Kathyayini Sivasubramanian

Recent Developments in Vascular Imaging Techniques in Tissue Engineering and Regenerative Medicine

Adequate vascularisation is key in determining the clinical outcome of stem cells and engineered tissue in regenerative medicine. Numerous imaging modalities have been developed and used for the visualization of vascularisation in tissue engineering. In this review, we briefly discuss the very recent advances aiming at high performance imaging of vasculature. We classify the vascular imaging modalities into three major groups: nonoptical methods (X-ray, magnetic resonance, ultrasound, and positron emission imaging), optical methods (optical coherence, fluorescence, multiphoton, and laser speckle imaging), and hybrid methods (photoacoustic imaging). We then summarize the strengths and challenges of these methods for preclinical and clinical applications.

High frame rate photoacoustic imaging at 7000 frames per second using clinical ultrasound system

Photoacoustic tomography, a hybrid imaging modality combining optical and ultrasound imaging, is gaining attention in the field of medical imaging. Typically, a Q-switched Nd:YAG laser is used to excite the tissue and generate photoacoustic signals. But, such photoacoustic imaging systems are difficult to translate into clinical applications owing to their high cost, bulky size often requiring an optical table to house such lasers. Moreover, the low pulse repetition rate of few tens of hertz prevents them from being used in high frame rate photoacoustic imaging. In this work, we have demonstrated up to 7000 Hz photoacoustic imaging (B-mode) and measured the flow rate of a fast moving object. We used a ~140 nanosecond pulsed laser diode as an excitation source and a clinical ultrasound imaging system to capture and display the photoacoustic images. The excitation laser is ~803 nm in wavelength with ~1.4 mJ energy per pulse. So far, the reported 2-dimensional photoacoustic B-scan imaging is only a few tens of frames per second using a clinical ultrasound system. Therefore, this is the first report on 2-dimensional photoacoustic B-scan imaging with 7000 frames per second. We have demonstrated phantom imaging to view and measure the flow rate of ink solution inside a tube. This fast photoacoustic imaging can be useful for various clinical applications including cardiac related problems, where the blood flow rate is quite high, or other dynamic studies.

Near Infrared light-responsive liposomal contrast agent for photoacoustic imaging and drug release applications

Abstract. Photoacoustic imaging has become an emerging tool for theranostic applications. Not only does it help in in vivo, noninvasive imaging of biological structures at depths but it can also be used for drug release and therapeutic applications. We explore near-infrared light-sensitive liposomes coated with gold nanostars (AuNSs) for both imaging and drug release applications using a photoacoustic imaging system. Being amphiphilic, the liposomes lipid bilayer and the aqueous core enable encapsulation of both hydrophobic and hydrophilic drugs. The AuNSs on the surface of the liposomes act as photon absorbers due to their intrinsic surface plasmon resonance. Upon excitation by laser light at specific wavelength, AuNSs facilitate rapid release of the contents encapsulated in the liposomes due to local heating and pressure wave formation (photoacoustic wave). Herein, we describe the design and optimization of the AuNSs-coated liposomes and demonstrate the release of both hydrophobic and hydrophilic model drugs (paclitaxel and calcein, respectively) through laser excitation at near-infrared wavelength. The use of AuNSs-coated liposomes as contrast agents for photoacoustic imaging is also explored with tissue phantom experiments. In comparison to blood, the AuNSs-coated liposomes have better contrast (approximately two times) at 2-cm imaging depth.

In situ synthesis of gold nanostars within liposomes for controlled drug release and photoacoustic imaging

This report describes the design and synthesis of gold nanostars (AuNSs) containing liposomes by the in situ reduction of gold precursor, HAuCl4 (pre-encapsulated within the liposomes) through HEPES diffusion and reduction. Compared with the conventional process that encapsulates the pre-synthesized gold nanoparticles into liposomes during the thin-film hydration step, this facile and convenient method allows the formation and simultaneous encapsulation of AuNSs within liposomes. The absorption spectra of AuNSs can be tuned between visible and near infra-red (NIR) regions by controlling the size and morphology of AuNSs through varying the concentrations of HAuCl4 and HEPES. As a proof of concept, we demonstrate the synthesis of AuNSs with a maximum absorbance at 803 nmwithin the temperature-sensitive liposomes. These liposomes can produce stronger photoacoustic signals (1.5 fold) in the NIR region than blood. Furthermore, when there are drugs (i.e., doxorubicin) within these liposomes, the irradiation with the NIR pulse laser will disrupt the liposomes and trigger the 100% release of these pre-encapsulated drugs within 10 seconds. In comparison, there is neglectable contrast enhancement or minor release (10%) of drugs for the pure liposomes under the same conditions. Finally, cell experiment shows the potential therapeutic application of this system.摘要本文通过在脂质体内还原金的前体HAuCl4原位合成了金纳米星. 这种设计跟常用的在脂质体内装载金纳米球的方法相比, 优点是方 便快捷的同时实现了金纳米材料的形成和装载. 通过改变实验条件, 合成的金纳米星具有可控的尺寸, 以及在可见区到近红外区之间的可 控吸收光谱. 作为一个例子, 我们在脂质体内合成了最大吸收在803nm的金纳米星. 这种材料具有温度敏感性, 在近红外区可以产生比血液 好1.5倍的光声造影信号. 当我们把抗癌药物阿霉素装载到这种脂质体内时, 近红外区的激光照射可以在10秒内触发药物100%的释放. 相对应的, 不含纳米星的脂质体在同等条件下只能释放10%的药物, 也不具备光声造影的信号增强. 最后, 我们在癌细胞内测试了该脂质体的疗效, 初步验证了该体系的应用前景.

Optimizing light delivery through fiber bundle in photoacoustic imaging with clinical ultrasound system: Monte Carlo simulation and experimental validation

Abstract. Translating photoacoustic (PA) imaging into clinical setup is a challenge. We report an integrated PA and ultrasound imaging system by combining the light delivery to the tissue with the ultrasound probe. First, Monte Carlo simulations were run to study the variation in absorbance within tissue for different angles of illumination, fiber-to-probe distance (FPD), and fiber-to-tissue distance (FTD). This is followed by simulation for different depths of the embedded sphere (object of interest). Several probe holders were designed for different light launching angles. Phantoms were developed to mimic a sentinel lymph node imaging scenario. It was observed that, for shallower imaging depths, the variation in signal-to-noise ratio (SNR) values could be as high as 100% depending on the angle of illumination at a fixed FPD and FTD. Results confirm that different light illumination angles are required for different imaging depths to get the highest SNR PA images. The results also validate that one can use Monte Carlo simulation as a tool to optimize the probe holder design depending on the imaging needs. This eliminates a trial-and-error approach generally used for designing a probe holder.

Non‐invasive sentinel lymph node mapping and needle guidance using clinical handheld photoacoustic imaging system in small animal

Translating photoacoustic imaging (PAI) into clinical setup is a challenge. Handheld clinical real-time PAI systems are not common. In this work, we report an integrated photoacoustic (PA) and clinical ultrasound imaging system by combining light delivery with the ultrasound probe for sentinel lymph node imaging and needle guidance in small animal. The open access clinical ultrasound platform allows seamless integration of PAI resulting in the development of handheld real-time PAI probe. Both methylene blue and indocyanine green were used for mapping the sentinel lymph node using 675 and 690 nm wavelength illuminations, respectively. Additionally, needle guidance with combined ultrasound and PAI was demonstrated using this imaging system. Up to 1.5 cm imaging depth was observed with a 10 Hz laser at an imaging frame rate of 5 frames per second, which is sufficient for future translation into human sentinel lymph node imaging and needle guidance for fine needle aspiration biopsy.

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.

High speed photoacoustic tomography system with low cost portable pulsed diode laser

Photoacoustic tomography (PAT) is a potential hybrid imaging modality that has attracted great attention in the fields of medical imaging. In order to generate photoacoustic signal efficiently Q-switched Nd:YAG pump lasers capable of generating tens of millijoules of nanosecond laser pulses have been widely used. However, PAT systems using such lasers have limitations in clinical applications because of their high cost, large size, and cooling requirements. Furthermore, the low pulse repetition rate (PRR) of tens of hertz is not suitable for real-time PAT. So, there is a need for inexpensive, compact, simple, fast imaging system for clinical applications. Nanosecond pulsed laser diodes could meet these requirements. In this work, we present a high-speed photoacoustic tomography imaging system that uses a compact and yet relatively powerful near-infrared pulsed laser diode. The PAT system was tested on phantoms to verify its potential imaging speed. Photoacoustic reconstructed images at different scanning speeds are presented. With single ultrasound detector scanning, the system could provide PA image ~10 times faster than the Nd:YAG laser based systems.

On-chip generation of microbubbles in photoacoustic contrast agents for dual modal ultrasound/photoacoustic in vivo animal imaging

Dual-modal photoacoustic (PA) and ultrasound (US) contrast agents are becoming increasingly popular in recent years. Here, a flow-focusing junction based microfluidic device is used for the generation of nitrogen microbubbles (<7 μm) in two photoacoustic contrast agents: methylene blue (MB) and black ink (BI). The microbubble diameter and production rate could be precisely controlled in both MB and BI solutions. Microbubbles were collected from the outlet of the microfluidic device and optical microscope was used to study the size distributions in both solutions. Next, the microbubbles in both solutions were injected into tubes for phantom imaging experiments. Signal to noise ratio (SNR) of both US, PA imaging experiments were calculated to be 51 dB, 58 dB in MB + microbubbles and 56 dB, 61 dB in BI + microbubbles, respectively. Finally, the microbubbles were injected into the urinary bladder of rats for in vivo animal imaging. The SNR in US imaging with MB + microbubbles and BI + microbubbles were 41 dB and 48 dB, respectively. Similarly, the SNR in PA imaging with the same solutions were 32 dB and 36 dB, respectively. The effect of size and concentration of microbubbles in both MB and BI solutions, on the US and PA signals, has been examined.

Photoacoustic cystography using handheld dual modal clinical ultrasound photoacoustic imaging system

Vesicoureteral reflux is the abnormal flow of urine from your bladder back up the tubes (ureters) that connect your kidneys to your bladder. Normally, urine flows only down from your kidneys to your bladder. Vesicoureteral reflux is usually diagnosed in infants and children. The disorder increases the risk of urinary tract infections, which, if left untreated, can lead to kidney damage. X-Ray cystography is used currently to diagnose this condition which uses ionising radiation, making it harmful for patients. In this work we demonstrate the feasibility of imaging the urinary bladder using a handheld clinical ultrasound and photoacoustic dual modal imaging system in small animals (rats). Additionally, we demonstrate imaging vesicoureteral reflux using bladder mimicking phantoms. Urinary bladder imaging is done with the help of contrast agents like black ink and gold nanoparticles which have high optical absorption at 1064 nm. Imaging up to 2 cm was demonstrated with this system. Imaging was done at a framerate of 5 frames per second.