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.      

Indocyanine green labeling for optical and photoacoustic imaging of mesenchymal stem cells after in vivo transplantation

The transplantation of mesenchymal stem cells (MSCs) holds great promise for the treatment of a plethora of human diseases, but new noninvasive procedures are needed to monitor the cell fate in vivo. Already largely used in medical diagnostics, the fluorescent dye indocyanine green (ICG) is an established dye to track limited numbers of cells by optical imaging (OI), but it can also be visualized by photoacoustic imaging (PAI), which provides a higher spatial resolution than pure near infrared fluorescence imaging (NIRF). Because of its successful use in clinical and preclinical examinations, we chose ICG as PAI cell labeling agent. Optimal incubation conditions were defined for an efficient and clinically translatable MSC labeling protocol, such that no cytotoxicity or alterations of the phenotypic profile were observed, and a consistent intracellular uptake of the molecule was achieved. Suspensions of ICG‐labeled cells were both optically and optoacoustically detected in vitro, revealing a certain variability in the photoacoustic spectra acquired by varying the excitation wavelength from 680 to 970 nm. Intramuscular engraftments of ICG‐labeled MSCs were clearly visualized by both PAI and NIRF over few days after transplantation in the hindlimb of healthy mice, suggesting that the proposed technique retains a considerable potential in the field of transplantation‐focused research and therapy. Stem cells were labeled with the Food and Drug Administration (FDA)‐approved fluorescent dye ICG, and detected by both PAI and OI, enabling to monitor the cell fate safely, in dual modality, and with good sensitivity and improved spatial resolution.      

Near-infrared photoacoustic spectroscopy of proteins

A major problem encountered with the use of electronic spectroscopy in the analysis of biological materials in the ultraviolet, visible, and infrared region involves the limited range of the physical state of samples that can be examined. In an attempt to expand this range, photoacoustic spectra of both solid- and solution-state proteins have been obtained in the near-infrared region. Solid proteins generate detailed spectra in the region 1.0–2.6 μm, resulting primarily from hydrogenic overtone and combinational modes. Harmonics and combinations of amide group frequencies which display significant spectral complexity are observed between 1.4 and 1.7 μm, although they appear to manifest only limited conformational sensitivity. Solution spectra in D₂O are of much lower resolution. Assignments of peaks for both solution- and solid-state proteins are presented and the advantages and disadvantages of the use of near-infrared photoacoustic spectroscopy with proteins are discussed.     

Energy compensated synthetic aperture focusing technique for photoacoustic microscopy

We report an adaptive energy-compensated synthetic aperture focusing technique (eC-SAFT) for improving the imaging performance of photoacoustic microscopy (PAM) in terms of depth of field (DOF), spatial resolution (both axial and lateral), and SNR. In addition to coherency and time-delay (in conventional SAFT), our beamforming-based reconstruction algorithm takes into account acoustic energy loss—a primary physical parameter in acoustic wave propagation—following Beer-Lambert's law. Experimental validation studies were performed in tissue-mimicking (Agar) phantoms, complex leaf veins, and chicken breast tissues. Results demonstrate that our proposed eC-SAFT+CF outperforms conventional SAFT+CF to improve axial resolution (up to $\sim 5\%$), lateral resolution (up to $\sim 5\%$), SNR (up to $\sim 6\%$) and CR (up to $\sim 8\%$).     

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)     

Wearable scanning photoacoustic brain imaging in behaving rats

A wearable scanning photoacoustic imaging (wPAI) system is presented for noninvasive brain study in behaving rats. This miniaturized wPAI system consists of four pico linear servos and a single transducer‐based PAI probe. It has a dimension of 50 mm × 35 mm × 40 mm, and a weight of 26 g excluding cablings. Phantom evaluation shows that wPAI achieves a lateral resolution of ～0.5 mm and an axial resolution of ～0.1 mm at a depth of up to 11 mm. Its imaging ability is also tested in a behaving rat, and the results indicate that wPAI is able to image blood vessels at a depth of up to 5 mm with intact scalp and skull. With its noninvasive, deep penetration, and functional imaging ability in behaving animals, wPAI can be used for behavior, cognition, and preclinical brain disease studies.

     

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.      

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.      

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.     

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.     

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.      

Integrating photoacoustic microscopy with other imaging technologies for multimodal imaging

As a hybrid optical microscopic imaging technology, photoacoustic microscopy images the optical absorption contrasts and takes advantage of low acoustic scattering of biological tissues to achieve high-resolution anatomical and functional imaging. When combined with other imaging modalities, photoacoustic microscopy-based multimodal technologies can provide complementary contrast mechanisms to reveal complementary information of biological tissues. To achieve intrinsically and precisely registered images in a multimodal photoacoustic microscopy imaging system, either the ultrasonic transducer or the light source can be shared among the different imaging modalities. These technologies are the major focus of this minireview. It also covered the progress of the recently developed penta-modal photoacoustic microscopy imaging system featuring a novel dynamic focusing technique enabled by OCT contour scan.     

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.      

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.     

Spherical-matching hyperbolic-array photoacoustic computed tomography

Linear-array photoacoustic computed tomography (LA-PACT), for its flexibility and simplicity, has great potential in providing anatomical and functional information of tissues. However, the limited coverage view impedes the LA-PACT obtaining high-quality images. In this study, a photoacoustic tomographic system with a hyperbolic-array transducer was developed for stereoscopic PA imaging of carotid artery. The hyperbolic-array PACT increases the receiving sensitivity for PA signal detection due to its transducer's geometric structure matching with the spherical wave. The control phantom experiment shows that the proposed system can expand the angular coverage of ∼1/3 more than that of the LA-PACT system, and the volumetric PA images of rat's carotid artery demonstrates the potential of the system for carotid artery imaging. Furthermore, volumetric imaging of the human forearm verifies that the system has significant capability in human imaging, which indicates that it has bright prospect for assisting diagnosis in the vascular disease.     

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.     

Detecting hemodynamic changes in the foot vessels of diabetic patients by photoacoustic tomography

Limb perfusion monitoring is critical for diabetes mellitus (DM) patients as they are vulnerable to vascular complications due to prolonged hyperglycemia. However, current clinical approaches are ineffective in vascular imaging and in assessing vascular function in lower limbs. In this work, a concave ultrasound transducer array‐based photoacoustic tomography (PAT) system was used to image the foot dorsal section of a subject, and a total of seven DM patients and seven healthy volunteers were enrolled in this study. Hemodynamic changes in foot vessels during vascular occlusion as well as oxygen saturation (SO₂) in rest were analyzed for both groups. The results obtained showed that DM patients have a unique peripheral hemodynamic response to occlusion and a lower level SO₂, compared to that for healthy subjects. This suggests that PAT has the potential to detect vascular dysfunction in DM patients and to measure the effect of treatment.     

近红外光谱技术在烟草行业中的应用进展

介绍了近红外光谱分析技术在烟草常规化学成分分析、烟气分析、卷烟配方设计、不同产地模式识别和真假烟鉴别等方面的应用,认为近红外光谱分析技术在烟草行业中正扮演着越来越重要的角色,而且具有十分广阔的应用前景.     

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.