A microvascular image analysis method for optical-resolution photoacoustic microscopy

A novel combined photoacoustic tomography (PAT), optical resolution photoacoustic microscopy (ORPAM) and optical coherence tomography (OCT) instrument has been developed for imaging biological tissues. The system is based on the use of a Fabry-Perot (FP) polymer film ultrasound sensor. This is designed to be transparent to wavelengths between 590nm and 1200nm so that photoacoustic excitation laser pulses in this spectral range can be transmitted through the sensor into the underlying tissue to allow backward mode operation. The dual PAT-ORPAM capability of the system was demonstrated by imaging a tissue phantom composed of 7μm diameter carbon fibres immersed in an optically scattering liquid. The lateral and vertical spatial resolutions in ORPAM mode are approximately 7μm and 10μm respectively for sub-mm depths. In PAT mode, the lateral spatial resolution is less than 50μm for depths up to 5mm and the vertical resolution is approximately 10μm. The transparent nature of the FP polymer film ultrasound sensor offers a convenient platform for combining other optical imaging modalities with PAT and ORPAM. To illustrate this, a frequency-domain OCT system operating at 1060nm was integrated into the system and combined PAT/OCT images of the skin of a mouse were obtained in vivo.

We present a dual modality ultra-thin imaging system based on a optical multimode fiber and a optical fiber hydrophone that combines optical resolution photoacoustic and fluorescence microscopy.

In this Letter, reflection-mode optical resolution photoacoustic microscopy (OR-PAM) using glancing angle-deposited (GLAD) nanostructured Fabry-Perot interferometers (FPI) for in vivo applications is reported. GLAD is a single-step physical vapor deposition (PVD) technique used to fabricate porous nanostructured thin films. Using titanium dioxide, a transparent semiconductor with a high refractive index (n=2.4), the GLAD technique can be employed to fabricate samples with tailored nano-porosity, refractive index periodicities, and high Q-factor reflectance spectra. The OR-PAM in vivo images of chorioallantoic membrane (CAM) of 5-day chicken embryo model are demonstrated. The phantom study shows lateral resolution and signal-to-noise ratio better than 7μm and 35dB, respectively. The sensitive GLAD FPI allows photoacoustic imaging down to a few-nJ pulse energy. To the best of our knowledge, this is the first time that a FPI-based reflection mode optical resolution photoacoustic imaging technique is demonstrated for in vivo applications.

Optical resolution photoacoustic microscopy (ORPAM) is a high-resolution hybrid imaging modality having potential for microscale in vivo imaging. Optical diffraction limits the lateral resolution of ORPAM. A photonic nanojet (PNJ) was used to break this diffraction limit. A single round microsphere can generate a PNJ with subwavelength waist, but its short axial length limits its applications to surface imaging only. We investigate different sphere designs to achieve ultralong nanojets that will make the nanojet more viable in far-field applications, such as photoacoustic imaging. The PNJ properties, including effective length, waist size, working distance, and peak intensity, can be tuned and controlled by changing the sphere design and its refractive index. A truncated multilayer microsphere design could generate an ultraelongated PNJ with length larger than ∼172λ (∼138 μm) while retaining a large working distance ∼32λ (∼26 μm). Through simulation study, we observed ∼11-fold enhancement in lateral resolution with 5 μm round sphere (refractive index 2.2) when used in a conventional ORPAM setup with NA=0.1 and λ=800 nm.

Photoacoustic imaging is an emerging imaging modality that combines the high contrast of optical imaging and the high penetration of acoustic imaging. However, the strong focusing of the laser beam in optical-resolution photoacoustic microscopy (OR-PAM) leads to a limited depth-of-field (DoF). Here, a volumetric photoacoustic information fusion method was proposed to achieve large volumetric photoacoustic imaging at low cost. First, the initial decision map was built through the focus detection based on the proposed three-dimensional Laplacian operator. Majority filter-based consistency verification and Gaussian filter-based map smoothing were then utilized to generate the final decision map for the construction of photoacoustic imaging with extended DoF. The performance of the proposed method was tested to show that our method can expand the limited DoF by a factor of 1.7 without the sacrifice of lateral resolution. Four sets of multi-focus vessel data at different noise levels were fused to verify the effectiveness and robustness of the proposed method. The proposed method can efficiently extend the DoF of OR-PAM under different noise levels.

As a functional imaging technique, photoacoustic imaging has the ability to produce high contrast and high resolution image. However, photoacoustic microscopy shows the disadvantage of limited depth of field, due to the strong focusing process. Only some part of the whole image is clear while some other information might be lost or misleading. To achieve a large volumetric and high resolution photoacoustic, we propose a depth-of-field expansion motivated multi-focus photoacoustic microscopy image fusion algorithm which fuses the information from imaging of different focal positions into single one to obtain all-in-focus image. To achieve this goal, the volumes obtained at different focus positions are sliced into 2D and the 2D image fusion based on cross bilateral filter is performed for correspond slices. Finally, 3D reconstruction can be performed on these fused slices. Experiment results show that our method can produce a 3D fusion result which maintains useful photoacoustic signal information for further analysis and visualization.

Optical resolution photoacoustic microscopy (OR-PAM) can achieve an optical-dependent high lateral resolution (<; 1 μm) and visualize photoacoustic (PA) property at single-cell level. The spatial resolvability of the PA imaging is expected to precisely characterize biological activities of cells such as their interactions with drugs and contrast agents by comparing the acquired PA characteristics with the cell's morphology. However, since the conventional OR-PAM systems only visualize objects that generate PA signals, it was difficult to acquire a PA image and an optical morphological image simultaneously. Here, we report a novel system of the hybrid optical / PA microscopy that can acquire morphological and PA characteristics of cells at the same time. The performance of the developed system was validated by measuring a USAF1951 target and bovine red blood cells.

Photoacoustic (PA) imaging is rapidly progressing imaging modality in which very short pulsed laser causes thermal expansion to generate PA signal. In previous PA microscope systems, relatively long time was required for image acquisition because they required mechanical scan of the PA transducer. The objective of the present study is to develop fast laser scanning optical resolution photoacoustic microscopy (OR-PAM) with fixed PA signal detector to realize very high frame rate PA imaging. Q-switched Nd:YAG laser with the wavelength of 532 nm, pulse width of 5.5 ns and pulse repetition rate of up to 50 kHz was equipped in the system. Low frequency PA detector was comprised of a glass prism configuring acoustic focusing and a polymethyl methacrylate (PMMA) prism with 5 MHz PZT (lead zirconate titanate) transducer. High frequency PA detector was comprised of the same glass prism and a glass prism with ZnO (zinc oxide) thin film transducer. Galvano scanner operating in the air was controlled by a microcomputer to scan laser beam. PA signal was detected with the fixed PA detector thus realized the frame rate of 5 fps for C-mode equivalent to 500 fps for B-mode. The lateral resolution of the low frequency system was found to be 11.6 μm and the blood vessel on the surface of cod roe was clearly visualized. The system may be applicable for imaging blood flow and vascular dynamics of micro vessel.

Intravascular ultrasound (IVUS) plays a vital role in assessing the severity of atherosclerosis and has greatly enriched our knowledge on atherosclerotic plaques. However, it mainly reveals the structural information of plaques. In contrast, spectroscopic and molecular photoacoustic imaging can potentially improve plaque composition identification, inflammation detection, and ultimately the stratification of plaque vulnerability and risk. In this work, we developed an integrated intravascular ultrasound and optical-resolution photoacoustic microscopy (IVUS-PAM) system with a single catheter as small as 1 mm in diameter, comparable to that of existing clinical IVUS catheters. In addition, by using a GRIN lens to focus the excitation laser pulse, the system provides an optical-diffraction limited photoacoustic lateral resolution as fine as 19.6 micrometers, ~10-fold finer than that of conventional intravascular photoacoustic imaging and existing IVUS technology. The system employs a custom-made miniaturized single-element ultrasonic transducer with a dimension of ~0.5 mm, a centre frequency of ~40 MHz, and a fractional bandwidth of ~60%. The IVUS-PAM can simultaneously acquire co-registered IVUS images with an axial resolution of ~40 micrometers and a lateral resolution of ~200 micrometers. In the future, IVUS-PAM may open up new opportunities for improved high-resolution vulnerable plaque imaging and image-guided stent deployment.

Super-resolution ultrasound (SRUS) has become a tool for in vivo microvascular imaging. Most of the SRUS methods are based on microbubble localization: namely, ultrasound localization microscopy (ULM). The aim of this study was to develop a nonlocalization SRUS method and verify its feasibility in microvascular imaging. We introduce a new super-resolution strategy based on the postprocessing of contrast-enhanced ultrasound. The proposed method, which is termed ultrasound diffraction attenuation microscopy (UDAM), uses super-resolution radial fluctuations instead of microbubble localization to overcome acoustic diffraction limits. Biceps of Japanese long-ear white rabbits were adopted to validate its feasibility on muscle vascular imaging, using a clinical accessible ultrasound system at a frame rate of 30 Hz under a single bolus injection of SonoVue (Bracco SpA, Milan, Italy). The super-resolution image was compared with the maximum-intensity projection and ULM. The animal study illustrates that the proposed UDAM can obtain super-resolution microvascular images of rabbits' muscles under a single bolus injection of SonoVue with a 150-second contrast-enhanced ultrasound video. Both ULM and UDAM can achieve a very similar vascular structure with the maximum-intensity projection but much higher spatial resolution. The measurement of 1-dimensional signals shows that UDAM can distinguish the subwavelength structures and substantial reduce the full width at half-maximum of microvessels. We conclude UDAM provides a noninvasive tool for in vivo super-resolution microvascular imaging.

Intracranial microvascular imaging at 7 T MRI with transceiver RF coils

Optical-resolution photoacoustic microscopy (OR-PAM) can achieve submicron lateral resolution by tightly focusing the excitation light, while the axial resolution is still limited by the frequency bandwidth of the ultrasonic transducer. The Grueneisen relaxation effect, in which the Grueneisen parameter changes within the thermal relaxation time following a laser impulse heating, can provide excellent axial resolution due to its optical sectioning property. Based on this effect, Grueneisen relaxation photoacoustic microscopy (GR-PAM) was developed and demonstrated ex vivo. Here, we present for the first time in vivo imaging of mouse brains with improved axial resolution based on GR-PAM. An intensity-modulated continuous-wave (CW) 532 nm laser thermally heated the in-focus absorber. Another 532 nm pulsed laser, which is aligned confocally with the CW laser, generated the photoacoustic (PA) signal from the absorber. The difference between the amplitudes of the photoacoustic signals with and without heating was used for image reconstruction. The achieved axial resolution is ~12.5 µm, which is fivefold better than the acoustically determined value for a 20 MHz-bandwidth ultrasound transducer. The system was demonstrated by imaging a blood-filled tube ex vivo and blood vessels of mouse brains in vivo. The blood-filled tube diameter obtained from the PA image by GR-PAM is 105 µm, which is much closer to its actual diameter (100 µm) than the value from conventional OR-PAM (160 µm). This axial resolution improvement was further validated in imaging mouse brains in vivo, and yielded significantly narrower axial profiles of the vessels. This in vivo demonstration of imaging by GR-PAM might inspire more applications in PA biomedical imaging and sensing.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Photoacoustic imaging and optical coherence tomography are two emerging imaging modalities which provide complementary optical absorption and scattering contrasts for biological tissues. While photoacoustic imaging provides tissue vasculature information, optical coherence tomography offers micron-scale morphological imaging with penetration depths of 1~3 mms. Pulse-echo ultrasound is readily available from photoacoustic system and it provides tissue structure information at deeper depths with resolutions scalable with the transducer frequency. We present a prototype endoscope that consists of a ball lensed optical coherence tomography probe, a right-angled multimode fiber for delivering the laser beam for photoacoustic imaging, and a high frequency ultrasound transducer of 35 MHz center frequency. The overall diameter is 5mms. Porcine ovaries were imaged ex vivo to demonstrate the capability of this new combined endoscopy. The microvascular and high resolution structural images at subsurface and deeper tissue range demonstrate the synergy of the combined endoscopy over each modality alone.

Photoacoustic tomography (PAT), a hybrid technology combining optical excitation and ultrasonic detection, senses functional or molecular optical absorption contrasts and enables highhresolution imaging as deep as the optical diffusive regime. PAT for labelhfree microvascular imaging is highly desirable because of the presence of hemoglobin as an endogenous chromophore. In this chapter, we first review two major embodiments of PAT (photoacoustic microscopy and photoacoustic computed tomography). Then, we introduce methods for in vivo quantification of total hemoglobin concentration, blood oxygenation, and blood flow. After that, we show a few recent biomedical applications, including angiogenesis monitoring, brain and ocular imaging, and in vivo detection of circulating tumor cells. We end by summarizing the current status of microvascular applications of PAT and proposing several future research directions.