A Forked Microvascular Phantom for Ultrasound Localization Microscopy Investigations.

In the development of ultrasound localization microscopy (ULM) methods, appropriate test beds are needed to facilitate algorithmic performance calibration. Here, we present the design of a new ULM-compatible microvascular phantom with a forked, V-shaped wall-less flow channel pair ( 250 µ m channel width) that is bifurcated at a separation rate of 50 µ m/mm. The lumen core was fabricated using additive manufacturing, and it was molded within a polyvinyl alcohol (PVA) tissue-mimicking slab using the lost-core casting method. Measured using optical microscopy, the lumen core's flow channel width was found to be 252 ± 15 µ m with a regression-derived flow channel separation gradient of 50.89 µ m/mm. The new phantom's applicability in ULM performance analysis was demonstrated by feeding microbubble (MB) contrast flow (1.67 to 167 µ L/s flow rates) through the phantom's inlet and generating ULM images with a previously reported method. Results showed that, with longer acquisition times (10 s or longer), ULM image quality was expectedly improved, and the variance of ULM-derived flow channel measurements was reduced. Also, at axial depths near the lumen's bifurcation point, the current ULM algorithm showed difficulty in properly discerning between the two flow channels because of the narrow channel-to-channel separation distance. Overall, the new phantom serves well as a calibration tool to test the performance of ULM methods in resolving small vasculature.

Improvement of spatial resolution in photoacoustic microscopy using transmissive adaptive optics with a low-frequency ultrasound transducer.

Maintaining a high spatial resolution in photoacoustic microscopy (PAM) of deep tissues is difficult due to large aberration in an objective lens with high numerical aperture and photoacoustic wave attenuation. To address the issue, we integrate transmission-type adaptive optics (AO) in high-resolution PAM with a low-frequency ultrasound transducer (UT), which increases the photoacoustic wave detection efficiency. AO improves lateral resolution and depth discrimination in PAM, even for low-frequency ultrasound waves by focusing a beam spot in deep tissues. Using the proposed PAM, we increased the lateral resolution and depth discrimination for blood vessels in mouse ears.

Suppression of reflected signals from substrate as clutters for cell measurements using acoustic impedance microscopy.

Recently, a method for estimating three-dimensional acoustic impedance profiles in cultured cells and human dermal organs was proposed by interpreting the reflected ultrasonic signal based on a 1-D transmission line model for acoustic impedance microscopy (AIM). However, AIM has a disadvantage that reflected signals from cells overlap with that from a reference substrate. Additionally, the amplitudes of the reflected signals from the specimens are significantly weaker than that from the substrate. In this paper, we proposed a new method for separation of those signals based on a concept of clutter filter, which had been developed for a color Doppler method in medical ultrasonic imaging. The proposed filter using singular value decomposition (SVD) could separate original signals into desired signals such as those from the substrate and cells. Additionally, an effect from a tilt of the substrate was investigated in this study. Separability of the proposed filter was evaluated by two investigations. First one was to evaluate the separability by estimating a correlation coefficient between the filtered signal and signal reflected from a position only with the substrate. Second one was to compare a slope of the substrate estimated from the original signal with that estimated from the filtered signals from the substrate. The experimental results showed that the proposed filter could separate signals from the substrate, and the compensation of the tilt of the substrate could improve the performance of the proposed filter.

Fourier transform acousto-optic imaging with off-axis holographic detection.

Acousto-optic (AO) imaging is an in-depth optical imaging technique of highly scattering media. One challenging end-application for this technique is to perform imaging of living biological tissues. Indeed, because it relies on coherent illumination, AO imaging is sensitive to speckle decorrelation occurring on the millisecond time scale. Camera-based detections are well suited for in vivo imaging provided their integration time is lower than those decorrelation time scales. We present Fourier transform acousto-optic imaging combined with off-axis holography, which relies on plane waves and long-duration pulses. We demonstrate, for the first time to the best of our knowledge, a two-dimensional imaging system fully compatible with in vivo imaging prerequisites. The method is validated experimentally by performing in-depth imaging inside a multiple scattering sample.

Optical resolution photoacoustic computed microscopy.

Optical resolution photoacoustic microscopy (ORPAM) has demonstrated both high resolution and rich contrast imaging of optical chromophores in biologic tissues. To date, sensitivity remains a major challenge for ORPAM, which limits the capability of resolving biologic microvascular networks. In this study, we propose and evaluate a new ORPAM modality termed as optical resolution photoacoustic computed microscopy (ORPACM), through the combination of a two-dimensional laser-scanning system with a medical ultrasonographic platform. Apart from conventional ORPAMs, we record multiple photoacoustic (PA) signals using a 128-element ultrasonic transducer array for each pulse excitation. Then, we apply a reconstruction algorithm to recover one depth-resolved PA signal referred to as an A-line, which reveals more detailed information compared with conventional single-element transducer-based ORPAMs. In addition, we carried out both in vitro and in vivo experiments as well as quantitative analyses to show the advanced features of ORPACM.

Transparent High-Frequency Ultrasonic Transducer for Photoacoustic Microscopy Application.

We report the development of an optically transparent high-frequency ultrasonic transducer using lithium niobate single-crystal and indium-tin-oxide electrodes with up to 90% optical transmission in the visible-to-near-infrared spectrum. The center frequency of the transducer was at 36.9 MHz with 33.9%, at -6 dB fractional bandwidth. The photoacoustic imaging capability of the fabricated transducer was also demonstrated by successfully imaging a resolution target and mouse-ear vasculatures in vivo, which were irradiated by a 532 nm pulse laser transmitted through the transducer.

Noninvasive ultrasound imaging for assessment of intact microstructure of extracellular matrix in tissue engineering.

Development of artificial tissues or organs is one of the actual tasks in regenerative medicine that requires observation and evaluation of intact volume microstructure of tissue engineering products at all stages of their formation, from native donor tissues and decellularized scaffolds to recipient cell migration in the matrix. Unfortunately in practice, methods of vital noninvasive imaging of volume microstructure in matrixes are absent. In this work, we propose a new approach based on high-frequency acoustic microscopy for noninvasive evaluation and visualization of volume microstructure in tissue engineering products. The results present the ultrasound characterization of native rat diaphragms and lungs and their decellularized scaffolds. Verification of the method for visualization of tissue formation in the matrix volume was described in the model samples of diaphragm scaffolds with stepwise collagenization. Results demonstrate acoustic microscopic sensitivity to cell content concentration, variation in local density, and orientation of protein fibers in the volume, micron air inclusions, and other inhomogeneities of matrixes.

Scanning Acoustic Microscopy and Time-Resolved Fluorescence Spectroscopy for Characterization of Atherosclerotic Plaques.

Atherosclerotic plaques constitute the primary cause of heart attack and stroke. However, we still lack a clear identification of the plaques. Here, we evaluate the feasibility of scanning acoustic microscopy (SAM) and time-resolved fluorescence spectroscopy (TRFS) in atherosclerotic plaque characterization. We perform dual-modality microscopic imaging of the human carotid atherosclerotic plaques. We first show that the acoustic impedance values are statistically higher in calcified regions compared with the collagen-rich areas. We then use CdTe/CdS quantum dots for imaging the atherosclerotic plaques by TRFS and show that fluorescence lifetime values of the quantum dots in collagen-rich areas are notably different from the ones in calcified areas. In summary, both modalities are successful in differentiating the calcified regions from the collagen-rich areas within the plaques indicating that these techniques are confirmatory and may be combined to characterize atherosclerotic plaques in the future.

A rotary scanning method to evaluate grooves and porosity for nerve guide conduits based on ultrasound microscopy.

Grooved nerve guide conduits (NGCs) have been effective in the clinical treatment of peripheral nerve injury. They are generally fabricated from a micro-structured spinneret using a spinning process, which easily can cause a variety of pores and morphological deviation. The topography of internal grooves as well as the porosity can greatly influence the therapeutic effect. Traditional optical or scanning electron microscopy (SEM) methods can be used to image the grooves; however, these methods are destructive and require slicing NGCs to prepare specimens suitable for imaging. Moreover, lengthy experiments and large batches of NGCs are required to ensure reliable results from both in vitro experiments and clinical studies. In this paper, a non-destructive method for evaluating the grooves and porosity of NGCs is proposed using ultrasonic imaging combined with rotary scanning and an image analysis algorithm. Two ultrasonic methods were used: a 25-MHz point-focus ultrasonic transducer applied to observe axial cross sections of the conduits and a 100-MHz point-focus ultrasonic transducer to detect large pores caused by defects. Furthermore, a theoretical algorithm for detecting the local porosity of a conduit based on density is proposed. Herein, the proposed acoustic method and traditional optical methods are evaluated and compared. A parameter representing the specific surface area of the internal grooves is introduced and computed for both the optical and acoustic methods, and the relative errors of the computed parameter values for three different NGCs were 7.0%, 7.9%, and 15.3%. The detected location and shape of pores were consistent between the acoustic and optical methods, and greater porosity was observed in the middle of the conduit wall. In this paper, the results of the acoustic and optical methods are presented and the errors relating to the acoustic factors, device characteristics, and image processing method are further analyzed.

Stomach wall structure and vessels imaging by acoustic resolution photoacoustic microscopy.

AIM: To image stomach wall blood vessels and tissue, layer-by-layer. METHODS: We built up the acoustic resolution photoacoustic microscopy (AR-PAM) system for imaging layered tissues, such as the stomach wall. A tunable dye laser system was coupled to a fiber bundle. The fibers of the bundle were placed in nine directions with an incident angle of 45° around a high-frequency ultrasound transducer attached to the acoustic lens. This structure formed a dark field on the tissue surface under the acoustic lens and the nine light beams from the fibers to be combined near the focal point of the acoustic lens. The sample piece was cut from a part of the porcine stomach into a petri dish. In order to realize photoacoustic depth imaging of tumor, we designed a tumor model based on indocyanine green (ICG) dye. The ICG solution (concentration of 129 µM/mL) was mixed into molten gel, and then a gel mixture of ICG (concentration of 12.9 µM/mL) was injected into the stomach submucosa. The injection quantity was controlled by 0.1 mL to make a small tumor model. RESULTS: An acoustic resolution photoacoustic microscopy based on fiber illumination was established and an axial resolution of 25 µm and a lateral resolution of 50 µm in its focal zone range of 500 µm has been accomplished. We tuned the laser wavelength to 600 nm. The photoacoustic probe was driven to do B-scan imaging in tissue thickness of 200 µm. The photoacoustic micro-image of mucosa and submucosa of the tissue have been obtained and compared with a pathological photograph of the tissue stained by hematoxylin-eosin staining. We have observed more detailed internal structure of the tissue. We also utilized this photoacoustic microscopy to image blood vessels inside the submucosa. High contrast imaging of the submucosa tumor model was obtained using ICG dye. CONCLUSION: This AR-PAM is able to image layer-by-layer construction and some blood vessels under mucosa in the stomach wall without any contrast agents.