WO2024176239A1

WO2024176239A1 - Frequency mixing ultrasound imaging of nanoscale contrast agents

Info

Publication number: WO2024176239A1
Application number: PCT/IL2024/050215
Authority: WO
Inventors: Tali ILOVITSH; Keren Tchelet KARLINSKY
Original assignee: Ramot at Tel Aviv University Ltd
Current assignee: Ramot at Tel Aviv University Ltd
Priority date: 2023-02-26
Filing date: 2024-02-26
Publication date: 2024-08-29
Anticipated expiration: 2025-08-26

Abstract

Provided herein are nonlinear frequency mixing ultrasound imaging methods and systems for enhancing contrast of nanoscale contrast agents and improving image quality.

Description

FREQUENCY MIXING ULTRASOUND IMAGING OF NANOSCALE CONTRAST AGENTS

FIELD OF THE INVENTION

The present disclosure is directed to systems and methods for ultrasound imaging, using frequency mixing, of nanoscale contrast agents.

BACKGROUND

Ultrasound (US) is a widely used medical imaging modality due to its advantages of being safe, available, cost effective, and deep penetrating. US is typically used for soft tissue anatomical imaging where high contrast of internal organs is obtained. The development of microbubbles (MBs) contrast agents with an average diameter of 1.5 - 4 pm, has expanded the utility of US to functional intravascular imaging, and to therapeutic applications, with compelling studies of elicited biological effects. MBs are gas spheres encapsulated by a shell that concurrently serve as contrast and therapeutic agents. Upon US excitation, MBs cavitate and pulsate volumetrically. This together with the MB high impedance mismatch compared to the surrounding tissue, enables the detection of a single MB in vivo. Although MBs are efficient for intravascular ultrasonography, their large diameter confines them into the vasculature, and their size is an obstacle in a variety of applications involving capillaries. For example, MB oscillations were reported to be constrained in capillaries. Further, in super resolution US localization microscopy of the brain microvasculature, acquisition time was significantly enhanced in capillaries due to limited MB access and slower blood flow. In tumor imaging, efficient delivery into tumor tissues via systemic injection requires the transvascular passage of the particles from the bloodstream into the tumor interstitial space. Due to the enhanced permeability and retention (EPR) effect, the tumor vasculature is leakier than normal blood vessels, thus larger particles can be delivered into the tumor. Particles up to 400 nm were reported to be delivered. Therefore, MBs are too big for extravasation from the bloodstream into the tumor. In addition, MBs are cleared from circulation ~10 minutes post injection.

In response to these limitations, nanobubbles (NBs) with a diameter of less than 500 nm were developed as an enhanced class of US theranostic contrast agents. NBs expand gasbubbles effects beyond the vascular compartment and enables efficient tumor uptake through the leaky tumor vasculature. In addition, the small size of NBs may make them less susceptible to aggregation and more stable in the bloodstream, which can improve their performance as a contrast agents and expand their role in molecular imaging and theranostic applications. NBs were shown to be efficient for tumor imaging, either using free or targeted NBs, and also for imaging conditions that alter vascular permeability such as inflammation or typel diabetes. In addition, NBs can be used for therapeutic applications such as gene and drug delivery, blood brain barrier opening, and low energy ablation.

Contrast harmonic imaging (CHI) method enhances MB contrast and sensitivity in intravascular and molecular imaging applications. MBs are nonlinear contrast agents, and their echoes contain in addition to the fundamental transmitted frequency also integer harmonics thereof. At low peak negative pressures, the tissue echoes are mainly linear and contain the fundamental frequency. CHI eliminates the linear frequency components, leaving the nonlinear MB harmonics and separating them from the surrounding tissue. This yields a CHI image with high contrast that is widely used for imaging and image guidance Implementation of CHI is typically done by transmitting multiple pulses with positive or inverse polarities such that their summation or subtraction will cancel the linear components. For example, in pulse inversion (PI) two consecutive transmissions are used. The first is a single pulse with a positive polarity (+1), and the second is the same pulse with a negative polarity (-1), and both echoes are summed by the transducer. As a result, the linear components are canceled (0), leaving only the nonlinear components. Contrast pulse sequence (CPS) and amplitude modulation are additional pulse sequence types that can be used in CHI. CHI imaging with NBs is challenging due to weaker signals that lead to poor contrast. In addition, due to the NBs small size, their imaging is performed with higher frequencies that have higher attenuation which further degrades image quality.

Yet, due to the NBs smaller diameter, their scattering cross section is significantly lower than MBs, which reduces their echogenicity.

Therefore, there is a need in the art for ultrasound (US) imaging methods and systems that are cost effective, easy to implement and that can enhance the contrast of nanoscale contrast agents, such as, nanobubbles (NB), thereby improving image quality.

SUMMARY

According to some embodiments, there are provided herein systems and methods for enhancing ultrasound images, by enhancing contrast imaging of nanoscale contrast agents, such as, nanobubbles (NBs), by utilizing nonlinear frequency mixing for ultrasound imaging. According to some embodiments, the systems and methods disclosed herein are advantageous, as they are cost effective, easy to implement using currently employed ultrasound imaging systems, and can provide enhanced images, having improved contrast and significantly improved quality, rendering the images useful for a variety of imaging applications, as detailed below. Advantageously, the imaging systems and methods disclosed herein are implemented using an imaging array, in real time, and do not essentially require additional hardware or post-processing steps, for obtaining high quality images.

According to some embodiments, the systems and methods disclosed herein, make use of a dual frequency mixing effect, whereby a dual frequency ultrasound pulse (wave) is transmitted towards a target body region, and nanoscale ultrasound contrast agents (NUCA), such as, nanobubbles (NBs), that have been administered to the subject, return (echo) ultrasound signal(s) at a sum and difference frequencies, in addition to harmonic frequencies, thereby enhancing the contrast of the NUCA over the tissue, resulting in improved image quality. Thus, according to some embodiments, a dual-frequency pulse is transmitted (for example, using a programmable ultrasound imaging system), wherein the dual-frequency includes a superposition of two frequencies, fi and f2. Accordingly, due to the frequency mixing effect, the NUCAs echoes include the sum and difference frequencies (fi+f2, f2-fi), in addition to the standard harmonics of these frequencies. Using a broadband ultrasound transducer, all of these frequency components can be captured, and are used to enhance the resulting image contrast. Further, advantageously, the frequency mixing method and image enhancement may be incorporated in a common contrast harmonic imaging (CHI) sequence that is used for ultrasound contrast agents imaging, as detailed below.

According to some embodiments, nanoscale ultrasound contrast agents (NUCA) have a higher stability and ability to pass through blood vessels compared to standard microbubbles, making them an option for use in diagnostic and therapeutic applications. However, their small size limits their use because of their weaker echogenicity. Accordingly, the inventors have overcome such shortcoming, by providing herein an ultrasound imaging system and method, which includes a broadband US transducer that can capture both the sum and difference of the returned frequencies to thereby enhance contrast and improve image quality. Additionally, advantageously, in order to further enhance the imaging, NUCA with a relatively small average diameter (for example, in the range of about 120-180nm), that can extravasate through the vascular endothelium into interstitial space (such as that of a tumor), and circulate for extended periods in the bloodstream are used. According to some embodiments, the contrast improvement disclosed herein may be used, under certain conditions, to facilitate individual NB detection. According to some embodiments, the contrast improvement disclosed herein may be used, under certain conditions, to improve the imaging of functionalized NBs that target specific tissues or cells. According to some embodiments, the contrast improvement disclosed herein may be used for image guidance of various therapeutic procedures, including, for example, those involving NUCAs as therapeutic agents, such as, tumor therapy, breaching of the blood brain barrier, and drug delivery to the vasculature.

According to some embodiments, there is provided a method for performing enhanced ultrasound (US) contrast imaging of a region of interest, using systemically administered nanoscale ultrasound contrast agents (NUCA), the method includes: transmitting, using an ultrasonic transducer, a dual-frequency excitation pulse comprising a superposition of a first and second ultrasound frequencies; to thereby generate a frequency mixing effect configured to generate non-linear frequency of the nanoscale ultrasound contrast agents; and receiving, by an ultrasonic transducer, frequency components of echoes generated by the nanoscale ultrasound contrast agents, wherein the frequency components comprise a sum of the first and second ultrasound frequencies, a difference of the second and the first ultrasound frequencies, and harmonic frequencies of the first and second ultrasound frequencies; to thereby enhance the nonlinear response of the nanoscale ultrasound contrast agents in real-time to result in an image having enhanced contrast and/or resolution.

According to some embodiments, a single transducer is configured to transmit and receive the ultrasound frequencies.

According to some embodiments, the transducer is a broadband transducer.

According to some embodiments, the transducer comprises a bandwidth in the range of about 2-22 MHz, wherein the sum and difference frequencies thereof and their harmonics are configured to be within the bandwidth of the transducer.

According to some embodiments, the first ultrasound frequency is in the range of about 3-6 MHz and the second ultrasound frequence is in the range of about 6-12 MHz. According to some embodiments, the first ultrasound frequency is about 4 MHz and the second ultrasound frequence is in the range of about 8 MHz.

According to some embodiments, the sum of the first and second ultrasound frequencies is about 12 MHz, a difference of the second and the first ultrasound frequencies is about 4MHz, and harmonic frequencies of the first and second ultrasound frequencies are about 4, 8 and 16MHz.

According to some embodiments, the method is incorporated in a contrast harmonic imaging (CHI) sequence.

According to some embodiments, the transmitting may include a plurality of transmission cycles.

According to some embodiments, the transmitting may include one or more pulse inversion (PI) cycle.

According to some embodiments, the NUCAs may be selected from nanobubbles, gas vesicles and nanodroplets.

According to some embodiments, the NUCA may have an average diameter in the range of about 120-200nm.

According to some embodiments, the NUCA may have an average diameter in the range of about 160nm.

According to some embodiments, the NUCA may include nanobubbles, comprising one or more lipids.

According to some embodiments, the NUCA may include a targeting moiety on a shell thereof.

According to some embodiments, the targeting moiety may include a cell type-specific antibody conjugated to the shell.

According to some embodiments, the NUCAs may be administered systemically, at a concentration of about 5x109-5X1012 NUCA/ml.

According to some embodiments, the NUCA may include nanobubbles, having an average diameter of about 150-170 nm, being administered systemically, at a concentration of about 5x109-5X1012 NBs/ml. According to some embodiments, the contrast enhancement using a dual frequency excitation pulse, may exhibit an improvement of at least about 100% in a contrast ratio (CTR) determined at a designated mechanical index (MI), compared to using a single frequency excitation pulse at the same MI.

According to some embodiments, the contrast enhancement using a dual frequency excitation pulse, may exhibit an improvement of at least about IdB in a contrast ratio (CTR) determined at a designated mechanical index (MI), compared to using a single frequency excitation pulse at the same MI.

According to some embodiments, the contrast enhancement using a dual frequency excitation pulse, may include an improvement of at least about 2dB in a contrast ratio (CTR) determined at a designated mechanical index (MI), compared to using a single frequency excitation pulse at the same MI.

According to some embodiments, the contrast enhancement using a dual frequency excitation pulse including 4Mhz and 8Mhz, may include an improvement of at least about 4dB in a contrast ratio (CTR) determined at a mechanical index (MI) of 0.1, compared to using a single frequency 4MHz excitation pulse at an MI of 0.1.

According to some embodiments, the method includes systemically administering the NUCA, prior to performing the imaging.

According to some embodiments, the method is for use in real-time, in-vivo imaging applications, including: image-guided medical procedures, diagnostic imaging of blood vessels, diagnostic imaging of tissues, diagnostic imaging of tumor, diagnostic imaging of inflammation, diagnostic imaging of diabetes, or any combinations thereof.

According to some embodiments, there is provided a method for utilizing dual frequency ultrasound for imaging a region of interest in a body of a subject, using nanoscale ultrasound contrast agents (NUCAs), the method includes the steps of: generating dual frequency ultrasound waves using an ultrasonic transducer; transmitting the dual frequency ultrasound waves into the region of interest within, wherein said region of interest comprises the NUCAs; receiving reflected ultrasound waves components from the NUCAs utilizing the ultrasonic transducer, wherein the ultrasound waves components comprise the sum and difference of the dual-frequency waves, and harmonic frequencies thereof; processing, the received ultrasound waves components, to generate an image of said target area; and analyzing said image to assess the condition of the region of interest.

According to some embodiments, the method may further include systemically administering the NUCAs, prior to performing the imaging.

According to some embodiments, the transducer is a broadband transducer.

According to some embodiments, the NUCAs are selected from nanobubbles, gas vesicles and nanodroplets.

Certain embodiments of the present disclosure may include some, all, or none of the above advantages. One or more other technical advantages may be readily apparent to those skilled in the art from the figures, descriptions, and claims included herein. Moreover, while specific advantages have been enumerated above, various embodiments may include all, some, or none of the enumerated advantages

BRIEF DESCRIPTION OF THE FIGURES

Some embodiments of the disclosure are described herein with reference to the accompanying figures. The description, together with the figures, makes apparent to a person having ordinary skill in the art how some embodiments may be practiced. The figures are for the purpose of illustrative description and no attempt is made to show structural details of an embodiment in more detail than is necessary for a fundamental understanding of the disclosure. For the sake of clarity, some objects depicted in the figures are not to scale.

Fig. 1- A schematic illustration of a dual frequency imaging system and method, using NUCAs, according to some embodiments. A broadband transducer transmits a dualfrequency waveform that contains two frequencies for the imaging of NUCA (for example, NBs). Upon interaction with the ultrasound pulse, the NUCA pulsate. The frequency mixing effect generates the sum and difference frequencies components in the NUCA receive spectrum, in addition to standard harmonics. This effect is then used to improve image contrast and enhance image quality;

Figs. 2A-E. Dual-frequency combination optimization using microbubbles (MB) in a tissuemimicking phantom. Fig. 2A- Setup illustration. Fig. 2B- Hydrophone peak negative pressure measurements of the L12-3v transducer as a function of the transmitted frequency. Fig. 2C, panels i.-vi.- Exemplary images of the MB filled inclusion for MI=0.04. Axes are common to these subfigures. Panel i. - Control image using a single plane wave at 4 MHz without pulse inversion (PI). The dashed (originally green) and solid (originally red) circles mark the regions for contrast calculation. PI images using frequencies of Panel ii.- 4 MHz and Panel iii. 5 MHz, Dual-frequency transmit of: Panel iv. 4&12 MHz, Panel v. 5&12 MHz, and Panel vi. 4&8 MHz. Fig. 2D- MB contrast results for the different transmit frequencies and two Mis: 0.04 (blue), 0.1 (orange). The results are plotted as mean ± STD (N=4) with the level of significance between different groups, *p<0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, based on two-way ANOVA with Tukey’s multiple comparisons test. Fig. 2E- Average contrast improvement for the dual-frequency method compared to single-frequency transmit, calculated based on Fig. 2D;

Figs. 3A-D. Spectra validation and concentration optimization of nanobubbles (NBs) in a tissue-mimicking phantom. Fig. 3A- NBs size distribution. Insert shows Representative transmission electron microscopy (TEM) image of a NB. Fig. 3B- Contrast results of NB imaging at four NB concentrations for two single-cycle transmits at frequencies of 4 MHz and 4&8 MHz, as indicated by arrows. Fig. 3C and Fig. 3D- Spectra validation experiments for frequencies of 4 MHz (darker line, originally orange) and dual-frequency of 4&8 MHz (brighter line, originally green). Axes are common to these subfigures. Fig. 3C - The received spectrum following 10 cycles single plane-wave transmission and Fig. 3E- 10 cycles pulse inversion (PI) sequence;

Figs. 4A-C- Frequency mixing-based contrast harmonic imaging of nanobubbles (NBs). Fig. 4A, panels (a)-(h) Example images of the NB filled inclusion within the tissue mimicking phantom, imaged with two different frequencies: Panels (a)-(d) 4 MHz and Panels (c)-(h) dual-frequency 4&8 MHz. Each frequency was imaged with four mechanical indices (MI): Panels (a) and (c) 0.04; Panels (b) and (f) 0.06, Panels (c) and (g) 0.08, and Panels (d) and (h) 0.1. All Axes are common to these panels; Fig. 4B- Bar graphs showing Contrast results, plotted as mean ± STD (N=3) in dB scale with the level of significance between the two transmits, **p<0.01, ****p <0.0001, based on two-way ANOVA with Sidak’s multiple comparison test; Fig. 4C- Summary of average contrast values displayed in Fig. 4B;

Figs. 5A-C. Frequency mixing-based contrast harmonic imaging of breast cancer tumors in vivo following a systemic nanobubbles (NBs) injection. Fig. 5A- schematic illustration of ultrasonic imaging of a tumor. Fig. 5B- Bar graphs showing Intensity improvement with the dual-frequency transmit, 4&8 MHz, over a single -frequency transmit with 4 MHz. Results are plotted as mean ± STD for three mechanical indices (MI): 0.04 (originally bourdeaux), 0.08 (originally green) and 0.1 (originally turquoise). Fig. 5C, panels i.-ix: Example tumor images presented at a 40 dB scale: (Panel i., Panel iv and Panel (vii)) before NB injection, Panels ii. , v. and vii following NB injection and imaging at a single 4 MHz frequency; Panels iii., vi, and ix, following NB injection and imaging at a 4&8 MHz dual-frequency. Arrows indicate tumor borders. Scale bar is common to these subfigures; and

Fig. 6 - A block diagram of a method for dual frequency imaging of nanoscale non-linear contrast agents (NUCAs), according to some embodiments.

DETAILED DESCRIPTION OF THE INVENTION

The principles, uses and implementations of the teachings herein may be better understood with reference to the accompanying description and figures. Upon perusal of the description and figures present herein, one skilled in the art will be able to implement the teachings herein without undue effort or experimentation.

In the following description, various aspects of the invention will be described. For the purpose of explanation, specific details are set forth in order to provide a thorough understanding of the invention. However, it will also be apparent to one skilled in the art that the invention may be practiced without specific details being presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the invention.

In the following description, numerous details are set forth for the purpose of explanation. However, one of ordinary skill in the art will realize that the invention may be practiced without the use of these specific details.

According to some embodiments, there are provided herein ultrasound systems and methods utilizing a frequency mixing effect with administered NUCA (such as, NBs), to enhance and improve obtain image quality, by enhancing contrast and/or resolution of the image, due to the dual frequency mixing effect on the administered NUCAs.

As used herein, the term “Contrast-enhanced ultrasound” and “CEUS imaging” are directed to ultrasound imaging that make use of contrast agents to enhance the visualization of blood flow and tissue perfusion in real-time ultrasound images. Unlike traditional ultrasound imaging, which relies on the reflection of sound waves off tissue interfaces, CEUS involves the use of contrast agents (such as, microbubbles), that contain gas encapsulated within a shell Generally, the method includes administering of the contrast agents (for example, by systemic administration|), ^an^ ^once in the target area (such as a blood vessel or organ of interest), ultrasound imaging is performed using an suitable ultrasound probe having contrast- specific imaging modes. The ultrasound system can detect and enhance signals from the contrast agents circulating within the blood vessels or perfusing the target tissue. As the contrast agent within reflect (echoes) ultrasound waves differently from surrounding tissue, a contrast effect is created, thereby aiding in formation of a real time image of the target area, providing various information, including, for example, but not limited to dynamic assessment of blood flow and perfusion patterns, contrast, signal to noise ratio, and contrast agent concentration with high spatial and temporal resolution.

As used herein, the terms "Contrast harmonic imaging” and “CHI” is a CEUS, which is based on the nonlinear emission of harmonics by contrast agents, pulsating in an ultrasound field. Any frequency above or below the fundamental frequency could be detected. CHI can utilize, for example, Pulse inversion sequence, contrast pulse sequence and amplitude modulations.

As used herein, the terms “PI sequence ultrasound”, “Pulse Inversion sequence” and “PI” are directed to an ultrasound technique that involves transmitting two successive ultrasound pulses with opposite polarities and then subtracting the received echoes to enhance the contrast of moving structures (such as blood), while suppressing static tissue signals PI is a technique used to improve tissue contrast and reduce artifacts, particularly in imaging blood flow and perfusion. Typically, the two successive ultrasound pulses, may have identical parameters such as frequency, amplitude, and duration, however, the second pulse has an opposite polarity (phase inversion) compared to the first pulse. The echoes received after the second pulse (with inverted polarity) are summed with the echoes received after the first pulse. This summation process effectively cancels out the signals from, for example, static tissue, as the echoes from static tissue will have the same amplitude and polarity in both pulses and will cancel each other out. The PI sequence is used with CHI imaging to improve the visualization of blood vessels, blood flow, and tissue perfusion. In cased where the contrast agent is directly injected into a target organ, their concentration is imaged. Targeted nanobubbles will thus highlight the targeted region in terms of enhanced contrast and resolution.

As used herein, the terms “contrast pulse sequences” and “CPS” are directed to an ultrasound technique that involves transmitting three successive ultrasound pulses with different amplitudes of ( , -1, ^). Such a technique can be used with CHI imaging.

As used herein, the terms “ultrasound contrast agents”, “Nonlinear ultrasound contrast agents” and “UCA” refer to specialized microbubble contrast agents that exhibit nonlinear behavior when exposed to ultrasound waves. This nonlinear behavior may include, for example, harmonic generation, subharmonic emission, inertial cavitation, and the like. Such agents can be used in contrast-enhanced ultrasound imaging to enhance, for example, the visualization of blood flow and tissue perfusion.

As used herein, the terms “Nanoscale ultrasound contrast agents”, “Nanoscale Nonlinear ultrasound contrast agents” and “NUCA” refer to specialized nanometric contrast agents that exhibit nonlinear behavior when exposed to ultrasound waves. In some embodiments, NUCA may include nanobubbles (NBs), gas vesicles, and/or nanodroplets, as further detailed below.

As used herein, the term “Echogenicity” relates to the ability of a tissue, structure, or contrast agent, to produce echoes or reflect ultrasound waves. It relates to the brightness or intensity of the echoes produced by a particular tissue, structure or agent, relative to surrounding tissues on the ultrasound image. Highly echogenic structures appear bright white, while hypoechoic structures appear darker or grayish. Anechoic structures are entirely black because they do not reflect any ultrasound waves.

According to some embodiments, there is provided herein a nonlinear frequency mixing ultrasound imaging method for improving NUCA imaging, by enhancing the nonlinear acoustic response thereof, to a dual-frequency excitation. According to some embodiments, a suitable broadband transducer (for example, an L12-3v transducer) may be used. The transducer may be a single broadband transducer, capable of emitting and receiving all range of frequences. In some embodiments, the US transducer is controlled by a programmable ultrasound controller system, and can transmit a dual-frequency single-cycle wavefront using its built-in arbitrary waveform generator. Due to the frequency mixing effect, the sum and difference frequencies generated by the NUCAs, are capable of being captured by the same transducer, to enhance the nonlinear response, without the need for additional hardware or postprocessing and in real-time. In some embodiments, the dual-frequency wavefront can be incorporated into a standard contrast harmonic pulse inversion imaging protocol, by replacing the single-frequency wavefront.

Reference is now made to Fig. 1, which is a schematic illustration of a dual frequency imaging system and method, using NUCAs, according to some embodiments. As shown in Fig. 1, a broadband transducer 100, transmits a dual-frequency waveform 102, that contains two frequencies fl and f2, for the imaging of NUCA (exemplary NUCA 106 is marked). Upon interaction with the ultrasound pulse, the NUCA pulsate/oscillate (as shown in panel 108). The frequency mixing effect generates the sum (fl+f2) and difference (f2-fl) frequencies components in the NUCA receive spectrum, in addition to standard harmonics (fl, f2, 2fl and 2f2). This effect is then used to improve image contrast and enhance image quality, as detailed below.

According to some embodiments, there is provided herein a frequency mixing method to enhance NUCA-based CHI. The harmonic frequencies generated by bubbles provide more detailed images because they are less absorbed and scattered by tissues. Therefore, CHI can be used to produce images with improved resolution and contrast compared to traditional US imaging.

According to some embodiments, by exciting the NUCAs with a dual-frequency excitation instead of a single-frequency, new frequency components are shown herein to be generated in their echo spectrum (e.g. the sum and difference of the transmitted frequencies). Consequently, the NUCAs nonlinear frequency components increase, resulting in a contrast improvement. In some embodiments, the real-time frequency mixing imaging method is transmitted using a single broadband array transducer in a programmable US system, by transmitting a superposition of two frequencies. This implementation is different than MBs imaging methods that divided the transducer elements into subgroups, where each transmits a different frequency, or methods that used complex mechanically aligned setups that utilize multiple single element transducers. Herein, the dual-frequency wavefront is incorporated into a standard CHI sequence. In some embodiments, the method makes use of PI, including the transmitting of two polarity opposite pulses. In some embodiments, the method may make use of CPS, where three pulses are used. In some embodiments, PI may be preferred so as to reduce the number of transmitted pulses. According to some embodiments, the methods disclosed herein can be useful to improve the accuracy and usefulness of US imaging in a variety of medical applications that involve NUCAs imaging. The NUCAs small size allows them to penetrate into smaller blood vessels and provide improved imaging of the microvasculature, which can be used for identifying abnormalities or evaluating the perfusion of tissues, while providing more detailed and accurate images of small structures and tissues. Some conditions can include, for example, but not limited to: cancer, inflammation, and diabetes imaging. In some embodiments, the methods disclosed herein can also be used for super resolution imaging and the visualization of blood flow in the microvasculature. In some embodiments, the methods disclosed herein can also be used for improving the imaging of functionalized NUCAs, that target specific tissues or cells. In further embodiments, the methods disclosed herein can be used for image guidance in applications involving NUCAs also as therapeutic agents, such as, tumor therapy, blood brain barrier opening, and drug delivery to the vasculature.

According to some embodiments, the methods may be used for various clinical applications, including liver imaging (e.g., characterization of liver lesions, assessment of hepatic blood flow), vascular imaging (e.g., assessment of peripheral vascular disease, detection of abdominal aortic aneurysms), assessment of focal lesions in other organs (e.g., kidneys, spleen, pancreas), and the like, or any combinations thereof. Each possibility is a separate embodiment.

According to some embodiments, there is provided a NUCA-based frequency mixing technique for enhanced CHI. The method may include transmitting a single-cycle dualfrequency waveform using a standard PI sequence, which triggers a nonlinear frequency mixing mechanism that amplifies the NUCA signals through the generation of new frequency components. The technique can be implemented in real-time in programmable ultrasound systems, and improve NB contrast with no tradeoffs or drawbacks.

According to some embodiments, the method may improve contrast of the obtained images. In some embodiments, contrast improvement may be determined based on relative improvement in contrast, calculated as CTR. As used herein, the term “CTR” is directed to the contrast ratio between images obtained at different frequencies, or before or after ultrasound wave(s) transmission. In some embodiments, CTR is the relative ratio between intensity or pixels of an image obtained at dual frequency excitation and images obtained at a single frequency excitation (under otherwise similar conditions, including, for example, Mechanical index( MI) and/or peak negative pressure (PNP)). According to some embodiments, such a CTR may be calculated according to the general equation:

where CTR is a contrast ratio, pn&fMHz is the mean pixel values of an area within the tumor region imaged with the dual-frequency f 1 &f2 MHz transmit and pn \n iz is the mean pixel values on the same area within the tumor region that was imaged with a single-frequency fl MHz transmit.

For example, the relative contrast improvement between the dual-frequency 4&8 MHz imaging compared to a single-frequency 4 MHz PI imaging was calculated by:

where CTR is a contrast ratio, p4&8MHz is the mean pixel values of an area within the tumor region imaged with the dual-frequency 4&8 MHz transmit and p4MHz is the mean pixel values on the same area within the tumor region that was imaged with a single-frequency 4 MHz transmit.

In some embodiments, CTR (dB) is used to determine the contrast between a contrast image and the surrounding background and may be calculated by the following equation:

U_;

CTR[dB] = 20 to g₁₀(— ) (2)

Ro where CTR[dB] is a contrast ratio, pi is the mean of the pixels in a region inside the contrast agent cavity and p₀ is the mean of the pixels in a background region (in particular, with respect of a phantom experimental setting).

According to some embodiments, the method can result in a CTR improvement of at least about 100%, at least about 200%, at least about 500%, or at least about 1000%. Each possibility is a separate embodiment.

According to some embodiments, the method can result in a CTR improvement of at least about IdB, 2dB, 3dB, 4dB, 5dB, 6dB, 7dB, 8dB, 9dB, or at least about lOdB. Each possibility is a separate embodiment. According to some embodiments, the method may thus improve the contrast by at least about 10%, 50%, 100%, 200%, 500%, 750%, at least about 1000%, when compared to a single frequency transmission. Each possibility is a separate embodiment.

According to some embodiments, the method may thus improve the contrast by at least about IdB, 2dB, 3dB, 4dB, 5dB, 6dB, 7dB, 8dB, 9dB, lOdB, or at least about 12dB, when compared to a single frequency transmission. Each possibility is a separate embodiment.

According to some embodiments, the contrast enhancement using a dual frequency excitation pulse, include an improvement of at least about 2dB in a contrast ratio (CTR) determined at a designated mechanical index (MI), compared to a CTR determined at the same MI, using a single frequency excitation pulse.

According to some embodiments, the method may thus improve the contrast by at least about IdB, at least about 2 dB, at least about 3dB, at least about 4dB, at least about 5 dB, at least about 6dB, at least about 7dB, at least about 8dB, at least about 9dB, at least about 9B, for a designated MI. In some embodiments, the MI may be in the range of about 0.01-1.9.

According to some embodiments, the method may improve the contrast by about IdB, 2 dB, 3dB, 4dB, 5 dB, 6dB, 7dB, 8dB in tissue mimicking phantoms, at an MI of 0.01-1.9. According to some embodiments, the method may improve the contrast by about IdB, 2 dB, 3dB, 4dB, 5 dB, 6dB, 7dB, 8dB in in-vivo tissue, at an MI of 0.1. In some embodiments, the method may enhance the contrast in the range of about l-10dB, for a designated MI (for example, in the range of 0.01-1.9).

In some embodiments, an increase in MI may increase the contrast enhancement using the method.

According to some embodiments, the method may improve the contrast by about 6-8 dB in tissue mimicking phantoms and by about 4-6 dB in vivo in breast cancer tumor imaging, at an MI of 0.01-1.9.

According to some embodiments, when utilized at essentially similar conditions, the dual frequency method enhances the contrast using NUCA, as compared to contrast enhancement using MBs.

According to some embodiments, using NUCA compositions, the suspension including the same are more stable (over a range of time) as compared to MBs compositions. According to some embodiments, the concentration of NUCA may be in the range of about O.l-2*1O¹⁰ NUCAs/ml. In some embodiments, the concentration may be in the range of about 0.5-lxl0¹⁰ NUCAs/ml. In some embodiments, the concentration may be in the range of about 10⁷-10¹³ NUCAs/ml. In some embodiments, an optimal concentration may be determined, such that values that exceed this concentration may cause a reduction in contrast due to US beam blockage; whereas value below this concentration do not provide enough contrast.

According to some embodiments, the average diameter (size) of the NUCA may be in the range of about 30-250nm. In some embodiments, the average diameter may be in the range of about 60-200nm. In some embodiments, the average diameter may be in the range of about 100-180nm. In some embodiments, the average diameter may be in the range of about 120- 170nm. In some embodiments, the average diameter may be about 160nm. In some embodiments, the average size may be determined during the fabrication of the NUCA.

In some embodiments, the NUCA are monodispersed.

According to some embodiments, as exemplified herein, the bubbles NUCA may exhibit a spherical morphology.

In some embodiments, the NUCA may be nanobubbles (NBs).

According to some embodiments, the NBs may have an average diameter in the range of about 25-250nm. In some embodiments, the NBs may have an average diameter in the range of about 50-230nm. In some embodiments, the NBs may have an average diameter in the range of about 75-200. In some embodiments, the NBs may have an average diameter in the range of about 90-150nm. In some embodiments, the NBs may have an average diameter in the range of about 100-250nm. In some embodiments, the NBs may have an average diameter of about 110-230 nm. In some embodiments, the NBs may have an average diameter in the range of about 150-200nm. In some embodiments, the NBs may have an average diameter of about 160nm. In some embodiments, the NBs may have an average diameter of less than about 300nm, less than about 250nm, less than about 200nm. each possibility is a separate embodiment.

According to some embodiments, the amount/concentration/number of the nanobubbles may be determined according to the target tissue, size of tissue, type of tumor, size of tumor, location of the tumor, and the like. In some embodiments, the amount of NBs administered may be about IxlO¹³ NBs. In some embodiments, the amount of NBs administered may be about IxlO¹² NBs. In some embodiments, the amount of NBs administered may be about IxlO¹¹ NBs. In some embodiments, the amount of NBs administered may be about 5xlOⁿ NBs. In some embodiments, the amount of NBs administered may be about 6xlOⁿ NBs. In some embodiments, the amount of NBs administered may be at least about IxlO⁶ NBs. In some embodiments, the amount of NBs administered may be at least about IxlO⁷ NBs. In some embodiments, the amount of NBs administered may be at least about IxlO⁸ NBs. In some embodiments, the amount of NBs administered may be at least about IxlO⁹ NBs.

According to some embodiments, the NUCA are lipid bubbles, having an external lipid shell. In some embodiments, the shell may include such components as, but not limited to: disteroylphosphatidylcholine (DSPC), 2-dibehenoyl-sn-glycero-3-phosphocholine (C22), 1,2- dipalmitoyl-sn-glycero-3-phosphate (DPPA), l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE), 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-

[methoxy(poly ethylene glycol)-2000] (ammonium salt) (DSPE-PEG2K) and 1,2- distearoylsnglycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol) 2000] (DSPE- PEG2000-Biotin). In some embodiments, the bubbles include a fluid core. In some embodiments, the fluid is gas. In some embodiments, the gas is selected from: perfluorobutane (C4F10), octafluoropropane C3F8, perfluorocarbons, sulfur hexafluoride, air and nitrogen.

In some embodiments, the nanobubbles have an external lipid shell. In some embodiments, the lipid shell may include phospholipids. In some embodiments, the lipid shell may include: l,2-dibehenoyl-sn-glycero-3-phosphocholine (C22), 1,2-dipalmitoyl-sn- glycero- 3 -phosphate (DPPA), l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)- 2000] (ammonium salt) (DSPE-mPEG 2000). In some embodiments, the lipids may be in a molar ratio of 18.8:4.2:8.1:1 with a final lipid concentration of 10 mg/mL. In some embodiments, the lipid mixture may be sonicated.

According to some embodiments, the NUCA may further include a targeting moiety on an external region thereof. In some embodiments the targeting moiety may be on the shell of the NUCA. In some embodiments, the targeting moiety may be a cell-type specific targeting moiety. In some embodiments, the targeting moiety may be a cell-type specific antibody.

According to some embodiments, NUCAs may be administered by systemic administration. In some embodiments, systemic administration may include, for example, parenteral administration, including, for example: intravenously, intra-arterially, intramuscularly, intraperitoneally, intradermally, intravitreally, or subcutaneously administration. In some embodiments, the systemic administration is by injection.

According to some embodiments, the application of US may be performed at a time period after administration of the NUCA. In some embodiments, the time period may be at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 60 minutes. Each possibility is a separate embodiment.

According to some embodiments, the maximal frequency response may be at the transducer’s center frequency. In some embodiments, in order to maximize the transmitted energy, each frequency component in the dual-frequency wavefront may be transmitted without apodization, which can cause amplitude variations due to the reduced transducer sensitivity. Moreover, the sum and difference frequencies may fall at the edges of the transducer bandwidth and therefore may be attenuated, which weakens the obtained effect, compared to the ideal case of flat frequency response. In some embodiments, the bandwidth may limit the number of possible frequency combinations that can produce a sum or difference within the bandwidth range. In some instances, a difference frequency, (for example, 4 MHz when using 4&8MHz dual frequency), is also one of the transmitted frequencies, and the sum frequency is the third harmonic of 4 MHz. Accordingly, in some embodiments, the contribution of the fundamental frequency may be eliminated using PI, thus the frequency component that is observed at 4 MHz is a result of the difference frequency.

In some embodiments, the imaging transducer may include an array of transducers. In some embodiments, the transducer may have a broadband in the range of about l-50MHz. In some embodiments, the transducer may have a broadband of l-22MHz. In some embodiments, the transducer may have a broadband of 2-18MHz. In some embodiments, the transducer may have a broadband of 3-12MHz. In some embodiments, the MI of the transducer may be below about 1.9, below about 1.85, below about 1.7, below about 1.5, below about 1.2, below about 1. In some embodiments, the transmission may include a single cycle or a plurality of transmission cycles. In some embodiments, the transmission is performed, such as to maximize axial resolution.

According to some embodiments, advantageously, the method disclosed herein utilizes a mechanical index (MI) within the safety limits. The MI parameter is defined as the peak negative pressure (PNP) divided by the square root of frequency, is a parameter that determines the likelihood of creating mechanical damage as a result of US application.

Reference is now made to Fig. 6, which is a block diagram of a steps in method for non-linear frequency mixing ultrasound imaging of nanoscale contrast agents (NUCAs), according to some embodiments. As shown in Fig. 6, method 200, includes at step 210, administration of NUCAs to a tested subject. The administration may be systemic, and may be performed at any desired time point prior to imaging. The NUCAs may include any type of NUCA, including, for example, nanobubbles (NBs). The NUCA may be administered at any suitable concentration, in particular, such a concentration enabling enhanced contrast, but without hindering ultrasonic transmission. In some embodiments, the NUCA may have a desired average diameter, such as, for example, in the range of about 150-170nm. Next, at step 220, a dual frequency ultrasonic pulse is transmitted, at fl and f2 frequencies, using an ultrasonic transducer. The dual frequency may be, for example, 4 and 8MHZ, at a designated MI, for a designated duration and power, but may include any desired combination of frequencies. The transmission may include a single cycle, or a plurality of cycles, that may be identical or different therebetween. At step 230, echoed signal components from the NUCAs, including, the sum of frequencies (fl+f2), difference of frequencies (f2-fl), as well as harmonics of the frequencies (for example, 2f 1, 2f2) are received by a transducer. In some embodiments, the transducer for transmitting (emitting/generating) the dual frequency pulse and for receiving the echoed signals, in the same transducer. Next, at step 240, an image is constructed, based on the received signals, wherein the image exhibits enhanced contrast and/or resolution.

According to some embodiments, there is provided a method for utilizing dual frequency ultrasound in medical imaging, using NUCAs, the method includes the steps of: generating dual frequency ultrasound waves with a transducer; transmitting the dual frequency ultrasound waves into a target area of interest within a subject body, said area includes the NUCAs; receiving reflected ultrasound waves components from the NUCAs in the target area using the (same or different) transducer, wherein the waves components include the sum and difference of the dual-frequency waves, as well as harmonic frequencies thereof; processing the received ultrasound waves components, to generate an image of said target area; and analyzing the image to assess the condition of the target area.

According to some embodiments, as exemplified herein below, optimization experiments were performed in tissue mimicking phantoms, in order to determine mixing effect of NUCAs. Among the dual-frequency combinations that were tested, the highest contrast was obtained using 4&8 MHz, where both the sum and difference frequencies could be captured by the transducer. NUCAs contrast improved with increased mechanical index, reaching a maximal contrast improvement of 8.4+0.5 dB compared to 4 MHz single-frequency pulse inversion imaging. In vivo in a breast cancer tumor mouse model, tumor imaging following a systemic nanobubbles injection resulted in a higher contrast for the frequency mixing method over standard single-frequency imaging. The contrast was improved by 3.4 + 1.7, 4.8 + 1.8, and 6.3 + 1.6 dB for mechanical indices of 0.04, 0.08, and 0.1, respectively. Nonlinear frequency mixing thus significantly improves nanobubbles contrast, which facilitates their imaging in-vivo, and their utilization in various other diagnostic applications.

According to some embodiments, there is provided a system for performing enhanced ultrasound (US) contrast imaging of a region of interest, using systemically administered nanoscale ultrasound contrast agents (NUCA), the system includes an ultrasonic broadband transducer configured to transmit a dual-frequency excitation pulse comprising a superposition of a first and second ultrasound frequencies; and configured to receive frequency components of echoes generated by the nanoscale ultrasound contrast agents, wherein the frequency components comprise a sum of the first and second ultrasound frequencies, a difference of the second and the first ultrasound frequencies, and harmonic frequencies of the first and second ultrasound frequencies; and a processing unit configured to generate enhanced image, based on the nonlinear response of the nanoscale ultrasound contrast agents in real-time, the image having enhanced contrast and/or resolution.

In the description and claims of the application, the words “include” and “have”, and forms thereof, are not limited to members in a list with which the words may be associated.

One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. EXAMPLES

Materials and Methods:

CHI imaging with UCAs

Imaging was performed using a broadband linear array transducer (L12-3v, Verasonics, USA), controlled by a programmable research US system (Verasonics Vantage, Kirkland, WA, USA). The system’s built-in arbitrary waveform generator was used to transmit the dualfrequency engineered pulses. The L12-3v transducer has 196 elements, a center frequency of 7.54 MHz, and an aperture width of 38.4 mm. For CHI imaging, a PI sequence of single-cycle pulses was used. In addition, coherent compounding with 9 steered PWs was implemented with 128 elements out of 196 for each PW (shown in Fig. 2A). One full frame was the combination of 18 transmit/receive events. To compare the different imaging techniques and transmits, images were acquired with the same MI, defined according to:

where PNP is the peak negative pressure in MPa, and f is the transmitted frequency in MHz.

Transducer calibration

The transducer PNP and frequency response across its 3-12 MHz bandwidth, were evaluated and calibrated in a distilled and degassed water tank using a needle hydrophone (NH0200, Precision Acoustics, Dorchester, UK) with an active aperture of 0.2 mm. The hydrophone probe was mounted on a three-dimensional motorized stage (Newport motion controller ESP 301, Newport M-443 series). The pressure signals received by the hydrophone were first displayed on a digital oscilloscope (MDO3024, Tektronix, OR, USA), and further recorded for offline calculations. The transducer frequency response was evaluated for 5 frequencies of 3, 4, 5, 8, and 12 MHz with the same output voltage of 9 V.

Tissue mimicking phantom

Tissue-mimicking phantoms were prepared by boiling a mixture of 1.5% agarose powder (A10752, Alfa Aesar, MA, USA) in distilled water. Before being allowed to cool down, 1% silicon carbide (357391, Sigma Aldrich, MO, USA), was added to the mixture and thoroughly mixed using a magnetic stirrer. The solution was poured into a custom mold and allowed to congeal. The mold was a box with dimensions of 90 x 60 x 50 mm³ that was milled in aluminum. The mold had a cylindrical 6 mm rod at its center, with a height of 2.5 cm. The extracted phantom contained a cylindrical cavity, located at a distance of 48 mm from the transducer. Diluted bubble suspension was injected into this cavity and used for imaging (See Fig. 2A). preparation of Nanobubbles (NB) and measurements thereof

Microbubbles (MBs) were prepared as reported previously using a thin film hydration method (Karlinsky sky and Ilovitsh, IEEE Trans. Ultrason. Ferroelectr. Freq. Control, vol. 69, no. 8, pp. 2414-2424, 2022). Nanobubbles (NB) synthesis was performed based on Bismuth et.al., (Nanoscale, 2022, 14, 13614-13627). Briefly, four lipids (l,2-dibehenoyl-sn-glycero-3- phosphocholine (C22), l,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA), 1,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine (DPPE), and l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE- mPEG 2000) (Sigma-Aldrich)) at a molar ratio of 18.8:4.2:8.1:1, respectively, were dissolved into propylene glycol by heating to 80°C and sonicating. Then, a mixture of Glycerol with phosphate buffer solution (PBS), at 80°C, was added to the lipid solution to a final lipid concentration of 10 mg/mL, and 10 min of sonication at room temperature was applied. The resulting lipid solution was divided into portions of 1 mL in 3 mL headspace vials and saturated with octafluoropropane (CsFs) gas. Vials were sealed and stored at 4°C until usage. Prior to each experiment, a vial was activated by mechanical shaking for 45 sec in a Vialmix shaker (Bristol-Myers Squibb Medical Imaging Inc., N. Billerica, MA) and then centrifuged at 50 relative centrifugal force for 5 min with the vial placed flipped in the centrifuge (581 OR centrifuge, Eppendorf AG, Hamburg, Germany). 200 pL of the NB solution was pulled out with a 21G needle, at a distance of 1-5 mm from the cap. A particle sizing system (AccuSizer FX-Nano, Particle Sizing Systems, Entegris, MA, USA) was used to measure the size and concentration of the purified MBs and NBs. The average bubble diameters were 1.55 pm and 0.16 pm for the MBs and NBs, respectively. In addition, NBs were also imaged with transmission electron microscopy (TEM), (JEM-1400Plus, JEOL, Tokyo, Japan) that was operated at 120 kV to examine their structure and morphology. The bubbles were used within 3 hours of their preparation.

Tissue mimicking phantom experiments Initial experiments were performed in a tissue-mimicking phantom that contained a rod-shaped cavity that the UCA suspension was injected into. The transducer was placed perpendicularly to the phantom and the UCA cavity was located at a distance of z = 48 mm from the transducer surface (Fig. 2A). MB contrast experiments were used to identify the frequency combinations that maximize contrast for the L12-3v transducer. In these experiments, a diluted MB suspension of 4.5 x 10⁶ MB/ml was injected into the cavity and imaged using two MI of 0.04 and 0.1. Five different transmits frequency combinations were compared: 4 MHz, 5 MHz, 4&12 MHz, 5&12 MHz, and 4&8 MHz. Imaging was performed with a CHI sequence of PI that contained single-cycle transmission for each of the frequencies that were tested. Each experiment was performed in triplicates and the phantom was washed between measurements. For quantitative evaluations and optimization of the proposed imaging technique in the tissue-mimicking phantom experiments, the contrast between the UCA cavity to the surrounding background was calculated by:

Ui

CTR[dB] = 20log₁₀(— ) (2)

Bo where CTR[dB] is a contrast ratio, pi is the mean of the pixels in a region inside the UCAs cavity and p₀ is the mean of the pixels in a background region in the phantom.

Next, NB concentration optimization was performed in the same tissue-mimicking phantom. A NB mixture was injected into the cavity and imaged as described for the MBs. The frequency combinations that yielded the highest MB contrast were used for NB imaging (4 MHz and a dual-frequency of 4&8 MHz). The NB -filled inclusion contrast was evaluated for four different NB concentrations: IxlO⁹, 5xl0⁹, IxlO¹⁰, and 5xlO¹⁰ NBs/ml with the same MI of 0.06. For the optimal concentration of 5xl0⁹ NBs/ml, the effect of MI on the contrast was also evaluated for four different Mis: 0.04, 0.06, 0.08, and 0.1.

Radio-frequency spectrum validation

NB frequency response for the different transmit methods was examined and validated using the same tissue-mimicking phantom and a NB-filled cavity. To facilitate frequency components isolation, a 10-cycle transmission pulse in a fixed angle PW, was transmitted (as opposed to single-cycle transmission for the contrast experiments). The sequence included the submission of a positive pulse followed by a negative pulse. The positive pulse was used for standard linear imaging, while the coherent summation of the echoes from both positive and negative pulses yielded a PI image. Two different transmit frequencies were compared: 4 MHz and 4&8 MHz, with MI=0.06. The recorded echoes were captured by the same transducer, and a Fourier transform was applied to the received radio frequency signals and averaged over all channels to yield the frequency spectrum.

Animal model

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Tel Aviv University and were carried out in compliance with institutional guidelines for the care and use of animal models (IACUC Protocol TAU-MD-IL- 2205-157-2). Three bilateral tumor-bearing mice were used for the in-vivo studies. Met-1 mouse breast carcinoma cells were subcutaneously injected into 8 -week-old female FVB/NHanHsd mice (Envigo, Jerusalem, Israel), into 4th and 9th inguinal mammary fat pads. The cells were cultured at 37°C in a humidified 5% CO2 incubator in Dulbecco modified Eagle medium (DMEM, Biological Industries [BI] Ltd., Cat # 01-055-1A, Kibbutz Beit-Haemek, Israel) and collected at the day of injection using TrypLE Express dissociation reagent, as described in Bismuth et.a. (Nanoscale, 2022, 14, 13614-13627). The cells' final concentration was 10¹⁶ cells in 25 pL of PBS+/+. Following cell injections, tumor sizes were measured 7, 14, and 17 days post-injection, until they reached ~4 mm in the smallest diameter. Tumor sizing was performed using an ultrasound system (Vevo 3100, Fujifilm VisualSonics, Toronto, Canada).

In vivo tumor imaging experiments

The tumor-bearing mice were anesthetized with 1.5% isoflurane using a low-flow vaporizer system (SomnoFlo, Kent Scientific). The tumor area was completely shaved and US gel was applied. A NB mixture containing 3.7xlO¹²NBs/ml was diluted in 200 pL of PBS and systemically injected. 5 minutes post-injection, the tumors were imaged using PI at a single 4 MHz frequency or the dual 4&8 MHz method using three Mis of 0.04, 0.08, and 0.1. Each tumor was imaged using both methods and for each MI (N=3 tumors per MI). The relative contrast improvement between the dual-frequency 4&8 MHz imaging compared to a singlefrequency 4 MHz PI imaging was calculated by:

where CTR is a contrast ratio, p4&8MHz is the mean pixel values of an area within the tumor region imaged with the dual-frequency 4&8 MHz transmit and p4MHz is the mean pixel values on the same area within the tumor region that was imaged with a single-frequency 4 MHz transmit. Statistics

Statistical analyses were performed using Prism9 software (GraphPad Software Inc.). Results are presented as mean ± standard deviation (STD). Statistical tests are reported in the relevant captions. P values smaller than 0.05 were considered significant.

Example 1- Dual-frequency combination optimization using microbubbles (MB) in a tissuemimicking phantom

Reference is made to Fig. 2A, which shows a schematic illustration of the experimental setup. As shown in Fig. 2A, a tissue mimicking phantom is administered with MBs, and a dual frequency ultrasound pulse if provided by a corresponding US transducer.

Imaging was performed with a broadband L12-3v transducer, which has the advantage of a large 3-12 MHz bandwidth. This made it possible to maximize the frequency combinations such that both their sum and difference frequencies and their harmonics fell within its bandwidth. The transducer frequency response was evaluated across its bandwidth (Fig. 2B).

Five different transmit frequencies were tested: two single-frequency PI imaging at center frequencies of 4 and 5 MHz, and three dual-frequency PI imaging with combinations of 4&12 MHz, 5&12 MHz and 4&8 MHz. The single frequencies were selected such that their second harmonic remained within the transducer bandwidth (8 and 10 for 4 and 5 MHz transmission, respectively), as compared to 8 and 12 MHz whose second harmonic was beyond the transducer’s bandwidth. The dual-frequency combinations were selected such that either the difference frequency (7 and 8 MHz for the 5&12 and 4&12 MHz, respectively), or both the sum and difference frequencies (4 and 12 MHz for the 4&8 MHz) fell within the bandwidth. All of the imaging experiments utilized single-cycle transmissions to maximize the axial resolution. The MB contrast was evaluated for each transmission at two MI of 0.04 and 0.1 (Fig. 2).

The results presented in Fig. 2B show a line graphs of the Hydrophone peak negative pressure (PNP) measurements of the L12-3v transducer, as a function of the transmitted frequency, for a 2-cycle pulse excitation with the same output voltage of 9v.

The results presented in Fig. 2C, panels i.-vi, show images of the MB filled inclusion for MI=0.04. Axes are identical for all these panels. Panel i. shows a Control image using a single plane wave at 4 MHz without pulse inversion (PI). The indicated circles mark the regions used for contrast ratio (CTR) calculation. Panels ii. And iii. Show PI images using frequencies of 4 MHz and 5 MHz, respectively. Panels iv., v. and vi. show images of dual-frequency transmit of: 4&12 MHz, 5&12 MHz, and 4&8 MHz, respectively.

The results shown in Fig. 2D show the MB contrast results for the different transmit frequencies and two Mis: 0.04 and 0.1. The CTR results are plotted as the mean ± the STD (represented by the error bars, N=4) with the level of significance between different groups, *p<0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, based on a two-way ANOVA with Tukey’s multiple comparison test.

The results shown in Fig. 2E present the Average CTR improvement for the dualfrequency method compared to single-frequency transmit, calculated based on the results presented in Fig. 2D.

The results show that MB cavity contrast was the lowest when imaged using single PW imaging, compared to the PI with the same 4 MHz transmit. The contrast was calculated as the logarithmic ratio between the MB region circled, and the background region circled. For an MI of 0.04, the contrast was higher for all three dual-frequency combinations compared to the single-frequency PI transmission. Increasing the MI to 0.1 decreased the contrast, possibly due to the onset of MB fragmentation. Of the two single-frequency PI transmits, the 4 MHz yielded a higher contrast than the 5 MHz for both MI (a maximal contrast of 13.4 ± 1.2dB, 12.3 + 0.8 dB, respectively. Therefore, the 4 MHz frequency was used as the control for the subsequent experiments. The improvements in contrast with the dual-frequency combinations as compared to single-frequency PI imaging at 4 and 5 MHz, which are summarized in Fig. 2E. The average contrast improvement was calculated by computing the difference between the mean contrast of the 5 transmit options (N=4 samples for each transmit), and the mean contrast for the dualfrequency technique. A positive value indicates that the mean contrast of the dual-frequency method was higher. The 4&8 MHz outperformed the other combinations and achieved a maximal contrast improvement of about 3dB over 4 MHz. Therefore, this dual-frequency combination was used in the next experiments with NBs.

Taken together, the results show that strongest amplitude was found to be at 8 MHz, which was the closest to the transducer’s center frequency (7.5 MHz). The relative amplitudes at 3 MHz, 4MHz, 5 MHz and 12 MHz were 29%, 55%, 81%, and 44% of the maximal value at 8 MHz, respectively. Therefore, the most effective 3 dB bandwidth was 4-12 MHz, which was the range used for further experiments. Example 2- Spectrum validation and concentration optimization of nanobubbles (NBs) in a tissue-mimicking phantom.

Reference is made to Fig. 3A, which shows a graph of the size distribution (in microns) of the generated NBs. The insert shows a representative transmission electron microscopy (TEM) image of a NB .

Fig. 3B shows a Contrast ratio (CTR) results of NB imaging at four NB concentrations (NBs/ml), for two single-cycle transmits at frequencies of 4 MHz (orange) and dual frequency of 4&8 MHz. The CTR results are plotted as the mean ± the STD (represented by the error bars, N=4).

Fig. 3C shows Spectrum validation experiments for frequencies of 4 MHz (single frequency) and dual-frequencies of 4&8 MHz, after 10 cycles of single plane-wave transmission. The received spectrum after 10 cycles of the single plane-wave transmission.

Fig. 3D shows Spectrum validation experiments for frequencies of 4 MHz (single frequency) and dual-frequencies of 4&8 MHz, after 10 cycles of pulse inversion (PI) sequence.

NBs were then fabricated with an average diameter of 160 nm. The NB concentration optimization experiments were used to determine the optimal concentration for the subsequent experiments. Out of the four concentrations, the highest contrast was obtained at a concentration of 5xl0⁹ and lOxlO¹⁰ NBs/ml. A reduced signal was observed at lower concentrations because the NB solution had lower echogenicity. Conversely, higher concentrations of NBs increased the bulk attenuation of the medium. This led to the obstruction of the US signal and the creation of acoustic shadowing, ultimately resulting in a decrease in the overall signal. In order to verify the frequency mixing effect with NBs, a spectrum analysis of the NB echoes was conducted. Single PW imaging with a 10 cycle positive pulse resulted in receive spectra with a dominant fundamental frequency for each transmit frequency. The receive spectra for the 4 MHz transmitted frequencies showed lower amplitude second and third harmonics at 8 and 12 MHz in addition to the fundamental frequency. For the dual 4&8 MHz, harmonics and the sum and difference frequencies contributed to the effect where the amplitude at 12 MHz was higher than the single-frequency. When PI was used, the fundamental frequency was cancelled out, such that at 4 MHz, the second harmonic at 8 MHz was observed. In comparison, for the dual 4&8 MHz transmit, the second harmonic at 8 MHz was observed in addition to the sum (12 MHz) and difference (4 MHz) frequencies. Example 3- Frequency mixing-based contrast harmonic imaging of nanobubbles (NBs)

After optimizing the NB concentration and the transmitted frequencies, the impact of MI on the contrast was evaluated. The NB inclusion was PI imaged with either a single 4 MHz frequency or the dual 4&8 MHz transmit for four different Mis: 0.04, 0.06, 0.08, and 0.1.

The results are presented in Fig. 4A, panels (a)-(h).

Fig. 4A, shows exemplary images of NB filled inclusion, imaged with two different transmits: 4MHz (panels a-d) and dual frequency of 4&8MHz (panels e-h). Each frequency was imaged with four mechanical indices (MI): (a),(e) 0.04, (b),(f) 0.06, (c),(g) 0.08 and (d),(h) 0.1.).

Fig. 4B shows the contrast results, plotted as the mean ± STD (represented by the error bars, N=3) in a dB scale with the level of significance between the two transmits, based on a two-way ANOVA with Sidak’ s multiple comparison test.

Fig. 4C shows the Average CTR improvement for the dual-frequency of 4&8 MHz compared to single-frequency transmit of 4 MHz, and calculated based on Fig. 4B.

Taken together, the results demonstrate that the dual-frequency transmit contrast outperformed the 4 MHz single-frequency transmission for all the Mis, as shown in Fig 4B and 4C. Contrast improvement increased with MI, reaching an average of 8.4 ±0.5 dB for an MI of O.l.

Example 4- Frequency mixing-based contrast harmonic imaging of nanobubbles (NBs) in vivo

Frequency mixing based contrast harmonic imaging of breast cancer tumors in vivo following a systemic NB injection was tested.

Reference is made to Fig. 5A, which shows a schematic illustration of the experimental setting. Briefly, a test animal was injected with tumor cells, as detailed above, and after tumor development, NBs were systemically administered and imaged using an ultrasonic transducer (E12-3v).

As shown in Fig. 5C, panels i., iv. and vii., the sham tumors were anechoic, with the tumor appearing dark. After NB injection, the tumors became echoic (Fig. 5C, panels, ii. , iii., v., vi., viii., and ix. The improvement in NB echogenicity as a result of the frequency mixing effect was expected to increase tumor brightness. Therefore, the improvement in tumor intensity for the dual-frequency transmit was assessed against a single 4 MHz frequency.

As shown in Fig. 5B, for all the tested Mis, the tumor intensity was higher for the dualfrequency 4&8 MHz transmit and increased with MI. The improvement in tumor intensity was 3.4 ± 1.7, 4.8 ± 1.8, and 6.3 ± 1.6 dB for Mis of 0.04, 0.08, and 0.1, respectively.

Thus, collectively, the results indicate that an intensity improvement was obtained with the dual-frequency transmit with 4&8 MHz over a single frequency transmit with 4 MHz.

Claims

CLAIMS What we claim is:

1. A method for performing enhanced ultrasound (US) contrast imaging of a region of interest, using systemically administered nanoscale ultrasound contrast agents (NUCA), the method comprising: transmitting, using an ultrasonic transducer, a dual-frequency excitation pulse comprising a superposition of a first and second ultrasound frequencies; to thereby generate a frequency mixing effect configured to generate non-linear frequency of the nanoscale ultrasound contrast agents; and receiving, by an ultrasonic transducer, frequency components of echoes generated by the nanoscale ultrasound contrast agents, wherein the frequency components comprise a sum of the first and second ultrasound frequencies, a difference of the second and the first ultrasound frequencies, and harmonic frequencies of the first and second ultrasound frequencies; to thereby enhance the nonlinear response of the nanoscale ultrasound contrast agents in real-time to result in an image having enhanced contrast and/or resolution.

2. The method of claim 1, wherein a single transducer is configured to transmit and receive the ultrasound frequencies.

3. The method according to any one of claims 1-2, wherein the transducer is a broadband transducer.

4. The method according to claim 3, wherein the transducer comprises a bandwidth in the range of about 2-22 MHz, wherein the sum and difference frequencies thereof and their harmonics are configured to be within the bandwidth of the transducer.

5. The method according to any one of claims 1-4, wherein the first ultrasound frequency is in the range of about 3-6 MHz and the second ultrasound frequence is in the range of about 6-12 MHz.

6. The method according to any one of claims 1-5, wherein the first ultrasound frequency is about 4 MHz and the second ultrasound frequence is in the range of about 8 MHz.

7. The method according to claim 6, wherein the sum of the first and second ultrasound frequencies is about 12 MHz, a difference of the second and the first ultrasound frequencies is about 4MHz, and harmonic frequencies of the first and second ultrasound frequencies are about 4, 8 and 16MHz.

8. The method of any one of claims 1-7, incorporated in a contrast harmonic imaging (CHI) sequence.

9. The method according to any one of claims 1-8, wherein the transmitting comprises a plurality of transmission cycles.

10. The method according to any one of claims 1-9, wherein the transmitting comprises a pulse inversion (PI) cycle.

11. The method according to any one of claims 1-10, wherein the NUCAs are selected from nanobubbles, gas vesicles and nanodroplets.

12. The method according to any one of claims 1-11, wherein the NUCA comprise an average diameter in the range of about 120-200nm.

13. The method according to any one of claims 1-12, wherein the NUCA comprise an average diameter in the range of about 160nm.

14. The method according to any one of claims 1-13, wherein the NUCA comprise nanobubbles, comprising one or more lipids.

15. The method according to any one of claims 1-14, wherein the NUCA comprise a targeting moiety on a shell thereof.

16. The method according to claim 15, wherein the targeting moiety comprises a cell type-specific antibody conjugated to the shell.

17. The method according to any one of claims 1-16, wherein the NUCAs are administered systemically, at a concentration of about 5xl0⁹-5X10¹²NUCA/ml.

18. The method according to any one of claims 1-17, wherein the NUCA comprise nanobubbles, having an average diameter of about 150-170 nm, being administered systemically, at a concentration of about 5xl0⁹-5X10¹²NBs/ml.

19. The method according to any one of claims 1-18, wherein the contrast enhancement using a dual frequency excitation pulse, comprises an improvement of at least about 100% in a contrast ratio (CTR) determined at a designated mechanical index (MI), compared to using a single frequency excitation pulse at the same MI.

20. The method according to any one of claims 1-19, wherein the contrast enhancement using a dual frequency excitation pulse, comprises an improvement of at least about IdB in a contrast ratio (CTR) determined at a designated mechanical index (MI), compared to using a single frequency excitation pulse at the same MI.

21. The method according to any one of claims 1-20, wherein the contrast enhancement using a dual frequency excitation pulse, comprises an improvement of at least about 2dB in a contrast ratio (CTR) determined at a designated mechanical index (MI), compared to using a single frequency excitation pulse at the same MI.

22. The method according to any one of claims 1-21, wherein the contrast enhancement using a dual frequency excitation pulse comprising 4Mhz and 8Mhz, comprises an improvement of at least about 4dB in a contrast ratio (CTR) determined at a mechanical index (MI) of 0.1, compared to using a single frequency 4MHz excitation pulse at an MI of 0.1.

23. The method according to any one of claims 1-22, comprising systemically administering the NUCA, prior to performing the imaging.

24. The method according to any one of claims 1-23, for use in real-time, in-vivo imaging applications, comprising: image-guided medical procedures, diagnostic imaging of blood vessels, diagnostic imaging of tissues, diagnostic imaging of tumor, diagnostic imaging of inflammation, diagnostic imaging of diabetes, or any combinations thereof.

25. A method for utilizing dual frequency ultrasound for imaging a region of interest in a body of a subject, using nanoscale ultrasound contrast agents (NUCAs), the method comprising the steps of: generating dual frequency ultrasound waves using an ultrasonic transducer; transmitting the dual frequency ultrasound waves into the region of interest within, wherein said region of interest comprises the NUCAs; receiving reflected ultrasound waves components from the NUCAs utilizing the ultrasonic transducer, wherein the ultrasound waves components comprise the sum and difference of the dual-frequency waves, and harmonic frequencies thereof; processing, the received ultrasound waves components, to generate an image of said target area; and analyzing said image to assess condition of the region of interest.

26. The method according to claim 25, further comprising systemically administering the NUCAs, prior to performing the imaging.

27. The method according to any one of claims 25-26, wherein the transducer is a broadband transducer.

28. The method according to any one of claims 25-27, wherein the NUCAs are selected from nanobubbles, gas vesicles and nanodroplets.

29. The method according to any one of claims 25-28, wherein the NUCA comprise a targeting moiety on a shell thereof.

30. The method according to any one of claims 25-29, wherein the contrast enhancement using a dual frequency excitation pulse, comprises an improvement of at least about 2dB in a contrast ratio (CTR) determined at a designated mechanical index (MI), compared to using a single frequency excitation pulse at the same MI.