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.      

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.      

Wide‐field color imaging of scatter‐based tissue contrast using both high spatial frequency illumination and cross‐polarization gating

This study characterizes the scatter‐specific tissue contrast that can be obtained by high spatial frequency (HSF) domain imaging and cross‐polarization (CP) imaging, using a standard color imaging system, and how combining them may be beneficial. Both HSF and CP approaches are known to modulate the sensitivity of epi‐illumination reflectance images between diffuse multiply scattered and superficially backscattered photons, providing enhanced contrast from microstructure and composition than what is achieved by standard wide‐field imaging. Measurements in tissue‐simulating optical phantoms show that CP imaging returns localized assessments of both scattering and absorption effects, while HSF has uniquely specific sensitivity to scatter‐only contrast, with a strong suppression of visible contrast from blood. The combination of CP and HSF imaging provided an expanded sensitivity to scatter compared with CP imaging, while rejecting specular reflections detected by HSF imaging. ex vivo imaging of an atlas of dissected rodent organs/tissues demonstrated the scatter‐based contrast achieved with HSF, CP and HSF‐CP imaging, with the white light spectral signal returned by each approach translated to a color image for intuitive encoding of scatter‐based contrast within images of tissue. The results suggest that visible CP‐HSF imaging could have the potential to aid diagnostic imaging of lesions in skin or mucosal tissues and organs, where just CP is currently the standard practice imaging modality.      

Volumetric hand‐held optoacoustic angiography as a tool for real‐time screening of dense breast

Existing mammographic screening solutions are generally associated with several major drawbacks, such as exposure to ionizing radiation or insufficient sensitivity in younger populations with radiographically‐dense breast. Even when combined with ultrasound or magnetic resonance imaging, X‐Ray mammography may still attain unspecific or false positive results. Thus, development of new breast imaging tools represents a timely medical challenge. We report on a new approach to high‐resolution functional and anatomical breast angiography using volumetric hand‐held optoacoustic tomography, which employs light intensities safe for human use. Experiments in young healthy volunteers with fibroglandular‐dominated dense breasts revealed the feasibility of rendering three‐dimensional images representing vascular anatomy and functional blood oxygenation parameters at video rate. Sufficient contrast was achieved at depths beyond 2 cm within dense breasts without compromising the real‐time imaging performance. The suggested solution may thus find applicability as a standalone or supplemental screening tool for early detection and follow‐up of carcinomas in radiographically‐dense breasts.

Volumetric handheld optoacoustic tomography scanner uses safe pulses of near‐infrared light to render three‐dimensional images of deep vascular anatomy, blood oxygenation and breast parenchyma at video rate.     

Prostate‐specific membrane antigen‐targeted photoacoustic imaging of prostate cancer in vivo

A sensitive, noninvasive method to detect localized prostate cancer, particularly for early detection and repetitive study in patients undergoing active surveillance, remains an unmet need. Here, we propose a molecular photoacoustic (PA) imaging approach by targeting the prostate‐specific membrane antigen (PSMA), which is over‐expressed in the vast majority of prostate cancers. We performed spectroscopic PA imaging in an experimental model of prostate cancer, namely, in immunocompromised mice bearing PSMA+ (PC3 PIP) and PSMA? (PC3 flu) tumors through administration of the known PSMA‐targeted fluorescence agent, YC‐27. Differences in contrast between PSMA+ and isogenic control tumors were observed upon PA imaging, with PSMA+ tumors showing higher contrast in average of 66.07‐fold with 5 mice at the 24‐hour postinjection time points. These results were corroborated using standard near‐infrared fluorescence imaging with YC‐27, and the squared correlation between PA and fluorescence intensities was 0.89. Spectroscopic PA imaging is a new molecular imaging modality with sufficient sensitivity for targeting PSMA in vivo, demonstrating the potential applications for other saturable targets relevant to cancer and other disorders.      

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.      

Multispectral photoacoustic imaging of cancer with broadband CuS nanoparticles covering both near‐infrared I and II biological windows

In this study, CuS nanoparticles with optical absorption covering both near‐infrared I (NIR‐I) and NIR‐II biological windows were prepared and served as the contrast agents for multispectral photoacoustic imaging. The physiological parameters including concentrations of deoxyhemoglobin and oxyhemoglobin as well as the water content in the tumor location were quantified based on the multispectral photoacoustic reconstruction method. More importantly, the concentration of CuS nanoparticles/drugs accumulated in the tumor was also recovered after intravenously injection, which are essential for image‐guided cancer theranostics. In addition, phantom and in vivo experimental tests were performed to inspect and compare the imaging depth and signal‐to‐noise ratio (SNR) between the two NIR biological windows. Interestingly, we discovered that a higher SNR was obtained in the NIR‐II window than that in the NIR‐I window. Meanwhile, the multispectral imaging results also demonstrated that the imaging contrast and penetration depth in the NIR‐II window were also significantly improved as compared to those from the NIR‐I window.      

Large‐depth‐of‐field full‐field optical angiography

A large‐depth‐of‐field full‐field optical angiography (LD‐FFOA) method is developed to expand the depth‐of‐field (DOF) using a contrast pyramid fusion algorithm (CPFA). The absorption intensity fluctuation modulation effect is utilized to obtain full‐field optical angiography (FFOA) images at different focus positions. The CPFA is used to process these FFOA images with different focuses. By selecting high‐contrast areas, the CPFA can highlight the characteristics and details of blood vessels to obtain LD‐FFOA images. In the optimal case of the proposed method, the DOF for FFOA is more than tripled using 10 differently focused FFOA images. Both the phantom and animal experimental results show that the LD‐FFOA resolves FFOA defocusing issues induced by surface and thickness inhomogeneities in biological samples. The proposed method can be potentially applied to practical biological experiments.      

1064‐nm‐resonant gold nanorods for photoacoustic theranostics within permissible exposure limits

Therapeutic and diagnostic methods based on photomechanical effects are attracting much current attention in contexts as oncology, cardiology and vascular surgery, for such applications as photoacoustic imaging or microsurgery. Their underlying mechanism is the generation of ultrasound or cavitation from the interaction of short optical pulses with endogenous dyes or targeted contrast agents. Among the latter, gold nanorods are outstanding candidates, but their use has mainly been reported for photoacoustic imaging and photothermal treatments. Conversely, much less is still known about their value as a precision tool for photomechanical manipulations, such as to impart local damage with high spatial resolution through the expansion and collapse of microbubbles. Here, we address the feasibility of gold nanorods exhibiting a distribution of surface plasmon resonances between about 900 to above 1100 nm as a contrast agent for photoacoustic theranostics. After testing their cytotoxicity and cellular uptake, we discuss their photostability and use to mediate cavitation and the photomechanical destruction of targeted cells. We find that the choice of a plasmonic band peaking around 1064 nm is key to enhance the translational potential of this approach. With respect to the standard alternative of 800 nm, at 1064 nm, relevant regulations on optical exposure are less restrictive and the photonic technology is more mature.      

In vivo blind‐deconvolution photoacoustic ophthalmoscopy with total variation regularization

Photoacoustic ophthalmoscopy (PAOM) is capable of noninvasively imaging anatomic and functional information of the retina in living rodents. However, the strong ocular aberration in rodent eyes and limited ultrasonic detection sensitivity affect PAOM's spatial resolution and signal‐to‐noise ratio (SNR) in in vivo eyes. In this work, we report a computational approach to combine blind deconvolution (BD) algorithm with a regularizing constraint based on total variation (BDTV) for PAOM imaging restoration. We tested the algorithm in retinal and choroidal microvascular images in albino rat eyes. The algorithm improved PAOM's lateral resolution by around 2‐fold. Moreover, it enabled the improvement in imaging SNR for both major vessels and capillaries, and realized the well‐preserved blood vessels' edges simultaneously, which surpasses conventional Richardson‐Lucy BD algorithm. The reported results indicate that the BDTV algorithm potentially facilitate PAOM in extracting retinal pathophysiological information by enhancing in vivo imaging quality without physically modifying PAOM's optical configuration.      

Wavelength‐Modulated Differential Photoacoustic Spectroscopy (WM‐DPAS) for noninvasive early cancer detection and tissue hypoxia monitoring

This study introduces a novel noninvasive differential photoacoustic method, Wavelength Modulated Differential Photoacoustic Spectroscopy (WM‐DPAS), for noninvasive early cancer detection and continuous hypoxia monitoring through ultrasensitive measurements of hemoglobin oxygenation levels (StO₂). Unlike conventional photoacoustic spectroscopy, WM‐DPAS measures simultaneously two signals induced from square‐wave modulated laser beams at two different wavelengths where the absorption difference between maximum deoxy‐ and oxy‐hemoglobin is 680 nm, and minimum (zero) 808 nm (the isosbestic point). The two‐wavelength measurement efficiently suppresses background, greatly enhances the signal to noise ratio and thus enables WM‐DPAS to detect very small changes in total hemoglobin concentration (C_Hb) and oxygenation levels, thereby identifying pre‐malignant tumors before they are anatomically apparent. The non‐invasive nature also makes WM‐DPAS the best candidate for ICU bedside hypoxia monitoring in stroke patients. Sensitivity tunability is another special feature of the technology: WM‐DPAS can be tuned for different applications such as quick cancer screening and accurate StO₂ quantification by selecting a pair of parameters, signal amplitude ratio and phase shift. The WM‐DPAS theory has been validated with sheep blood phantom measurements.

Sensitivity comparison between conventional single‐ended signal and differential signal.     

Retinal imaging with optical coherence tomography and low‐loss adaptive optics using a 2.8‐mm beam size

As data acquisition for retinal imaging with optical coherence tomography (OCT) becomes faster, efficient collection of photons becomes more important to maintain image quality. One approach is to use a larger aperture at the eye's pupil to collect more photons that have been reflected from the retina. A 2.8‐mm beam diameter system with only seven reflecting surfaces was developed for low‐loss retinal imaging. The larger beam size requires defocus and astigmatism correction, which was done in a closed loop adaptive optics method using a Shack‐Hartmann wavefront sensor and a deformable mirror (DM) with 140 actuators and a ±2.75 μm stroke. This DM facilitates defocus correction ranging from approximately ?3 D to +3 D. Comparing the new system with a standard 1.2‐mm system on a model eye, a signal‐to‐noise gain of 4.5 dB and a 2.3 times smaller speckle size were measured. Measurements on the retinas of five subjects showed even better results, with increases in dynamic range up to 13 dB. Note that the new sample arm only occupies 30 cm × 60 cm, which makes it highly suitable for imaging in a clinical environment. Figure: B‐scan images obtained over a width of 8 deg from the right eye of a 31‐year‐old Caucasian male. While the left side was imaged with a standard 1.2‐mm OCT system, the right side was imaged with the 2.8‐mm system. Both images were collected with the same integration time and incident power, after correction of aberrations. Using the dynamic range within the images, which is determined by comparing the highest pixel value to the noise floor, a difference in dynamic range of 10.8 dB was measured between the two systems.      

Hand‐held,clinical dual mode ultrasound ‐ photoacoustic imaging of rat urinary bladder and its applications

Urinary bladder imaging is critical to diagnose urinary tract disorders, and bladder cancer. There is a great need for safe, non‐invasive, and sensitive imaging technique which enables bladder imaging. Photoacoustic imaging is a rapidly growing imaging technique for various biological applications. It can be combined with clinical ultrasound imaging system for hand‐held, dual modal ultrasound‐photoacoustic real‐time imaging. Structural (bladder wall) and functional (accretion of nanoparticles) bladder imaging is shown here with combined ultrasound and photoacoustic imaging in rats. Photoacoustic images of bladder wall is shown using black ink as the contrast agent. Chicken tissues were stacked on the abdomen of the animal to demonstrate the feasibility of photoacoustic imaging till a depth of 2 cm. Also, the feasibility of photoacoustic imaging for a common bladder disorder, vesicoureteral reflux is studied using urinary tract mimicking phantom. It is also shown that a clinical ultrasound system can be used for photoacoustic imaging of non‐invasive clearance study of gold nanorods from circulation by monitoring the gradual accumulation of the gold nanorods in the bladder. The time taken for accumulation of nanorods in the bladder can be used as an indicator of the clearance rate of the nanoparticle circulation from the body.      

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.      

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.      

A 15.6 frames per second 1‐megapixel multiple exposure laser speckle contrast imaging setup

A multiple exposure laser speckle contrast imaging (MELSCI) setup for visualizing blood perfusion was developed using a field programmable gate array (FPGA), connected to a 1000 frames per second (fps) 1‐megapixel camera sensor. Multiple exposure time images at 1, 2, 4, 8, 16, 32 and 64 milliseconds were calculated by cumulative summation of 64 consecutive snapshot images. The local contrast was calculated for all exposure times using regions of 4 × 4 pixels. Averaging of multiple contrast images from the 64‐millisecond acquisition was done to improve the signal‐to‐noise ratio. The results show that with an effective implementation of the algorithm on an FPGA, contrast images at all exposure times can be calculated in only 28 milliseconds. The algorithm was applied to data recorded during a 5 minutes finger occlusion. Expected contrast changes were found during occlusion and the following hyperemia in the occluded finger, while unprovoked fingers showed constant contrast during the experiment. The developed setup is capable of massive data processing on an FPGA that enables processing of MELSCI data in 15.6 fps (1000/64 milliseconds). It also leads to improved frame rates, enhanced image quality and enables the calculation of improved microcirculatory perfusion estimates compared to single exposure time systems.      

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.      

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.      

Back Cover: Large‐depth‐of‐field full‐field optical angiography (J. Biophotonics 5/2019)

A large‐depth‐of‐field full‐field optical angiography (LD‐FFOA) method is developed to expand the depth‐of‐field (DOF). The contrast pyramid fusion algorithm is used to fuse 10 FFOA images at different focus depth. Cover images of mouse ear shows LD‐FFOA image has higher contrast and more detailed features. The LD‐FFOA method solves the defocused problem caused by the limited DOF of lens, the curved surface and uneven thickness of the sample. Further details can be found in the article by Mingyi Wang, Nanshou Wu, Hongheng Huang, et al. ( e201800329 ).

     

Characterization and application of porous gold nanoparticles as 2‐photon luminescence imaging agents: 20‐fold brighter than gold nanorods

Two‐photon nonlinear microscopy with the aid of plasmonic contrast agents is an attractive bioimaging technique capable of generating high‐resolution images in 3 dimensions and facilitating targeted imaging with deep tissue penetration. In this work, physically synthesized gold nanoparticles containing multiple nanopores are used as 2‐photon contrast agents and are reported to emit a 20‐fold brighter 2‐photon luminescence as compared to typical contrast agents, that is, gold nanorods. A successful application of our porous gold nanoparticles is experimentally demonstrated by in vitro nonlinear optical imaging of adipocytes at subcellular level.