Yu-Hsin Wang

Photoacoustic/ultrasound dual-modality contrast agent and its application to thermotherapy

This study investigates a photoacoustic/ultrasound dual-modality contrast agent, including extending its applications from image-contrast enhancement to combined diagnosis and therapy with site-specific targeting. The contrast agent comprises albumin-shelled microbubbles with encapsulated gold nanorods (AuMBs). The gas-filled microbubbles, whose diameters range from submicrometer to several micrometers, are not only echogenic but also can serve as drug-delivery vehicles. The gold nanorods are used to enhance the generation of both photoacoustic and photothermal signals. The optical absorption peak of the gold nanorods is tuned to 760 nm and is invariant after microbubble encapsulation. Dual-modality contrast enhancement is first described here, and the applications to cellular targeting and laser-induced thermotherapy in a phantom are demonstrated. Photoacoustic imaging can be used to monitor temperature increases during the treatment. The targeting capability of AuMBs was verified, and the temperature increased by 26°C for a laser power of 980 mW, demonstrating the potential of combined diagnosis and therapy with the dual-modality agent. Targeted photo- or acoustic-mediated delivery is also possible.

Synergistic delivery of gold nanorods using multifunctional microbubbles for enhanced plasmonic photothermal therapy.

Plasmonic photothermal therapy (PPTT) using plasmonic nanoparticles as efficient photoabsorbing agents has been proposed previously. One critical step in PPTT is to effectively deliver gold nanoparticles into the cells. This study demonstrates that the delivery of gold nanorods (AuNRs) can be greatly enhanced by combining the following three mechanisms: AuNRs encapsulated in protein-shell microbubbles (AuMBs), molecular targeting, and sonoporation employing acoustic cavitation of microbubbles (MBs). Both in vitro and in vivo tests were performed. For molecular targeting, the AuMBs were modified with anti-VEGFR2. Once bound to the angiogenesis markers, the MBs were destroyed by ultrasound to release the AuNRs and the release was confirmed by photoacoustic measurements. Additionally, acoustic cavitation was induced during MB destruction for sonoporation (i.e., increase in transient cellular permeability). The measured inertial cavitation dose was positively correlated with the temperature increase at the tumor site. The quantity of AuNRs delivered into the cells was also determined by measuring the mass spectrometry and observed using third-harmonic-generation microscopy and two-photon fluorescence microscopy. A temperature increase of 20°C was achieved in vitro. The PPTT results in vivo also demonstrated that the temperature increase (>45°C) provided a sufficiently high degree of hyperthermia. Therefore, synergistic delivery of AuNRs was demonstrated.

A photoacoustic imager with light illumination through an infrared-transparent silicon CMUT array

A novel hardware design and preliminary experimental results for photoacoustic imaging are reported in this paper. This imaging system makes use of an infrared-transparent capacitive micromachined ultrasonic transducer (CMUT) chip for ultrasound reception and illuminates the image target through the CMUT array. The cascaded arrangement between the light source and transducer array allows for a more compact imager head and results in more uniform illumination. Taking advantage of the low optical absorption coefficient of silicon in the near infrared spectrum as well as the broad acoustic bandwidth that CMUTs provide, an infrared-transparent CMUT array has been developed for ultrasound reception. The center frequency of the polysilicon-membrane CMUT devices used in this photoacoustic system is 3.5 MHz, with a fractional bandwidth of 118% in reception mode. The silicon substrate of the CMUT array has been thinned to 100 -m and an antireflection dielectric layer is coated on the back side to improve the infrared-transmission rate. Initial results show that the transmission rate of a 1.06-μm Nd:Yag laser through this CMUT chip is 12%. This transmission rate can be improved if the thickness of silicon substrate and the thin-film dielectrics in the CMUT structure are properly tailored. Imaging of a metal wire phantom using this cascaded photoacoustic imager is demonstrated.

SNR-dependent coherence-based adaptive imaging for high-frame-rate ultrasonic and photoacoustic imaging

This paper introduces the SNR-dependent coherence factor (CF), which can be used for adaptive side lobe suppression in ultrasound (US) and photoacoustic (PA) imaging. Previous methods employed the minimum-variance distortionless response (MVDR)-based CF to achieve remarkable resolution improvement (by MVDR) and to suppress side lobes (by CF). However, the SNR is often low when using an unfocused acoustic beam (e.g., high-frame-rate imaging) and in PA imaging (limited laser energy), giving such an approach suboptimal performance in these applications because noise also lowers the coherence and thus affects the effectiveness of the side lobe suppression by these CF-based methods. To overcome this problem, the proposed method takes into account the local SNR in the CF formulation so that the contrast can be restored even when the SNR is low. We tested this method with both high-frame-rate US imaging and PA imaging. Simulations show that the proposed method performs well even when the SNR is as low as -10 dB. Compared with the conventional CF, the contrast (CR) and contrast-to-noise ratio (CNR) in clinical US imaging can be improved by an average of 27.2% in CR and 11.1% in CNR with the proposed method, whereas in PA imaging, the lateral resolution could be restored and the image contrast was elevated by 17 dB.

Buoyancy-Activated Cell Sorting Using Targeted Biotinylated Albumin Microbubbles

Cell analysis often requires the isolation of certain cell types. Various isolation methods have been applied to cell sorting, including florescence-activated cell sorting and magnetic-activated cell sorting. However, these conventional approaches involve exerting mechanical forces on the cells, thus risking cell damage. In this study we applied a novel isolation method called buoyancy-activated cell sorting, which involves using biotinylated albumin microbubbles (biotin-MBs) conjugated with antibodies (i.e., targeted biotin-MBs). Albumin MBs are widely used as contrast agents in ultrasound imaging due to their good biocompatibility and stability. For conjugating antibodies, biotin is conjugated onto the albumin MB shell via covalent bonds and the biotinylated antibodies are conjugated using an avidin-biotin system. The albumin microbubbles had a mean diameter of 2μm with a polydispersity index of 0.16. For cell separation, the MDA-MB-231 cells are incubated with the targeted biotin-MBs conjugated with anti-CD44 for 10 min, centrifuged at 10g for 1 min, and then allowed 1 hour at 4°C for separation. The results indicate that targeted biotin-MBs conjugated with anti-CD44 antibodies can be used to separate MDA-MB-231 breast cancer cells; more than 90% of the cells were collected in the MB layer when the ratio of the MBs to cells was higher than 70:1. Furthermore, we found that the separating efficiency was higher for targeted biotin-MBs than for targeted avidin-incorporated albumin MBs (avidin-MBs), which is the most common way to make targeted albumin MBs. We also demonstrated that the recovery rate of targeted biotin-MBs was up to 88% and the sorting purity was higher than 84% for a a heterogenous cell population containing MDA-MB-231 cells (CD44+) and MDA-MB-453 cells (CD44–), which are classified as basal-like breast cancer cells and luminal breast cancer cells, respectively. Knowing that the CD44+ is a commonly used cancer-stem-cell biomarker, our targeted biotin-MBs could be a potent tool to sort cancer stem cells from dissected tumor tissue for use in preclinical experiments and clinical trials.

Design, fabrication and testing of a dual-band photoacoustic transducer.

Combining photoacoustic and ultrasonic imaging allows both optical and acoustic properties to be displayed simultaneously. In this paper, we describe a dual-band transducer for implementing such a multimodality imaging setup. The transducer exhibits two frequency bands so that it matches the frequency of interest in both imaging methods. An optical fiber is included in the center so that it is inherently coregistered. The transducer was fabricated from lithium niobate and comprises two concentric rings whose center frequencies are 4.9 MHz and 14.8 MHz. Pulse-echo measurements and phantom imaging were performed to demonstrate its performance characteristics.

Study of male sterility in Taiwania cryptomerioides Hayata (Taxodiaceae)

Summary.A study of male sterility over a period of three consecutive years on a conifer species endemic to Taiwan, Taiwania cryptomerioides Hayata (Taxodiaceae), was done for this article. With the aids of fluorescence and electron microscopic observations, the ontogenic processes in the fertile and sterile microsporangia are compared, using samples collected from Chitou Experimental Forest and Yeou-Shoei-Keng Clonal Orchard of the National Taiwan University, Nantou, Taiwan. The development of male strobili occurred from August to the end of March. Microsporogenesis starts with the formation of the archesporium and ends with the maturation of 2-celled pollen grains within the dehiscing microsporangium. Before meiosis, there was no significant difference in ultrastructure between the fertile and sterile microsporangia. Asynchronous pollen development with various tetrad forms may occur in the same microsporangium of either fertile or sterile strobili. However, a callose wall was observable in the fertile dyad and tetrad, but not in the sterile one. After dissolution of the callose wall, the fertile microspores were released into the locule, while some sterile microspores still retained as tetrads or dyads with intertwining of exine walls in the proximal faces. As a result, there was no well developed lamellated endexine and no granulate ectexine or intine in the sterile microspores. Eventually, the intracellular structures in sterile microspores were dramatically collapsed before anthesis. The present study shows that the abortion in pollen development is possibly attributed to the absence of the callose wall. The importance of this structure to the male sterility of T. cryptomerioides is discussed.

Thermotherapy with a photoacoustic/ultrasound dual-modality agent

A microbubble-based imaging/therapeutic agent is introduced. Specifically, gold nanoparticles (AuNRs) are encapsulated in microbubbles (MBs) for both ultrasound (US) imaging and laser-induced thermotherapy (LIT). In addition, this agent, AuNR-MB, takes albumin microbubble as a carrier and includes the AuNRs that maintain the original absorption peak at around 760nm. AuNR-MBs in different sizes are synthesized. Imaging is first performed to evaluate its feasibility. The enhanced PA and US signals in polyacrylamide gel for in vitro study are measured. The PA spectroscopy is then performed and the results generally agree with the measured optical absorption although its peak is slightly broadened and shifted possibly due to mixing. In phantoms, the contrast is 1.531, 2.447, 2.085, 1.994, 0.768, and 0.573 at wavelength of 720, 760, 800, 860, 900, and 940 nm respectively. Finally, the application of the new agent to LIT is presented. A continuous wave laser at 800 nm is used to heat the samples with the power at 1W. The photoacoustic (PA) intensity in the region of interest (ROI) is increased by an average of 5.2dB. The increased signal level implies that the temperature in the ROI can be increased to 44.3°C in aqueous filled setup. Furthermore, the dual-modality agent has the potential to be used in HIFU therapy, drug delivery and loading of DNA for gene transfer.

SNR-dependent coherence weighting for minimum variance beamforming

Coherence factor (CF) is used to suppress sidelobes and to enhance contrast in ultrasound imaging. Recent research also introduces minimum-variance-distortionless-response (MVDR) based CF (CFmvdr) to achieve good resolution for high frame rate application. However, increasing frame rate by unfocused beams reduces signal-to-noise-ratio (SNR). CF-based weighting is susceptible to noise, which underestimates the acoustic strength and lowers the contrast supposed to provide. Wiener postfilter, with worse performance on noise reduction but better robustness, can be combined with CF by scaling the noise power. Hence, CF weighting can be modified so as to avoid the noise effect with the knowledge of local SNR. The proposed method is specifically applied to the high frame rate imaging that has synthetic transmit aperture (STA) and MVDR beamformer. Before finding the MVDR solution, the channel data are used to estimate SNR, which then determines the noise power weighting by a sigmoid function and modifies the CF value. Specifically, at mainlobe with relatively high SNR, the CF or the CFmvdr is adjusted by lowering its noise weighting. For the region requiring higher resolution, MVDR beamformer can retain its performance. Thus, the contrast can be restored when the noise level is high. Simulations were performed by a 128-element linear array with central frequency 4.37 MHz. The pulse-echo data were generated through STA method with eight subgroups. When the channel has SNR -10 dB, the CR and the CNR by original CF/CFmvdr were 39.01/34.71 dB and 2.00/1.74 respectively. After modified by SNR, the CR and the CNR were improved to 47.45/47.82 dB and 2.26/2.14 in scaled CF/CFmvdr cases. Simulations for point pairs was also included to confirm the ability to maintain resolution. In summary, a robust method designed for low SNR cases is proposed.

Enhanced delivery of gold nanoparticles by acoustic cavitation for photoacoustic imaging and photothermal therapy

Gold-nanorods incorporated with microbubbles (AuMBs) were introduced as a photoacoustic/ultrasound dual- modality contrast agent in our previous study. The application can be extended to theragnosis purpose. With the unique physical characteristics of AuMBs, we propose an enhanced delivery method for the encapsulated particles. For example, laser thermotherapy mediated by plasmonic nanoparticles can be made more effective by using microbubbles as a targeted carrier and acoustic cavitation for enhanced sonoporation. The hypothesis was experimentally tested. Firts, these AuMBs first act as molecular probes with binding to specific ligands. The improved targeting efficacy was macroscopically observed by an ultrasound system. The extended retention of targeted AuMB was observed and recorded for 30 minutes in a CT-26 tumor bearing mouse. Secondly, cavitation induced by time-varying acoustic field was also applied to disrupt the microbubbles and cause increased transient cellular permeability (a.k.a., sonoporation). Multimodal optical microscope based on a Cr:forsterite laser was used to directly observe these effects. The microscope can acquired third-harmonic generation (THG) and two-photon fluorescent (2PF) signals produced by the AuMBs. In vitro examination shows approximately a 60% improvement in terms of fluorescence signals from the cellular uptake of gold nanoparticles after sonoporation treatment. Therefore, we conclude that the controlled release is feasible and can further improve the therapeutic effects of the nanoparticles.