AR)‐PAM depending on the type of resolution achieved. Using microelectromechanical systems (MEMS) scanner, high‐speed OR‐PAM system was developed earlier. Depth of imaging limits the use of OR‐PAM technology for many preclinical and clinical imaging applications. Here, we demonstrate the use of a high‐speed MEMS scanner for AR‐PAM imaging. Lateral resolution of 84 μm and an axial resolution of 27 μm with ~2.7 mm imaging depth was achieved using a 50 MHz transducer‐based AR‐PAM system. Use of a higher frequency transducer at 75 MHz has further improved the resolution characteristics of the system with a reduction in imaging depth and a lateral resolution of 53 μm and an axial resolution of 18 μm with ~1.8 mm imaging depth was achieved. Using the two‐axis MEMS scanner a 2 × 2 .5 mm² area was imaged in 3 seconds. The capability of achieving acoustic resolution images using the MEMS scanner makes it beneficial for the development of high‐speed miniaturized systems for deeper tissue imaging.      

In vivo study of rat cortical hemodynamics using a stereotaxic‐apparatus‐compatible photoacoustic microscope

Brain imaging is an important technique in cognitive neuroscience. In this article, we designed a stereotaxic‐apparatus‐compatible photoacoustic microscope for the studies of rat cortical hemodynamics. Compared with existing optical resolution photoacoustic microscopy (ORPAM) systems, the probe owns feature of fast, light and miniature. In this microscope, we integrated a miniaturized ultrasound transducer with a center frequency of 10 MHz to detect photoacoustic signals and a 2‐dimensional (2D) microelectromechanical system (MEMS) scanner to achieve raster scanning of the optical focus. Based on phantom evaluation, this imaging probe has a high lateral resolution of 3.8 μm and an effective imaging domain of 2 × 2 mm². Different from conventional ORPAMs, combining with standard stereotaxic apparatus enables broad studies of rodent brains without any motion artifact. To show its capability, we successfully captured red blood cell flow in the capillary, monitored the vascular changes during bleeding and blood infusion and visualized cortical hemodynamics induced by middle cerebral artery occlusion.      

In vivo study of endometriosis in mice by photoacoustic microscopy

Endometriosis (EM) impacts the healthcare and the quality of life for women of reproductive age. However, there is no reliable noninvasive diagnosis method for either animal study or clinical use. In this work, a novel imaging method, photoacoustic microscopy (PAM) was employed to study the EM on the mouse model. Our results demonstrated the PAM noninvasively provided the high contrast and 3D imaging of subcutaneously implanted EM tissue in the nude mouse in vivo. The statistical study also indicated PAM had high sensitivity and specificity in the diagnosis of EM in this animal study. In addition, we also discussed the potential clinical application for PAM in the diagnosis of EM. (© 2014 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Indocyanine green-enhanced multimodal photoacoustic microscopy and optical coherence tomography molecular imaging of choroidal neovascularization

Photoacoustic microscopy (PAM) has great potential for visualization of the microvasculature with high spatial resolution and contrast. Early detection and differentiation of newly developed blood vessels named choroidal neovascularization (CNV) from normal vasculature remains a challenge in ophthalmology. Exogenous contrast agents can assist with improving PAM sensitivity, leading to differentiation of CNV. Here, an FDA-approved indocyanine green (ICG) was utilized as a PAM contrast agent. ICG was conjugated with RGD peptides, allowing the ICG to bind to the integrin expressed in CNV. Molecular PAM imaging showed that ICG-RGD can target CNV for up to 5 days post intravenous administration in living rabbits with a model of CNV. The PAM image sensitivity and image contrast were significantly enhanced by 15-fold at 24 h post-injection. Overall, the presented approach demonstrates the possibility of targeted ICG to be employed in PAM molecular imaging, allowing more precise evaluation of neovascularization.     

Integrated photoacoustic and hyperspectral dual‐modality microscopy for co‐imaging of melanoma and cutaneous squamous cell carcinoma in vivo

Skin carcinoma such as melanoma (MM) and cutaneous squamous cell carcinoma (cSCC) are considered as the highest mortality and the most aggressive skin cancers in dermatology. In view that early diagnosis and treatment can greatly improve the survival rate and life quality of the patients, developing noninvasive and effective evaluation methods is of great significance for the detection and identification of early stage cutaneous cancers. In this article, we propose a hybrid photoacoustic and hyperspectral dual‐modality microscopy to evaluate and differentiate skin carcinoma by structural and multiphysiological parameters. The proposed system's imaging abilities are verified by mimic phantoms and normal mice experiments. Furthermore, in vivo characterization and evaluation results of MM and cSCC mice are obtained successfully, which prove this novel method could be used as a reliable and useful method for skin cancer detection in early stages.     

Multicomposite super‐resolution microscopy: Enhanced Airyscan resolution with radial fluctuation and sample expansions

Either modulated illumination or temporal fluctuation analysis can assist super‐resolution techniques in overcoming the diffraction limit of conventional optical microscopy. As they are not contradictory to each other, an effective combination of spatial and temporal super‐resolution mechanisms would further improve the resolution of fluorescent images. Here, a super‐resolution imaging method called fluctuation‐enhanced Airyscan technology (FEAST) is proposed, which achieves ~40 nm lateral imaging resolution and is useful for a range of fluorescent proteins and organic dyes. It was demonstrated not only to obtain different subcellular super‐resolution images, but also to improve the accuracy of counting the average human epidermal growth factor receptor 2 (HER2) copy number for diagnosis in breast cancer. Furthermore, the combination of FEAST and sample expansion microscopy (Ex‐FEAST) improves the lateral resolution to ~26 nm.