WO2025075498A1

WO2025075498A1 - Method and device for imaging a medium

Info

Publication number: WO2025075498A1
Application number: PCT/NL2024/050535
Authority: WO
Inventors: David Maresca; Baptiste Guillaume HEILES; Hugues FAVRE
Original assignee: Technische Universiteit Delft
Current assignee: Technische Universiteit Delft
Priority date: 2023-10-04
Filing date: 2024-10-01
Publication date: 2025-04-10
Anticipated expiration: 2026-04-04

Abstract

A method for imaging a medium, comprising: providing a plurality of transmitters arranged to a transmitter surface; selecting a first set of transmitters of the plurality of transmitters for transmitting a first plane wave into the medium; selecting a second set of transmitters of the plurality of transmitters for transmitting a second plane wave into the medium; transmitting a first plane wave; transmitting a second plane wave; and transmitting a first plane wave and a second plane wave for creating an interference sheet in the medium; wherein the wave amplitude inside the interference sheet is above a threshold, and outside the interference sheet below the threshold.

Description

METHOD AND DEVICE FOR IMAGING A MEDIUM

FIELD OF THE INVENTION

The invention relates to a method for imaging a medium. The invention further relates to an imaging device for imaging a medium. The invention further relates to a computer-readable storage medium for controlling a controller of an imaging device for imaging a medium.

BACKGROUND OF THE INVENTION

An ultrasound wave may be sent into a medium for forming an image of the medium from the set of echoes of the ultrasound wave. In some applications, multiple ultrasound waves are combined for imaging the medium.

As an example, US 2019/0314001 A1 discloses certain embodiments pertaining to cross-amplitude modulation (xAM) ultrasound imaging methods and systems. A disadvantage of these embodiments is that the imaging of the medium is coarse which may be a cause of missing features.

As a further example, US 2016/113625 A1 discloses an ultrasonic diagnostic apparatus and an ultrasonic diagnostic method. The ultrasonic diagnostic apparatus includes: a transducer for performing interconversion between an acoustic wave and an electric signal; and a transmitter for controlling the transducer so as to allow a plurality of plane waves to be transmitted through a plurality of sub-apertures. A disadvantage of these embodiments is that the imaging of the medium is coarse which may be a cause of missing features.

SUMMARY OF THE INVENTION

An object of the invention is to overcome one or more of the disadvantages mentioned above.

According to a first aspect of the invention, a method for method for imaging a medium, comprising: providing a plurality of transmitters arranged to a transmitter surface; selecting a first set of transmitters of the plurality of transmitters for transmitting a first plane wave into the medium; selecting a second set of transmitters of the plurality of transmitters for transmitting a second plane wave into the medium; and transmitting a first plane wave and a second plane wave for creating an interference sheet in the medium; wherein the wave amplitude inside the interference sheet is above a threshold, and outside the interference sheet below the threshold; and wherein transmitting the

SUBSTITUTE SHEET (RULE 26) first plane wave and the second plane wave comprises timing the transmission of the first plane wave relative to the transmission of the second plane wave for positioning the interference sheet.

The medium to be imaged should be a medium suitable for propagating the plane waves transmitted by the transmitters. The medium may be a part of a living organism, such as a tree, an animal or even a human. The medium may be the soft tissue of an animal body, such as a human body. The medium typically attenuates the plane wave while propagating in the medium. Imaging the medium is typically based on receiving a set of echoes resulting from the plane wave propagating through the medium, more specifically changes of material or substance in the medium.

The method comprises providing a plurality of transmitters. The plurality of transmitters are arranged to a transmitter surface. The transmitter surface is shaped for allowing to transmit a plane wave. The transmitter surface may be flat, curved or even curled in two or three dimensions. The transmitter surface may be deformable. The transmitter surface is typically shaped to promote a good contact with the medium for allowing as much energy of the plane wave generated by the transmitters to propagate into the medium.

The method comprises selecting a first set of transmitters of the plurality of transmitters. This first set of transmitters is arranged for transmitting a first plane wave into the medium. The first plane wave may be angled relative to the first set of transmitters. The angle or refraction angle of the plane wave is based on the wave transmission or propagation velocity in the medium, and the time delay between the set of transmitters emitting or transmitting the plane wave. It is further noted that the transmission speed of the interference sheet, or propagation speed of the interference sheet is based on the angle of the plane wave and the transmission speed in the medium. Further, the interference sheet may be labelled as an X-wave.

The method comprises selecting a second set of transmitters of the plurality of transmitters. This second set of transmitters is arranged for transmitting a second plane wave into the medium. The second plane wave may be angled relative to the second set of transmitters. The second plane wave is typically angled differently from the first plane wave.

The method comprises transmitting a first plane wave and a second plane wave for creating an interference sheet in the medium. These first and second plane wave transmissions are timed such that the waves create the interference sheet, interference plane, or interference layer. The interference sheet is the volume in the

SUBSTITUTE SHEET (RULE 26) medium where the first and second wave propagate or co-propagate through the medium at the same time. Typically, the interference between the first and second wave does extend over time, thereby the interference may be seen as a wave propagating through the volume defined by the interference sheet. The interference propagating through the volume of the interference sheet is typically elongated in shape and propagates aligned to an elongated axis of the elongated shaped interference. In the ideal situation, the volume is a plane and the interference propagating is a line shifting over this plane.

The interference of the first and second waves cause the energy and/or amplitude of these waves to be added inside the interference sheet. This addition is over the threshold, typically a predefined threshold. The first and the second waves each have an energy and/or amplitude outside the interference sheet below the threshold, typically a predefined threshold. The method allows to have a particular amplitude and/or energy at a specific part of the medium, typically over time, which is inside the interference sheet.

The threshold is typically selected such that advantage is taken of non-linear material, non-linear matter, non-linear substances, or non-linear parts present in the medium. The result of the transmission, typically a set of echoes returning from the transmission, may be used, or combined to result in image information regarding the interference sheet. The image information for a whole interference sheet, which is a volume in the medium, is obtained having the advantage of allowing to quickly image the medium below the transmitter surface. The image information for a whole interference sheet, which is a volume in the medium, is typically fine-grained image information by advantageously purposefully selecting the sets and angles of the plane waves. The image information for a whole interference sheet, which is a volume in the medium, is typically imaging more of the medium by purposefully selecting the sets and angles of the plane waves for advantageously arranging the interference sheet closer to the perimeter of the transmitter surface.

The transmission of the first plane wave and the second plane wave comprises timing the transmission of the first plane wave relative to the transmission of the second plane wave. Timing the transmission of the first plane wave and the second plane wave allows to position the interference sheet. The timing is typically selected such that the positioning of the interference sheet is different, preferably a smaller difference, compared to the positioning when selecting the adjacent transmitters, or shifting the set of transmitters at least one position.

SUBSTITUTE SHEET (RULE 26) The interference sheet is a volume in the medium. The interference sheet may be defined as an amplitude inside the volume above the threshold. The interference sheet is positionable by changing the timing between the transmission of the first plane wave and the second plane wave. Thus, the technical effect is that the volume in the medium with an amplitude above the threshold is positionable based on the timing between the transmission of the first plane wave and the second plane wave.

According to another aspect of the invention, an imaging device for imaging a medium, comprising: a transmitter surface; a plurality of transmitters arranged to the transmitter surface; a controller arranged for: selecting a first set of transmitters of the plurality of transmitters for transmitting a first plane wave into the medium; selecting a second set of transmitters of the plurality of transmitters for transmitting a second plane wave into the medium; and providing instructions for transmitting a first plane wave and a second plane wave for creating an interference sheet in the medium; wherein the wave amplitude inside the interference sheet is above a threshold, and outside the interference sheet below the threshold; and wherein transmitting the first plane wave and the second plane wave comprises timing the transmission of the first plane wave relative to the transmission of the second plane wave for positioning the interference sheet. The imaging device provides the advantages also mentioned for the other aspects of the invention, specifically those provided by the imaging method.

According to another aspect of the invention, a computer-readable storage medium comprising instructions which, when executed by a computer, cause the computer to carry out the steps of any of the computer implemented methods mentioned, or the steps of the method for imaging mentioned. The computer-readable storage medium provides the advantages also mentioned for the other aspects of the invention, specifically provided by the computer implemented method.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

In an embodiment of the imaging method, timing comprises delaying the transmission of the first plane wave relative to the transmission of the second plane wave for positioning the interference sheet. Delaying advantageously provides a simplified implementation, specifically in hardware a delay relative to another event is easily implemented preferably by triggering on a clock signal. Further, as the first and second plane waves are freely selectable, through selecting the first and second set of transmitters, either of the plane waves may be delayed.

SUBSTITUTE SHEET (RULE 26) In an embodiment of the imaging method, positioning the interference sheet comprises a lateral shift of the interference sheet relative to the transmitter surface. A lateral shift of the interference sheet provides the advantage of easily imaging a volume of a medium without overlap of the different interference sheets. Thus, the lateral shift provides the advantage of efficient imaging of a volume of the medium.

In an embodiment of the imaging method, timing comprises timing with discrete time steps having a discrete step size. Using discrete time, such as a clock signal, advantageously provides improved reproducibility and improved controllability. In a further embodiment of the imaging method, the discrete step size is advantageously based on a clock frequency provided to at least the first set of transmitters and the second set of transmitters.

In an embodiment of the imaging method, the method further comprising the steps of: transmitting a first plane wave; and transmitting a second plane wave. The method comprises transmitting a first plane wave. The method comprises transmitting a second plane wave. The first and second plane wave are transmitted such that these transmissions do not have any interference with each other. This typically means that the second plane wave is transmitted at a different time compared to the first plane wave, such as after each other. It is also noted that typically the set of echoes of one of the plane waves has propagated through the medium before the other plane wave starts propagating through the medium.

It is noted that the transmitting of the first plane wave, the second plane wave, and the first plane wave and the second plane wave may be done in a different order. The order of transmitting is not limited to the order in the claimed method.

The threshold is typically selected such that advantage is taken of non-linear material, non-linear matter, non-linear substances, or non-linear parts present in the medium. The threshold may be a range. The threshold range is advantageously selected such that the medium or at least a volume of the medium, typically where the interference sheet is positioned, acts linear or non-linear. The threshold range is advantageously selected such that the medium or at least a volume of the medium, typically where the interference sheet is positioned, is excited with a wave, such as a plane wave, having an amplitude either in the linear or non-linear regime of the medium. Or phrased differently, the threshold range is advantageously selected such that the medium or at least a volume of the medium, typically where the interference sheet is positioned, is excited with a wave, such as a plane wave, having an amplitude outside where the medium transits between the linear and non-linear regimes of the medium.

SUBSTITUTE SHEET (RULE 26) The result of the transmissions, typically a set of echoes returning from the transmissions, may be combined to result in image information regarding the interference sheet. The image information for a whole interference sheet, which is a volume in the medium, is obtained having the technical effect of allowing to image the medium below the transmitter surface fast. The image information for a whole interference sheet, which is a volume in the medium, is typically fine-grained image information by advantageously purposefully selecting the sets and angles of the plane waves. The image information for a whole interference sheet, which is a volume in the medium, is typically imaging more of the medium by purposefully selecting the sets and angles of the plane waves for advantageously arranging the interference sheet closer to the perimeter of the transmitter surface.

In an embodiment of the imaging method, the method comprises: providing a plurality of receivers, wherein at least a set of receivers from the plurality of receivers is arranged for receiving echoes from the first plane wave and the second plane wave; after transmitting the first plane wave, receiving a first set of echoes from the first plane wave with the set of receivers; after transmitting the second plane wave, receiving a second set of echoes from the second plane wave with the set of receivers; and after transmitting the first plane wave and the second plane wave, receiving a third set of echoes from the first plane wave and the second plane wave with the set of receivers. This embodiment advantageously receives the sets of echoes for deducing an image from the sets of echoes. Receiving a third set of echoes from the first plane wave and the second plane wave with the set of receivers is typically bordered in time. The reception may start after the transmission of the first plane wave and the second plane wave. The reception may be stopped after a time calculated by adding the longest distance of the transmitter to the furthest point in the medium with the longest distance of the furthest point in the medium to the receiver, and divided by the transmission speed through the medium.

In a further embodiment of the imaging method, the method comprises deducing image information related to the interference sheet based on subtracting the first set of echoes and the second set of echoes from the third set of echoes. The threshold is typically advantageously selected such that a non-linear effect takes place in the interference sheet. Subtracting the first set of echoes and the second set of echoes from the third set of echoes allows to filter out linear effects resulting in exposing the non-linear effect in the interference sheet. This filtering for exposing the

SUBSTITUTE SHEET (RULE 26) non-linear effect in the interference sheet is advantageously used for creating an image of the interference sheet.

In an embodiment of the imaging method, selecting the first set of transmitters and selecting the second set of transmitters comprises selecting the first set of transmitters and selecting the second set of transmitters such that these transmitters are adjacent. The width of both sets of the transmitters combined is advantageously minimized. Further, having the sets of transmitters adjacent allows to create the interference sheet, preferably originating, from a position half way between the two sets of transmitters. This interference sheet is therefore advantageously arranged at half a pitch distance between the two adjacent sets of transmitters.

In an embodiment of the imaging method, transmitting the first plane wave and the second plane wave comprises transmitting the first plane wave and the second plane wave with the adjacent sets of transmitters such that the interference sheet originates from a separation line separating the adjacent sets of transmitters and into the medium. This interference sheet is therefore advantageously arranged at half a pitch distance between the two adjacent sets of transmitters.

In an embodiment of the imaging method, the separation line is a straight line or curved line. The separation line being a straight line allows for the set of transmitters to be arranged with a straight side, typically a rectangular arrangement of the set of transmitters, opposing the other set of transmitters for advantageously simplifying the arrangement or placement, and/or the control of the set of transmitters. The separation line being a curved line allows for advantageously obtaining image information from another not straight cross-section of the medium.

In an embodiment of the imaging method, a mesh comprises vertices and edges; wherein the plurality of transmitters are arranged to respective vertices; wherein the edges have a length; wherein the length of the edges is selected such that the mesh forms a regular or repeating pattern; wherein the mesh forms the transmitter surface; wherein preferably the vertices form a grid, more preferably a square grid, a rectangular grid, a triangular grid, or a hexagonal grid; and wherein selecting the first set of transmitters and selecting the second set of transmitters comprises selecting the first set of transmitters and selecting the second set of transmitters such that these sets boarder each other at one edge, two edges, three edges, four edges or more edges apart from each other. It should be understood that an edge in the mesh specifies a distance between vertices. The mesh is typically forming the transmitter surface. Arranging a transmitter to a vertex, may comprise arranging all transmitters at two or

SUBSTITUTE SHEET (RULE 26) more levels while the mesh may be considered as a surface. Arranging the transmitters at two or more levels advantageously allows for the transmitters to be compacted together. When the transmitters are arranged at two or more levels, the transmitter may be seen as projected onto the mesh in a projection direction perpendicular to the mesh. Arranging the transmitters to a mesh advantageously allows the transmitters to be arranged in a simple and repeating pattern for simplified manufacturing and compacting the device comprising the transmitters. Arranging the transmitters at different levels allows to arrange the transmitters more compact. A specific arrangement comprises arranging, such as stacking, the transmitters at two different levels, such as that one level or layer is formed by transmitters in rows and one layer or level is formed by transmitters in columns.

In a further embodiment of the imaging method, an area is defined between the first set of transmitters and the second set of transmitters; and wherein the area is straight. The resulting straight interference sheet advantageously allows for simplified calculation and visualisation. Typically, the area has a width. The interference sheet is typically formed in a layer extending perpendicular to the transmitter surface halfway the width of the transmitter surface.

In a further embodiment of the imaging method, selecting the first set of transmitters and selecting the second set of transmitters comprises selecting the first set of transmitters and selecting the second set of transmitters such that these sets boarder each other one edge apart from each other for transmitting the first plane wave, the second plane wave, and the first and the second plane wave; wherein the method comprises: selecting a third set of transmitters of the plurality of transmitters for transmitting a third plane wave into the medium; transmitting a third plane wave; and transmitting a first plane wave and a third plane wave for creating a second interference sheet in the medium; wherein the wave amplitude inside the second interference sheet is above the threshold, and outside the second interference sheet below the threshold; wherein the third set of transmitters is one edge shifted compared to the second set of transmitters, such that the third set of transmitters and the second set of transmitters boarder each other two edges apart from each other; and wherein at least at the boundary between the transmitter surface and the medium, preferably a part of the interference sheet, more preferably over the whole interference sheet, the interference sheet and the second interference sheet are shifted from each other over half an edge length. This method advantageously allows to shift the interference sheet with a half an

SUBSTITUTE SHEET (RULE 26) edge of the mesh for allowing the interference sheet to step through the medium for improved imaging of the medium.

In a further embodiment of the imaging method, the method comprises: selecting a fourth set of transmitters of the plurality of transmitters for transmitting a fourth plane wave into the medium; selecting a fifth set of transmitters of the plurality of transmitters for transmitting a fifth plane wave into the medium; transmitting a fourth plane wave; transmitting a fifth plane wave; and transmitting a fourth plane wave and a fifth plane wave for creating a third interference sheet in the medium; wherein the wave amplitude at the third interference sheet is above the threshold, and outside the third interference sheet below the threshold; and wherein the set of transmitters formed by the first set of transmitters and the second set of transmitters do not overlap with the set of transmitters formed by the fourth set of transmitters and the fifth set of transmitters. The interference sheets may be created relatively far apart, such that the first and second plane wave do not interfere with the fourth and fifth plane wave even when transmitted at the same time. This advantageously allows to image two interference sheets at the same time in the medium. Imaging two interference sheets at the same time allows for faster imaging of the whole medium under the transmitters.

In a further embodiment of the imaging method, the method comprises: selecting a fourth set of transmitters of the plurality of transmitters for transmitting a fourth plane wave into the medium; selecting a fifth set of transmitters of the plurality of transmitters for transmitting a fifth plane wave into the medium; transmitting a fourth plane wave; transmitting a fifth plane wave; and transmitting a fourth plane wave and a fifth plane wave for creating a third interference sheet in the medium; wherein the wave amplitude at the third interference sheet is above the threshold, and outside the third interference sheet below the threshold; and wherein the interference sheet and the third interference sheet intersect, preferably are perpendicular. The interference sheets give different cross-sections of the medium which may provide additional, improved and/or simplified image information of the medium. The interference sheet, typically providing a cross-section of the medium, may be aligned with particular structures inside the medium by aligning the third interference sheet based on the first interference sheet.

In a further embodiment of the imaging method, the transmitter surface is rectangular; selecting the first set of transmitters and selecting the second set of transmitters comprises selecting the first set of transmitters in a first rectangle and selecting the second set of transmitters in a second rectangle; the first plane wave has a first normal vector; the second plane wave has a second normal vector; the first

SUBSTITUTE SHEET (RULE 26) cross-propagation angle is defined as the angle between the first and second normal vectors; the interference sheet has an interference depth reaching into the medium; the method comprises: selecting a sixth rectangular set of transmitters of the plurality of transmitters for transmitting a sixth plane wave into the medium; selecting a seventh rectangular set of transmitters of the plurality of transmitters for transmitting a seventh plane wave into the medium; transmitting a sixth plane wave; transmitting a seventh plane wave; and transmitting a sixth plane wave and a seventh plane wave for creating a fourth interference sheet in the medium; the wave amplitude at the fourth interference sheet is above the threshold, and outside the fourth interference sheet below the threshold; the sixth plane wave has a sixth normal vector; the seventh plane wave has a seventh normal vector; the second cross-propagation angle is defined as the angle between the sixth and seventh normal vectors; the fourth interference sheet has a fourth interference depth reaching into the medium; and the second cross-propagation angle is adapted such that the fourth interference depth is substantially equal to the interference depth. Having a cross-propagation angle closer to 180 degrees requires selecting wider rectangular sets of transmitters. Wider rectangular sets of transmitters requires the energy of the further transmitters to reach the interference sheet, thus requiring a longer transmission time in the medium. On the other hand, the longer transmission time allows the echoes from the interference sheet to reach the receivers over a longer time providing more detailed echoes and thus more accurate image information. Selecting the sixth and seventh rectangular sets in combination with the second cross-propagation angle relative to the first and second rectangular sets in combination with the first cross-propagation angle advantageously provides exchanging acquisition speed with accuracy of the image information.

In a further embodiment of the imaging method, the sixth rectangular set of transmitters and the seventh rectangular set of transmitters are located closer to the perimeter of the transmitter surface compared to the first rectangular set of transmitters and the second rectangular set of transmitters.

In a further embodiment of the imaging method, the first rectangular set of transmitters, the second rectangular set of transmitters, the sixth rectangular set of transmitters and the seventh rectangular set of transmitters all have a width; the width of the sixth rectangular set of transmitters is smaller compared to the width of the first rectangular set of transmitters; and the width of the seventh rectangular set of transmitters is smaller compared to the width of the second rectangular set of transmitters. This embodiment further details to advantageously allow to image a part of

SUBSTITUTE SHEET (RULE 26) the medium located closer to the edges of the transmitter surface. This embodiment advantageously allows to image more volume of the medium relative to the surface area of the transmitter surface.

In a further embodiment of the imaging method, the first rectangular set of transmitters, the second rectangular set of transmitters, the sixth rectangular set of transmitters and the seventh rectangular set of transmitters all have a width; the width of the sixth rectangular set of transmitters is smaller compared to the width of the first rectangular set of transmitters; the width of the sixth rectangular set of transmitters and the width of the seventh rectangular set of transmitters are both smaller compared to the width of the first rectangular set of transmitters as well as the width of the second rectangular set of transmitters. This embodiment even further details to advantageously allow to image a part of the medium located closer to the edges of the transmitter surface. This embodiment details further to advantageously allow to image more volume of the medium relative to the surface area of the transmitter surface. When more volume of the medium may be imaged with the same transmitter surface, the part of the medium imaged, and thus of interest, may be imaged without moving the transmitter surface. As mechanically moving the transmitter surface is slower and less accurate than selecting another interference sheet with the transmitter surface at the same position, provides the advantage of faster and/or more accurate imaging a part of the medium of interest.

In an embodiment of the imaging method, the medium comprises a medium boundary, typically adjacent to or in contact with the transmitter surface; and the interference sheet originates adjacent to the medium boundary and propagates substantially away, preferably away, from the medium boundary. This advantageously allows to image the medium in depth starting from the medium.

In an embodiment of the imaging method, the medium comprises a medium boundary, typically adjacent to or in contact with the transmitter surface; and the interference sheet originates at a distance from the medium boundary and propagates substantially away, preferably away, from the medium boundary. This advantageously allows to image the medium in depth while skipping a first distance from the medium boundary. This may be advantageous in case of the medium being a skin, and the upper part or layer of the skin is not of interest for imaging or even disturbing the imaging.

In an embodiment of the imaging method, the transmitter surface is a 2D transmitter surface. The 2D transmitter surface is typically square or rectangular or

SUBSTITUTE SHEET (RULE 26) substantially square or rectangular. These shapes advantageously provide flexibility of positioning the interference sheet.

In a further embodiment of the imaging method, the 2D transmitter surface is a 2D flat transmitter surface or a 2D curved transmitter surface. The transmitter surface is advantageously selected such that the shape of the transmitter surface fits the application. As an example, if the medium is a skin, pressing a curved transmitter surface, such as a convex curved transmitter surface, on the skin provides an improved contact over the whole transmitter surface.

In an embodiment of the imaging method, the first plane wave has a first normal vector; the second plane wave has a second normal vector; a first crosspropagation angle is defined as the angle between the first and second normal vectors; and the cross-propagation angle is less than 150 degrees, preferably in a range from 120-150 degrees, more preferably 130-150 degrees, most preferably 140-150 degrees. Experiments have shown that the cross-propagation angle in this range balances precision and depth of the interference sheet for providing good results.

In a further embodiment of the imaging method, the interference sheet is substantially flat; the interference sheet defines a sheet normal vector normal to the interference sheet; a first angle is between the first normal vector and the sheet normal vector; a second angle is between the second normal vector and the sheet normal vector; and the first angle is minus the second angle. Experiments have shown that having symmetry in the cross-propagation angles advantageously optimizes precision and depth of the interference sheet.

In an embodiment of the imaging method, the medium comprises a contrast agent; and the threshold is one of a buckling threshold, a collapse threshold, and a cavitation threshold of the contrast agent. The buckling threshold, the collapse threshold, and the cavitation threshold of the contrast agent are typically non-linear effects. Due to this non-linear effect of the contrast agent the moving through the interference sheet of one plane wave relative to two or more plane waves interfering in the interference sheet provide different reactions or echoes. Thus, due to this non-linear effect the presence of a contrast agent may be detected inside the interference sheet.

In a further embodiment of the imaging method, the contrast agent is an engineered harmonic gas vesicle or a microbubble. These are advantageously used contrast agents providing the non-linear effect.

In an embodiment of the imaging method, the method comprises providing a plurality of transducers, wherein each transducer comprises one transmitter of the

SUBSTITUTE SHEET (RULE 26) plurality of transmitters and one receiver of the plurality of receivers. Transmitters and receivers may partly or in whole use the same hardware, thereby combing these to transducers. As an example, an antenna may be used for receiving and transmitting electromagnetic waves. As another example, a piezo electric element may be used to transmit ultrasound waves and to receive ultrasound waves. Reusing hardware for transmitting and receiving advantageously reduces the space used or provides for a more compact device typically used in the method.

In an embodiment of the imaging method, the plurality of transmitters and the plurality of receivers are arranged for transmitting and receiving ultrasound, respectively. Although ultrasound waves are claimed, other waves, such as electromagnetic waves are also possible.

In a further embodiment of the imaging method, the threshold is a pressure threshold. This pressure threshold advantageously typically provides for a non-linear effect allowing detecting inside the interference sheet.

In a further embodiment of the imaging method, the interference sheet is substantially flat; the interference sheet defines a sheet normal vector normal to the interference sheet; the transmitter surface is substantially flat; and the sheet normal vector is substantially parallel to the transmitter surface. Experiments have shown that having symmetry in the cross-propagation angles advantageously optimizes precision and depth of the interference sheet.

In an embodiment of the imaging method, the interference sheet is flat or substantially flat. Experiments have shown that having symmetry in the crosspropagation angles advantageously optimizes precision and depth of the interference sheet.

In an embodiment of the imaging device, the device comprises: a plurality of receivers, wherein at least a set of receivers from the plurality of receivers is arranged for receiving echoes from the first plane wave and the second plane wave; wherein the controller is arranged for: after transmitting the first plane wave, receiving a first set of echoes from the first plane wave with the set of receivers; after transmitting the second plane wave, receiving a second set of echoes from the second plane wave with the set of receivers; and after transmitting the first plane wave and the second plane wave, receiving a third set of echoes from the first plane wave and the second plane wave with the set of receivers. This embodiment advantageously receives the sets of echoes for deducing an image from the sets of echoes.

SUBSTITUTE SHEET (RULE 26) In a further embodiment of the imaging device, the controller is arranged for deducing image information related to the interference sheet based on subtracting the first set of echoes and the second set of echoes from the third set of echoes. The threshold is typically advantageously selected such that a non-linear effect takes place in the interference sheet. Subtracting the first set of echoes and the second set of echoes from the third set of echoes allows to filter out linear effects resulting in exposing the non-linear effect in the interference sheet. This filtering for exposing the non-linear effect in the interference sheet is advantageously used for creating an image of the interference sheet.

In an embodiment of the imaging device, the device comprises a plurality of transducers, wherein each transducer comprises one transmitter of the plurality of transmitters and one receiver of the plurality of receivers. Transmitters and receivers may partly or in whole use the same hardware, thereby combing these to transducers. As an example, an antenna may be used for receiving and transmitting electromagnetic waves. As another example, a piezo electric element may be used to transmit ultrasound waves and to receive ultrasound waves. Reusing hardware for transmitting and receiving advantageously reduces the space used or provides for a more compact device typically used in the method.

In an embodiment of the imaging device, the plurality of transmitters and the plurality of receivers are arranged for transmitting and receiving ultrasound, respectively. Although ultrasound waves are claimed, other waves, such as electromagnetic waves are also possible.

In an embodiment of the imaging device, the imaging device incorporates one or more features mentioned for the imaging method for providing a similar or substantially similar technical effect as was mentioned for the embodno iment of the method.

The imaging device is useable for nonlinear sound sheet imaging. Nonlinear sound sheet imaging -NSSI- detects nonlinear echoes arising from ultrasound contrast agents, such as microbubbles, gas vesicles or else, located in the sound sheet plane typically at kilohertz framerates. As detection relies on the nonlinear frequency content of contrast agent echoes rather than their motion in blood or tissues, NSSI can detect slowly-circulating or static contrast agents. To achieve super-resolution imaging of contrast agents, the controller of the imaging device is arranged for applying ultrasound localization microscopy algorithms to achieve sound sheet localization microscopy - SSLM-. NSSI retain contrast agents signals while suppressing surrounding tissue

SUBSTITUTE SHEET (RULE 26) background. The imaging device may retrieve the position of individual microbubble using a radial symmetry-based localization. The controller of the imaging device may apply Kuhn-Munkres minimization algorithms for tracking contrast agents positions over time. If vascular contrast agents, such as microbubbles, gas vesicles or else, are used, the controller may be arranged for reconstructing flow velocimetry or density maps of the vasculature.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be apparent from and elucidated further with reference to the embodiments described by way of example in the following description and with reference to the accompanying drawings, in which:

Figure 1 a-i schematically show the imagining device, working of the imagining device and method, and test results;

Figure 2a-c show in vitro volumetric sound sheet imaging of genetically encoded acoustic reporters;

Figure 3a-d show in vivo volumetric NSSI of mammalian ARG expressing tumors in a mouse model;

Figure 4a-e show selective-plane ultrafast NSSI of flow dynamics in the rat brain;

Figure 5a-c schematically show transmissions of planes waves;

Figure 6a-d schematically show transmissions of planes waves;

Figure 7a-c schematically show orthogonal interference sheets;

Figure 8 schematically shows an extended field of view for an transmitter surface;

Figure 9 schematically shows an embodiment of a computer program product, computer readable medium and/or non-transitory computer readable storage medium according to the invention;

Figure 10a-b schematically show transmissions of planes waves; and Figure 11 a-b schematically show transmissions of planes waves without the medium.

The figures are purely diagrammatic and not drawn to scale. In the figures, elements which correspond to elements already described may have the same reference numerals.

LIST OF REFERENCE NUMERALS

SUBSTITUTE SHEET (RULE 26)

DETAILED DESCRIPTION OF THE FIGURES

The following figures may detail different embodiments. Embodiments can be combined to reach an enhanced or improved technical effect. These combined

SUBSTITUTE SHEET (RULE 26) embodiments may be mentioned explicitly throughout the text, may be hint upon in the text or may be implicit.

Abstract

Mesoscale light-sheet microscopy provides a comprehensive view of cleared organs down to the cellular scale. The reliable detection of genetically expressed fluorescent reporters allows for the visualization of entire cell populations or the description of previously unknown anatomical structures. In small, optically translucent organisms, scanned light sheet microscopy can even observe dynamic cellular processes in three dimensions. However, the scattering of light in thick tissue, as well as photobleaching of fluorescent reporters limits the spatiotemporal resolution of this method to studying thin specimens. Here we show that non-diffractive sound sheet imaging of mammalian tissues labelled with acoustic reporters enables fast and volumetric imaging of cellular processes at the organ scale. We found that modulating acoustic pressure along the main lobe of ultrasonic Bessel beams generated with a row-column array, alternatively named transmitter surface, efficiently confines nonlinear scattering of acoustic reporters to a sound sheet, also named interference sheet, as thin as 89 pm x 8.4 mm x 12.8 mm. We present sound sheet imaging of the rat cerebral vasculature at 3.2 kHz in selected planes separated by 55 pm and orthogonally scanned 3D sound sheet imaging of tumor gene expression in a mouse model of cancer. Together, our results demonstrate that nonlinear sound sheet imaging can reveal targeted biological processes in living opaque organisms with a single 2D transducer array or a plurality of transmitters arranged to a transmitter surface. We anticipate that the parallel development of high-speed volumetric sound sheet imaging and acoustic reporter genes and biosensors will unlock previously inaccessible basic biology studies at the mesoscale in living opaque organisms.

Introduction

The most informative method for observing dynamic cellular processes in vivo in 3D uses light sheet microscopy that leverages genetically encoded fluorescent reporters. Successive advances in light sheet fluorescence microscopy (LSFM) now enable fast, large-volume, and high-resolution imaging of fluorescently labelled cells in transparent or cleared organisms. These capabilities had a tremendous impact on developmental biology by enabling long-term imaging of embryogenesis.

A next frontier would be to achieve non-toxic deep tissue imaging with cellular precision in living opaque organisms so that researchers can investigate properties emerging from complex biological systems in health and disease.

SUBSTITUTE SHEET (RULE 26) Unfortunately, limitations inherent to optical microscopy (optical penetration depth < 1 mm and phototoxicity) prevent large scale imaging in intact and opaque organisms, and these limitations are more pronounced in instances where high speed (~ 1 ms) is desirable. Despite recent advances, high-speed volumetric LSFM such as SCAPE microscopy does not yet reach 1 mm3s-1 volume rates in living tissue, which makes mesoscale dynamic imaging of living tissue challenging.

The discovery of a “GFP for ultrasound” was a first step towards this goal. The fortuitous physics of ultrasound enables centimeters deep scanning of opaque tissue while genetically encoded acoustic biomolecules interface ultrasound waves with cellular function. To unlock the potential of acoustic reporter genes (ARGs) and biosensors, there is a need for ultrasound imaging methods equivalent to LSFM that can explore in vivo cellular biology with high information content, resolution, coverage, and translatability. Ultrasound has recently gained further appeal for basic biological research with the development of volumetric imaging methods such as functional ultrasound neuroimaging and vascular ultrasound localization microscopy. However, it is currently impossible to visualize specific cell types in 3D or to perform high-resolution acoustic sectioning of specific biological structures within a large field of view.

Here we introduce selective plane nonlinear sound sheet imaging (NSSI) of acoustically labelled organisms to visualize specific cells and physiological signals in wavelength-thin tissue sections. NSSI implements a cross amplitude modulation imaging framework on large field-of-view, high-frequency row-column addressed arrays (RCA). Orthogonally scanned volumetric NSSI expands the field of view of biomolecular ultrasound from ~1 cm2 at 30 frames/s to ~1 cm3 at 300 volumes/s. We demonstrate the versatility of NSSI by performing in vivo acoustic sectioning of the living rat brain vasculature with sub-wavelength precision using intravascular reporters, and fast volumetric in vivo imaging of ARG-expressing cells in subcutaneous breast cancer tumors in mice.

The combination of acoustic probes with fast volumetric cellular imaging methods such as NSSI carries a wave of opportunities for deep tissue imaging of complex biology systems.

Results

Sound-sheet image formation

Figure 1. Sound-sheet imaging leverages row-column array configurations to deliver non-diffractive linear and non-linear imaging in 2D and 3D.

SUBSTITUTE SHEET (RULE 26) a) Row column array configuration or a plurality of transmitters arranged to a transmitter surface, b) Transmit sequence for sound-sheet generation, such as the transmit steps in the method for imaging the medium. Columns transmit simultaneously cross-propagating plane waves from two contiguous half-apertures Nap/2 at a and -a angles. The two transmitted plane waves, the first plane wave and the second plane wave, intersect along a 2D plane referred to as the sound-sheet plane or interference sheet of width dial and depth zcp In the orthogonal plane, this sound-sheet spans the whole width of the array, c) Description of sub-wavelength sound sheet micro scanning in 1 D and two orthogonal directions, d) Simulation of the acoustic pressure field generated by cross-propagating plane waves in lateral and elevation direction, e) Cross amplitude modulation pulse sequence used for nonlinear sound sheet imaging. Transmission with two contiguous apertures elicits a 2A0 in the sound sheet plane whereas transmissions with half apertures elicit an amplitude /10 in the sound sheet plane. Typically, the threshold is selected in the range of AO to 2A0. f) Simulated sound sheet images of four resonant microbubbles in water. The top and bottom row display linear (SSI) and non-linear (NSSI) results respectively. Intensity profiles through the 3rd microbubble are plotted in green, g) 3D (N)SSI XY B-mode images of the 3rd microbubble. The green and orange plots show the intensity taken through the center of the 1st and 3rd microbubble in the X direction. The XZ image is obtained from a single sound sheet position, whereas YZ and XY profiles are acquired via micro scanning along 208 adjacent sound sheet positions with a pitch/2 step, h) Experimental SSI and Orthogonal Plane Wave (OPW) linear imaging of a wire phantom. The first 2 columns are obtained by transmitting on each of the row and column array once (one single transmit per array). Images in the third column are obtained with two compounded angles for SSI and 128 angles for OPW. The fourth column shows zoom-ed in linear images of the middle phantom wire, i) Green and orange intensity profiles are plotted the SSI and OPW linear images respectively, j) Linear 3D SSI reconstruction of the wire phantom encompassing a 1.2cm3 volume.

Columns of a RCA (figure 1 .a) were used to transmit two tilted plane waves with opposite angles. These two plane waves cross-propagate along a plane of thickness d_iat (sound-sheet plane) until depth z_cp =

(figure 2.b). A scatterer contained in this sound-sheet at position (x_s = x_ss,z_s) will backscatter and the pressure received by the orthogonal transducers arranged in columns can be beamformed to determine the position (y_s,z_s). Sound-sheets can be generated by using either contiguous half-apertures or with a silent a middle element in between (figure 1 .c) to SUBSTITUTE SHEET (RULE 26) obtain a fine sound sheet scanning step of ^, p being the pitch of the array. With this approach, sound-sheets can be generated along the two orthogonal directions of the RCA leading to orthogonal volumetric micro scanning.

To get insights into the sound sheet acoustic pressure field, we simulated plane waves intersecting with a 20.7 degree angle using INCS and the resulting pressure field is plotted in figure 1.d). The modulation of the pressure in the soundsheet plane is visible in the lateral direction and shows a non-diffractive beam equivalent to a Bessel beam planar illumination in optics. The main lobe exhibits an elevational width of 131 pm, and reaches its peak intensity of 600 kPa at a depth of z = 1 .56 mm. Additionally, another local intensity peak of 589 kPa is observed at 6.14 mm. The cross-propagation interaction ceases at approximately 8 mm. This transmit can be complemented by two additional transmits using only half of the apertures each time (figure 1 .e) to modulate acoustic pressure in the sound sheet plane.

We simulated 2D Point-Spread Functions (PSF) obtained with Sound-Sheet Imaging of resonant microbubbles using k-Wave (figure 1.f). We report sound sheet images as well as intensity plots taken through the center of the microbubble positioned at depth z = 3 mm. For linear sound sheet imaging (first row), the FWHM at -10dB in the X direction extends 85 pm at z = 3 mm. For nonlinear sound-sheet imaging (second row), the FWHM at -10dB in the X direction extends 84 pm for z = 3 mm. Figure 1 .f images are all displayed with a 70 dB range.

By scanning the sound-sheets in two orthogonal directions and compounding the beamformed data, it is possible to obtain a volume. PSFs resulting from the orthogonal scanning operation are presented in figure 1.g together with intensity plots taken at the center location of the microbubble positioned at depth z = 1 mm and z = 3 mm. FWHM in the X direction spans 73 pm in the linear mode for z = 3 mm. FWHM in the X direction spans 47 pm in the non-linear mode for z = 3 mm, illustrating non-linear confinement. The NSSI PSF side-lobe level is reduced by as much as 17 dBs compared to the SSI case.

A 15 MHz RCA with 128+128 elements (Imasonic, France) was positioned over a phantom embedding 100 pm Nylon diameter wires (Model 055A, CIRS) and two ultrasound pulse sequences were transmitted subsequently, an SSI sequence and an Orthogonal Plane Wave (OPW) sequence. Transmitted pressure levels by each pixel were matched for both sequences. Linear images obtained from a single transmit (7 degree angle for SSI, -19 degree angle for OPW) are compared in figure 1.h). SSI detected all 4 wires whereas OPW did not detect any. To deliver a C-scan view in SSI

SUBSTITUTE SHEET (RULE 26) imaging mode (contained in the XY plane), we scanned sound-sheets with a p itch/2 step across 10.8 mm. For the OPW imaging mode a C-scan view can be obtained with a single transmission per array. The top wire was visible in SSI but not in OPW imaging.

SSI results clearly revealed the presence of sidelobes specific to Bessel beams. By using two successive sound-sheet transmissions at two different angles (7, 13°), the side lobe level of SSI could be lowered as shown in the 3rd column of figure 1 .h. We compared the two-angle SSI image to a best-case scenario for OPW imaging obtained using 128 angled plane waves transmissions along rows and columns (total of 256 transmissions to generate one OPW image), with angles ranging from [-21 ; 21] degrees. Zoomed in images focused on the wire situated in the center of the image are displayed in the last column. The intensity profiles taken at the center of the wires are plotted in figure 1.i. in green for the SSI case and in orange for the OPW case. The measured Full Width at Half Maximum for SSI and OPW were 151 pm and 245 pm respectively. Finally, a 3D rendering obtained via orthogonal sound-sheet micro scanning is displayed in figure 1.j). Four wires are clearly visible in the 1.2 cm3 scanned volume.

Large field of view sound sheet imaging of genetically encoded acoustic reporters

Figure 2. In vitro volumetric sound sheet imaging of genetically encoded acoustic reporters a) Schematic of the phantom configuration. An agar phantom (gray) immerged in phosphate buffered saline (PBS) contains wells filled with agar linearly scattering wild-type gas vesicles (wtGVs) and nonlinearly scattering harmonic gas vesicles hGVs). Top, cross-sectional view of the GV wells. Bottom, long axis view of the GV wells, b) Top, cross sectional SSI image of GVs. Middle, long axis image of GVs acquired with the orthogonal RC array. Bottom, NSSI of GV wells revealing harmonic GVs only, c) 1 .2 cm3 volumetric NSSI image of harmonic GVs. d) Contrast to Noise Ratio (CNR) of the linear (SSI) and non-linear (NSSI) modes.

Next, we report volumetric NSSI of genetically encoded acoustic reporter genes using a 15 MHz RCA (Figure 2). GVs embedded in a phantom were imaged over a sound sheet plane of 12.8 mm x 8 mm along the short-axis and long-axis of the wells. As expected, wells filled agar did not scatter ultrasound (except for specular reflection at agar/agar interfaces) whereas wtGVs and hGVs appeared in the linear SSI mode (Figure 2.b).

SUBSTITUTE SHEET (RULE 26) When switching to NSSI, hGV wells were preferentially detected with high specificity as one would expect from xAM imaging with a 1 D array. Figure 2.c), displays a 1 .2 cm volumetric NSSI image of hGVs acquired by electronically scanning sound sheets in two orthogonal directions (110 sound sheet positions along each array with a step of 55 pm). Volumetric NSSI clearly detected nonlinearly scattering GVs in a large field of view. The Contrast to Noise Ratios were measured for each of the soundsheets. In the SSI mode, linearly scattering wtGVs were detected with 22 dB and 21 dB CNRs for the top and bottom wells respectively and nonlinearly scattering hGVs were detected with 14 dB and 20 dB CNRs for the top and bottom wells respectively. The lower CNR of the upper well might be due to partial collapse or buckling of these hGVs. In the NSSI mode, hGVs were detected with CNRS of 15 dB and 16 dB respectively, whereas wtGVs could not be detected (1 dB CNRs).

Deep tissue volumetric NSSI of acoustic reporter genes in a cancer model Figure 3. In vivo volumetric NSSI of mammalian ARG expressing tumors in a mouse model a) Protocol for mARGAna expression in orthopic tumors. Schematic of MDA- MB-231 -mARGAna engineering. Mice were injected bilaterally in the 4th mammary fat pads with engineered MDA-MB-231 -mARGAna human breast adenocarcinoma cells on day 0. mARGAna expression was induced by regular intraperitoneal doxycycline injections starting from the day of tumor injections. Tumors were imaged with ultrasound (US) after 4 and 8 days of expression. After 4 days, the necrotic core develops and is clearly visible at day 8. b) SSI of mARGs in 4- and 8-days old tumors. Left, linear structural SSI of tumors at day 4 and 8Middle, NSSI revealing spatial mARG expression in tumors. Right, NSSI overlaid structural SSI in C-scan orientation (XY plane). Scalebars are 1 mm. c) Representative 3D reconstruction of an 8-days old tumor with 55 m orthogonal scanning step, revealing the volume of the tumor and its necrotic core. The colored NSSI volume is overlaid on the grayscale linear SSI volume, d) Results from automatic segmentation of the tumor and necrotic core for N=5 mice. Light gray plots the tumor volume without the necrotic core, dark gray plots the tumor volume including the necrotic core. Asterisk show statistical significance using multiple paired t- test (P values from left to right: 0.1628, 0.1871 , 0.4125,0.0030, 0.0075) (**P < 0.01 ). Below the graph are representative sound sheet slices of the tumors for each mouse. Scalebars indicate 1 mm. Right, automatic segmentation result at day 8. Scalebar is 1 mm.

SUBSTITUTE SHEET (RULE 26) To test in vivo capabilities of NSSI, we imaged mammalian acoustic reporter gene expression in a mouse model. Orthotopic tumors were induced by injecting MDA- MB-231-mARGAna cells bilaterally in the 4th mammary fat pads of female immunocompromised mice, following a methodology published by Hurt, Buss, Duan et al. (figure 3. a). Mice were induced with doxycycline every day until day 4. We imaged tumors of 3 mice at day 4 and of 2 mice at day 8 of daily doxycycline induction. Representative images are displayed in figure 3.b) for these two groups. Linear SSI reveals anatomical tissue structures irrespective of the cellular content of tissue as would conventional ultrasound do. Different interfaces are visible such as the skin, tumor core, and fat. The tumor clearly appears in these structural images as an abnormal tissue mass. The contrast within the tumor appears slightly less echogenic as in previous reports. In the NSSI mode, distinct information revealing the organization of mARG-expressing cancer cells in space is revealed. In the 8 days old mice, NSSI clearly reveals contrast confined to the tumor area as well as the absence of gene expression in the hypoxic necrotic core of the tumor. No nonlinear propagation artifacts were visible in the core of the tumor highlighting the specificity of NSSI. NSSI microscanning in the two orthogonal directions enables C-scan views of mARG expression (right column of figure 3.b). Tumors were clearly detected at both stages, NSSI did not reveal necrotic cores at day 4 but a homogeneous nonlinear mARG contrast instead. NSSI was displayed with a 20dB and 18dB range in the 4 days and 8 days old cases respectively. A volumetric image fusing SSI and NSSI modes is presented in figure 3.c) and constitutes to our knowledge the first volumetric ultrasound image of gene expression The total volume scanned was 9 x 8 x 8 mm with scanning steps of 55 m. The right column of figure 3.c reveals cross sectional views in XZ, YZ and XY planes to showcase navigation through the tumor volume. We can see that the two orthogonal scans map almost the entire footprint of the probe.

Next, we used NSSI to quantify tumor and necrotic core volumes with an automated segmentation tool. For the 4-days old group, the volume measure with and without necrotic core is consistent and shows no significant difference in its average value as expected from images. For the 8-days old group, the volume without the necrotic core was statistically significant in both cases with a P-value below 0.01 using a multiple paired t-test. NSSI was specific enough to allow quantification of necrosis of the tumor environment and tumor volume measurement. Representative NSSI images of the tumors are shown below the bar plot showing the average volumes measured. In the last column, we show a representative NSSI image of an 8-days old tumor overlaid

SUBSTITUTE SHEET (RULE 26) with the result of the segmentation algorithm used for quantification outlined with a solid white line.

Selective-plane ultrasound NSSI of cerebrovascular flow

Figure 4. Selective-plane ultrafast NSSI of flow dynamics in the rat brain, a) Experimental setup for NSSI of flow dynamics in the rat brain, b) Ultrasound insonification scheme for multi slice NSSI in consecutive cardiac cycles. Two sound-sheet positions are arbitrarily set at positions as far as d = 3.9 mm, with a pulse repetition frequency of 37 kHz that enables ultrafast Doppler imaging in two sound sheet planes, c) Ultrafast Doppler images of the cerebral vasculature generated of each individual pulse of the NSSI sequence, and NSS Doppler image resulting from the cross amplitude modulation operation. Scalebars are 1 mm. d) NSS Doppler images from three adjacent sound-sheet planes spaced by 55 / rn. Structure Similarity Index Matrix calculated from the 3 adjacent frames. Boxplot of the Structure Similarity Index Matrix per set. Asterisk show statistical significance using t-test of two independent samples (P values are all inferior to 10-21) (***P < 0.001 , ****P < 0.0001 ). Average SSIM in between Doppler frames of different sets is significantly different to average SSIM of Doppler frames in the same set. e) Frequency filtering of NSSI Doppler frames with 4 different filters. From left to right, low-pass filter showing the noise floor, bandpass filter showing blood perfusion (slow flows), high pass filter revealing intermediate flow velocities and high-pass filter showing fast flow velocities.

We demonstrate the versatility of NSSI by performing selective-plane ultrafast nonlinear ultrasound imaging of the rat brain vasculature using lipid shelled microbubbles administered intravenously in the blood stream. The schematic of the experimental setup is displayed in figure 3. a). A tail vein catheter was placed to inject a commercial ultrasound contrast agent and acoustically label the rodent vasculature. Here the NSSI sequence was modified to image two arbitrarily selected planes in the brain with a frame-rate as high as 3200 Hz, and with a plane separation of up to 6.7 mm (figure 3. b). From the 3-pulse sequence shown in Figure 1 .e 4 images can be reconstructed using each pulse of the sequence (i.e. using the left, right, both apertures, or the amplitude modulation operation). Ultrasound data for individual transmissions of the sequence (left aperture, right aperture and SSI) were processed using a singular value decomposition filter to separate blood echoes from tissue clutter and summed into a power Doppler image. The NSSI Doppler image (lower right image of figure panel 3.c) was generated by applying the cross amplitude modulation sequence to separate microbubble echoes from tissue clutter, and a temporal filter was applied to remove

SUBSTITUTE SHEET (RULE 26) static echoes (6th order Butterworth low-pass, band-pass or high-pass filter). Top row Doppler images of figure 3.c that were obtained from the left and right aperture display oblique vascular projections through the brain with opposite angles. Several vessels are common to both frames but a significant amount is not. The Doppler image formed by the x-wave (SSI transmit with both apertures) insonifies vessels with the pressure field present in figure 1 .d. The image reveals preferentially vessels in the sound sheet plane but is not completely free of vascular information arising from the oblique projections. The NSSI Doppler image relying on the nonlinear echoes of circulating microbubbles selectively reveals the vasculature confined in the sound sheet plane (sound sheet dimensions = 100 pm x 9.6 mm x 8.8 mm), as the NSSI sequence only modulates acoustic pressure along the main lobe of the Bessel beam. The NSSI vascular image in figure 3.c is reveals less vessels as this is a wavelength thin acoustic section of the brain, and artifacts arising from oblique transmissions that are visible in the cortex for the SSI case have now disappeared.

To highlight the relevance of sectioning the vasculature with sub-wavelength steps, we imaged 3 adjacent sound-sheet planes separated by 55 /zm. Four Doppler images were acquired for each sound sheet position. Representative Doppler frames are displayed in the top row of figure 3.d). Visually, one can see that vascular structures are largely overlapping but fine scale vascular features are changing. To quantify vascular changes in these adjacent planes, we calculated Structural Similarity Index Matrix values for each Doppler frame in the set. Intra-sets, the structural similarity index averages 0.99, but inter-sets, this index drops to 0.96. The averages of this index intra sets are significantly different statistically of the index inter-sets as shown in the box plot, confirming that Doppler frames rendered from NSSI sound-sheets spaced by the sub-wavelength scanning step of 55 /zm are different. This is on par with the subwavelength elevation resolution observed in the PSF simulated in figure 1 .f) in the nonlinear mode.

Last, we filtered the NSSI Doppler frames with 4 different filters to reveal various compartments of thin NSSI vascular brain sections based on their flow dynamics. By using a low-pass filter below 96 Hz, we mostly retained noise and/or quasi-static microbubble echoes as shown in the left Doppler frame of figure 4.e). Using a band-pass filter in between 96 Hz and 250 Hz, we revealed slow blood perfusion (second image of figure 4.e). A high-pass filter above 192 Hz captured both blood perfusion and higher flow velocities (third image of figure 4.e), whereas a high-pass

SUBSTITUTE SHEET (RULE 26) filter above 250 Hz mostly contained vessels resolved by the PSF of NSSI that exhibit fastest flow velocities (see last image of figure 4.e).

Discussion

We report NSSI, a method for the nonlinear confinement along thin sound sheets of echoes arising from specific living tissue compartments labelled with acoustic reporters. The study introduces the use of two dimensional non-diffractive beams for molecular ultrasound imaging. We show that NSSI is compatible with the two major classes of nonlinear acoustic probes for biological specimen i.e. synthetic lipid-shelled microbubbles used to label the vasculature and genetically encoded acoustic biomolecules used to label specific cell populations of interest.

NSSI achieves 3.2 kHz 2D imaging along thin sound sheets of 12,800 x 8,400 x 90 pm) that can be arbitrarily positioned within the large 3D field-of-view of RC arrays. We demonstrate this imaging approach in Figure 4 by performing selective insonification of whole vascular planes in living brains using vascular reporters. NSSI was successfully used to acquire deep acoustic tissue sections with sub-wavelength step precision of 55 pm.

Orthogonal NSSI micro scanning enabled volumetric imaging at 300 VPS imaging over a 1 .2 cm3 fields of view (12,000 x 10,000 x 10,000 pm as shown in Figures 1 .i and 2.d) with a nearly isotropic PSF of 100 x 100 x 100 pm (Fig 1 .g). We demonstrate this approach in Figure 3 by performing volumetric sound sheet image of ARG-expressing cancer cells in subcutaneous breast cancer tumor in mouse. This is to our knowledge the first volumetric ultrasound visualization of gene expression in a specific cell population.

Compared to conventional 2D ultrasound imaging with 1 D transducer arrays, NSSI has the tremendous advantage of generating a non-diffractive beams and therefore a constant lateral resolution of ~1A. In comparison, a 1 D array of the same frequency provide an elevational resolution > 5A.

Compared with LSFM, NSSI generates sound sheets with dimensions equal to 1A x 128 A x 100 A that can be electronically scanned with a half pitch precision in two orthogonal directions. NSSI operates with a single imaging array used for both ultrasound transmission and reception and is therefore as ergonomic as single objective approaches in optics. As current RCA arrays have the footprint of a handled clinical ultrasound probe, NSSI could potentially be developed clinically using commercially approved marked acoustic reporters.

SUBSTITUTE SHEET (RULE 26) NSSI successfully expands the capabilities of xAM imaging by providing faster, larger, similarly specific imaging of all classes of nonlinear acoustic reporters at our disposal. Previous xAM implementations can only form a single image line per ultrasound transmission. Because the transmission of cross-propagating plane waves requires two contiguous apertures of size Dap/2, only half the elements of a 1 D array with Vtotal elements are typically active (^active = Vtotal - 2 * £)ap/2), meaning that xAM images are only as wide as half of the array. The sound-sheet approach using RCA maximizes the field of view because the orthogonal array is used to digitized echoes backscattered by the sound-sheet transmission. Resulting images from one single xAM transmit span the full width of the RCA. With orthogonal NSSI volumetric scanning, sound-sheets transmissions in one direction are compounded with soundsheet transmissions from the orthogonal direction will form a crossed pattern visible in the C-scan of figure 3.b. Note that we further expanded imaging by using smaller apertures on the side of the array in combination with smaller angles to maintain imaging depth. In the end, the volume of view available from 3D SSI is only 3.9% smaller than that of conventional 3D imaging modes with a RCA, whereas the 2D field of view of xAM is 50% smaller than that of a conventional plane wave imaging mode.

A limitation of linear SSI arises from high sidelobe levels intrinsic to Bessel beams. Future work will investigate methods to reduce sidelobe level. In this work, we show that angular compounded is one potential solution that yields good results in vitro (see figure 1.h). Unlike SSI, NSSI successfully mitigates sidelobe level through nonlinear confinement along the sound sheet plane. The kHz frame-rate achieved by SSI and NSSI is similar to that of 2D ultrafast ultrasound. However, the ability to selectively image planes of interest spaced by as little as a half wavelength to as much as 7 millimeters is inaccessible with other ultrasound imaging methods. A drawback of 2D arrays used for 3D imaging is the amount of data generated by such approaches, which is known to cause significant problems in both data acquisition, transfer and processing times. NSSI allows researchers to acquire data in arbitrarily selected 2D planes within the large field of view of RC arrays, hence reducing data size compared to a full 3D acquisition. Imaging planes of interest can be locked electronically without physically moving the probe, which is the advantage compared to mechanical actuation of 1 D ultrasound probes.

The combination of NSSI with mARGs allowed to quantify the progression of tumor growth in a mouse model. Not only could we measure the size of the tumor at different stages, but the high specificity of NSSI to nonlinear mARG signals reduced

SUBSTITUTE SHEET (RULE 26) nonlinear propagation artifacts enough to measure the volume of necrotic cores. AM implementations that do not rely on cross-propagation would not enable tumor core volume quantification as they tend to misclassify tissue as nonlinear scatterers.

To summarize, we introduce NSSI, an ultrasound imaging method that might seem equivalent to LSFM at first face, that further enables selective plane and volumetric imaging of acoustically-labelled structures in mammalian tissues. NSSI achieves sub-wavelength resolution at kHz framerates and revealed the first volumetric ultrasound images of gene expression. We anticipate that NSSI will provide novel insights into previously inaccessible biological processes arising from organs in space and time. In the future, the combination of NSSI with next generation acoustic reporter genes and biosensors will unlock dynamic deep tissue imaging in living opaque organisms.

Methods

Sound-sheet generation and focus restoration:

Rows (and alternatively columns) were used to transmit simultaneous crosspropagating plane waves from two contiguous half-apertures Dap/2 at angles a and -a (see figure 1 .b)). The two transmitted plane waves span the whole elevation width of the RCA probe and intersect along a 2D plane, referred to as the sound-sheet. A double amplitude planar wave-front is generated at the plane waves intersection and propagates across the sound-sheet plane at a supersonic velocity for a

propagation depth z_cp = y cot (a). Image reconstruction of the echoes received by the columns of the RCA probe relies on delay-and-sum beamforming with the assumption that backscattering only arises from the sound-sheet plane. The receive volume spans the entire transducer field of view. Due to the containment of the transmitted field along the column direction (or alternatively row direction), and the beamforming using the orthogonal row array (or alternatively column array), the position in (x_s,y_s,z_s) of scatterers is retrieved. The focus of the RCA is restored in both dimensions through this transmit focusing.

Sub-pitch scanning and 3D imaging:

There are two ways to generate a sound-sheet from two apertures (see figure 1 .c)). The original method used two apertures of n elements separated by an inactive element. This focuses the cross-propagation plane at a position x_ss = x_p = np. We present here the generation of sound-sheets with two contiguous apertures with n elements. This focuses the cross-propagation plane at a position x_ss = x_p/2 =

SUBSTITUTE SHEET (RULE 26) (n - l)p + Since the sound-sheet is a non-diffractive beam we can consider that these two generated sound-sheets capture different backscattered pressure amplitudes from echoes p/2 apart. The thickness of the sound-sheet was estimated from the INCS simulation and the Full Width at Half Maximum of the main beam can be approximated with the following relation:

F WHM_SS = sin(a)

The FWHM is smaller than the wavelength for any angle a > 18.66°.

By scanning the sound-sheets in both directions, it is possible to deliver 3D imaging from SSI. The resulting volume has an isotropic PSF in both elevational and lateral directions that can be smaller than the wavelength depending on the angle. It is also possible to deliver 3D imaging by scanning in only one direction resulting in a non- isotropic resolution. Since sound-sheets can only be created using two half-apertures, the field of view is reduced compared to the transducers footprint: FoV = N_totca x p - 2 x Nap. The size of a sound-sheet is: z_cp x FWHM_SS x w_array, w_array = NtotaiP the width of the RCA array, and so the size of a scan in one direction is: z_cp ^x _SSFWHM_SS x w_array

By using varying aperture sizes, it is possible to extend the field of view further, but then the propagation depth z_cp is reduced. Two mechanisms can be put in place to mitigate this issue: firstly, the angle can be decreased to ensure that the propagation depth stays the same, secondly, since the sound-sheets span the entire width of the array, scanning with orthogonal sound-sheets will restore the field of view at least for the center of the array. Should the minimum half-aperture used to create sound-sheet be Nredp , the total field of view covered by a scan in one direction is:

and for a 3D scan from two orthogonal direction, this becomes:

Compared to the field of view covered by the footprint of the probe, this is a reduction of:

In our case, we use a reduced aperture of as little as 12 elements, leading to a loss of only 3.5% of the total volume.

Amplitude modulation for Non-linear Sound-Sheet Imaging:

SUBSTITUTE SHEET (RULE 26) For the non-linear mode, the same half-apertures are used for two additional transmits, each firing independently this time. Since no cross-propagation takes place in each of the cases, the pressure wave propagating through the medium has an amplitude two times lower than that of the sound-sheet. An amplitude-modulated signal can thus be obtained by subtracting the two received signals from the half-aperture transmits to the signal received from the sound-sheets. This operation is done on the radiofrequency data and the result is then beamformed and filtered for additional processing. A 3D amplitude modulated image can also be captured by sweeping these 3-pulse transmissions along the two directions of the array.

Beamforming of a sound-sheet transmit:

To beamform a sound-sheet, an essential assumption is made: the backscattering echoes are considered to come only from the sound-sheet plane. If the sound-sheet plane is oriented with its normal along the x direction, then all scatterers can be assumed to have the same y coordinate. The forward delay for a scatterer at a position

can be written:

(x_s,y_s, _Zs) = ^- = z_s cos(a) /c c ₀ ss

With c_ss the supersonic speed of the sound-sheet and cO the speed of sound in the medium.

The return delay for a scatterer at a position

is calculated for the array orthogonal to the transmitting array to restore focusing and is written:

It is important to notice that t_f only depends on z_s, x_s while t_r only depends on z_s,y_s.

INCS simulation:

To study the sound-sheet propagation, we employed the Iterative Nonlinear Contrast Source (INCS) method as our computational simulation tool. Originally, INCS was devised for the purpose of solving the four-dimensional spatiotemporal Westervelt equation. The incident field is generated through the convolution of the primary source term with the Green's function corresponding to the linear background medium. In this specific context, the primary source term is characterized by the volume injection rate density, and it is mathematically represented as a velocity discontinuity condition at the transducer plane located at z = 0 mm. The simulations were conducted within a computational domain spanning X x y x z = 6.5 x 13 x .5 mm. The analysed medium

SUBSTITUTE SHEET (RULE 26) corresponds to water and is characterized by a mass density p₀ = 1060kg.m~³ and a speed of sound c₀ = 1482 m. s^-1. The incident beam has a center frequency f_c = 15 MHz. The RCA array contains 64 individual elements, each with length 12.8 mm, and a pitch of 100 pm. The time-varying pressure at the surface of the elements is given by the expression:

where we have chosen T_w = — , which represents the duration of the fo

Gaussian envelope, and T_d = — I- A_n which is a total time delay. The latter consists of a fo fixed delay for keeping p(t=O)~O, plus a delay per element for the beam steering. The time delays are generated due to a 20.7° chosen transmission angle for the planewaves. The maximum surface pressure of the elements is P_Q = 400 kPa. A sampling frequency of 90 MHz has been used to discretize the spatiotemporal domain. k-Wave simulation:

We simulated the response of 4 microbubbles positioned at z = 1 ; 2; 3 and 4 mm in the centre of a domain containing water with no inhomogeneity using the k-Wave simulation toolbox. Each of the microbubbles are 1 .5 pm in diameter making them resonant at the central transmit frequency of 15.625 MHz. The geometry of the transducer consists of 43 elements with a height of 4.3 mm, a width of 100 pm and a pitch of 100 pm, and a bandwidth of [14 - 22] MHz. 22 sound-sheets were created with a scanning step of 50 pm from plane waves with a 21 ° angle and a transmit pulse of a Gaussian envelope sine-burst of 4 cycles and a maximum pressure amplitude of 400 kPa. The backscattered amplitude is calculated using a Gaussian Quadro RTX 6000 GPU (24 Gb memory) and an Intel ® Xeon ® Gold 5218 CPU. The radiofrequency data is then beamformed using a Delay And Sum algorithm and the resulting beamformed IQs are compounded to deliver a scan of the microbubble responses. To deliver 3D imaging, it is considered that the probe is entirely symmetrical and the beamformed IQs from the orthogonal array are calculated by permuting the initial IQs.

Wire phantom imaging:

A 15.625 MHz central frequency RCA probe with 128+128 elements (manufactured by Imasonic) was placed over a 3D wire phantom (model 055A from CIRS, VA, USA). The wires were oriented at an angle from the orientation of the rows and columns elements. Sound-sheets were created using a 7° and in the case of two angles, the combination chosen was [7, 13]°. For orthogonal plane wave compounding,

SUBSTITUTE SHEET (RULE 26) an angle of -19 degrees was used for the single transmit and for the 128 transmits, the angles ranged from [-21 ,21] degrees. A delay-and sum beamforming algorithm was used in both cases and the beamformed IQs are displayed in figure 1 . The 3D rendering is obtained using the Avizo rendering software (ThermoFisher ®).

GV phantom:

Anabaena flos aquae GVs were cultured and transferred to sterile separating funnels. Buoyant cells were separated from the growth media through natural flotation, and GVs were harvested after 48h of hypertonic lysis. A cycle of centrifugation and resuspension allows to purify the GVs further. A stock of wild-type GVs (wtGVs) was stripped of their GvpC protein layer with a 6-M urea solution to obtain GVs that scatter higher harmonics (hGVs). These two GV variants were either stored in phosphate buffered saline (PBS) or clustered to end up with 4 different imaging samples: wtGVs, aggregated wtGVs (wtGVs+), hGVs and aggregated hGVs (hGVs+). A 2% agar phantom comprising 2 mm in diameter wells was casted using custom-printed molds and imprints. Wells were filled with agar (control) or agar mixed with one of the 4 different GV variants prepared. The final GV concentration was measured optically at OD2.5 (285 ppMM). The GV phantom images are obtained using a 15.625 MHz central frequency RCA probe with 128+128 elements (manufactured by Imasonic).

Tumor imaging:

All in vivo experiments were performed under protocol 1761 , approved by the Institutional Animal Care and Use of Committee of the California Institute of Technology. Animals were housed in a facility maintained at 71-75 °F and 30-70% humidity, with a lighting cycle of 13 hours on and 11 hours off (light cycle 6:00-19:00). Tumor xenograft experiments were conducted in NSG mice aged 12 weeks and 6 days (Jackson Laboratory). To implement an orthotopic model of breast cancer, all the mice were female. MDA-MB-231-mARGAna cells were grown in T225 flasks in DMEM supplemented with 10% TET-free FBS and penicillin-streptomycin until confluency as described above. Cells were harvested by trypsinization with 6 ml of trypsin/EDTA for 6 minutes and quenched with fresh media. Cells were washed once in DMEM without antibiotics or FBS before pelleting by centrifugation at 300xg. Cell pellets were resuspended in a 1 :1 mixture of ice-cold Matrigel (HC, GFR) (Coming, 354263) and PBS (Ca2+, Mg2+-free) at 30 million cells per milliliter. Then, 50-pl Matrigel suspensions were injected bilaterally into the 4th mammary fat pads at 1 .5 million cells per tumor via subcutaneous injection. Twelve hours after tumor injection and every 12 hours thereafter (except the mornings of ultrasound imaging sessions), test mice

SUBSTITUTE SHEET (RULE 26) were intraperitoneally injected with 150 pl of saline containing 150 pg of doxycycline for induction of GV expression.

A 15.625 MHz RCA probe with 80+80 elements with a 110 /zm pitch (Verasonics®, WA, USA) was used to transmit 110 sound-sheets with [15; 21] degrees angles with a 1 degree step. The data collected is beamformed using a delay-and-sum algorithm and is displayed in figure 3. All 3D renderings are obtained using the Avizo rendering software (ThermoFisher ®). To segment and measure the tumor and hypoxic core, the data is first 3D-Gaussian filtered (standard deviation a = 0.6) and interpolated twice in each direction. The data is then normalized and log-compressed and tissue attenuation is taken into account with an average tissue attenuation factor of 0.54 dB X MHz-1 X cm-1 . Attenuation is further corrected to aim for a uniform noise contrast value through depth of the image, an additional custom Gaussian filtered is added prebinarization to yield better results. The open volume result is then closed and measured using the regionprop function in Matlab. This is done for each of the volumes per acquisition obtained in the two directions of scanning and in the 3D compounded volume. This allows to calculate an average value for the volume of the tumor and hypoxic core from 3 measurements.

Cerebrovascular flow imaging:

The RCA probe was then used to image the vascular function of a rat brain (Sprague Dawley, female, 280g). All experiments were performed under CCD license number AVD8010020209725 at the Koninklijke Nederlandse Akademie van Wetenschappen with Study Dossier number 213601 . Immediately after Isoflurane induction, carprofen, and butorphanol are delivered subcutaneously. The animal is then prepared (shaved, disinfected, placed in earbars etc.) and the surgery begins no sooner than 20 minutes after the injections. A catheter is placed in the tail vein. Heparin is injected to prevent blood clots forming in the catheter. The animals then undergoes 14x14 mm craniotomy surgery. Carprofen and butorphanol are delivered subcutaneously during surgery (respectively 5mg/kg and 2mg/kg). To prevent cerebral edema, Dexamethasone is given subcutaneously with a dosage of 2.5mg/kg.

After craniotomy, the probe (80+80 elements with a 110 /zm pitch (Verasonics®, WA, USA)) was placed over the anesthetized animal. A Sound-Sheet was generated using a 18° angle and a 2 cycle transmit at 15.625MHz central frequency. 38 elements were used for each half apertures. 2100 frames are collected with a framerate of 3200Hz, leading to a 700ms long acquisition. The average heart rate throughout the experiment is 419 bpm, leading to a cardiac cycle of 143 ms which is

SUBSTITUTE SHEET (RULE 26) small enough to be covered several times with our acquisition. 1 .0 x 108 microbubbles (Micromarker® Fujifilm, Bracco) were injected in a bolus through a tail vein catheter.

The data was beamformed using a Delay-and-Sum algorithm. Beamformed IQs are filtered with a 6th order Butterworth low-pass, band-pass or high-pass filter. The Power Doppler is then calculated for each pixel and the 4th order root is displayed in figure 4.d) and e).

Figure 5 schematically shows transmissions of planes waves. Figure 5a, 5b and 5c schematically shows the same cross-section of the transmitter surface 110 with the same set of the plurality of transmitters 120. Figure 5b shows the transmission of the first plane wave. Figure 5c shows the transmission of the second plane wave. Figure 5a shows the transmission of the first plane wave and the second plane wave together, such as at the same time or with a time difference such that these plane waves still interact or interfere. Introducing a time difference between the transmission of the two plane waves causes the interference sheet to be positioned at another location or in another volume compared to having no time difference in the transmission of the two plane waves. Further adapting the time difference advantageously provides to position at another location or in another volume the interference sheet. Below the copies of the imaging device are graphs shown schematically showing the amplitude of the respective transmitted plane wave. Furthermore, the maximum amplitude of the plane wave is shown as A0. A threshold may be positioned in the range of A0 to two times A0. Figures 5b and 5c show that the amplitude of the first and second plane waves does not exceed A0. Figure 5a shows that the amplitude of the first and second plane wave together at the location where these plane waves interact or interfere have an amplitude of two times A0.

Figure 6 schematically shows transmissions of planes waves. Figure 6a, 6b and 6c schematically shows the same cross-section of the transmitter surface with the same set of the plurality of transmitters, similar to figures 5a, 5b, and 5c, respectively. The medium 10 is shown below the transmitter surface 110. Figure 6a, 6b and 6c further show a circle showing the part of the medium to be imaged. Figure 6a and 6d show the place of the interference sheet 240 in medium.

Figure 6b shows with dots in the medium how the first plane wave propagates through the medium. Figure 6c shows with dots in the medium where the second plane wave propagates through the medium. Figure 6a shows with dots the propagation of the first and second plane waves. Further, figure 6a shows the area within the circle where the two plane waves interact or interfere with horizontal bars.

SUBSTITUTE SHEET (RULE 26) The volume of the medium within the circle comprising the horizontal bars is positionable, typically in a lateral or horizontal direction, by timing the transmission of the first and second plane waves and/or selecting the transmitters involved in the transmission of the plane waves.

Figure 7 schematically shows orthogonal interference sheets. Figure 7a schematically shows an imaging device 100. The imaging device comprises a transmission surface 110. The plane waves are not shown. Further shows are the interference sheet 240 and the second interference sheet 241 . The interference sheets are shown orthogonal, but may also be under any other angle.

Figure 7b schematically shows a cross-section of the transmitter surface with a set of transmitters of the plurality of transmitters. Further shown is the second interference sheet 241 and a cross-section of the interference sheet 240.

Figure 7c schematically shows a cross-section of the transmitter surface with another set of transmitters of the plurality of transmitters. Further shown is the interference sheet 240 and a cross-section of the second interference sheet 241 .

Figure 8 schematically shows an extended field of view for a transmitter surface according to the invention. Figure 8a schematically shows a cross-section of a transmission surface 110. Figure 8a further shows an interference sheet 240 an interference sheet with a second angle, and an interference sheet with a third angle. Figure 8a further shows the timing of the first and second plane waves with dotted lines above the transmitter surface. The dotted lines progress down over time. When a part of the dotted line coincides with a transmitter, the transmitter is activated. The triangular dotted line 230 generates the interference sheet 240 with a first angle. The triangular dotted line 235 generates the interference sheet 245 with a second angle. The triangular dotted line 236 generates the interference sheet 246 with a third angle. The angle is defined as the angle between normal vectors of the different sections of the dotted lines forming the triangle. Towards the edge, the angle becomes less. More specifically, the third angle is less compared to the second angle, and the second angle is less compared to the first angle. What can also be seen in Figure 4a is that the triangle with the third angle is less wide compared to the second angle.

Figure 8b shows the exchange of the angle and the aperture. The aperture is the width of the set of transmitters. The width of the set of transmitters activated for transmitting the plane wave is less when the location of the interference sheet approaches the edge of the transmitter surface. The width is shown as the dashed line in Figure 8b.

SUBSTITUTE SHEET (RULE 26) For graphical purposes the angle shown is the transmit angle. The transmit angle is the angle between the normal vector of the dotted line and the transmit surface. The transmit angle is shown as the dotted line in Figure 8b.

Horizontal is the location of the interference sheet. At the edge of the transmitter surface the transmit angle is exchanged for width of the set of transmitters. Further shown in figure 8b is the depth of the interference sheet as a solid line. The exchange between transmitter angle and the width of the set of transmitters results in interference sheets having the same depth over the whole image area.

Further, when looking back to figure 8a, the imaged area 20 of the medium is typically restricted to where the width and angle are kept constant. By exchanging the transmit angle and the width, additional parts 21 , 22 of the medium can be imaged. The additional parts of the medium imaged provide more medium imaged with a relative small transmitter surface.

Figure 9 schematically shows an embodiment of a computer program product 1000, computer readable medium 1010 and/or non-transitory computer readable storage medium according to the invention comprising computer readable code 1020. The compounding system typically comprises a controller arranged for executing one or more of the methods as specified throughout the description and claims as typically coded in software.

Figure 10a-b schematically show transmissions of planes waves. Both figures show transmissions of the first and second plane waves for creating an interference sheet in the medium 240. Figure 10a shows a timing with no delay between the transmission of the first and the second plane waves. Figure 10b shows a timing with a time delay or time shift for the transmission of the first plane wave relative to the transmission of the second plane wave. The time delay causes the interference sheet to be repositioned relative to the dotted line, wherein the dotted line indicates the position of the interference sheet when both plane waves are transmitted without delay relative to each other. In this example, the interference sheet in Figure 10b is laterally shifted compared to the interference sheet in Figure 10a. It is noted that the lateral shift is more than the distance between transmitters. The lateral shift is typically during experiments or implementations smaller than the distance between transmitters allowing for interpolating or positioning of the interference sheet between transmitters.

Figure 11a-b schematically show transmissions of planes waves without the medium. The figures 11 a-b provide detail of the transmissions also shown in Figure 10a-b, respectively.

SUBSTITUTE SHEET (RULE 26) The lateral shift Ax and time delay T are related though:

Ax = T - —

2 tan(0) , wherein c is the propagation speed of the plane wave through the medium, and 9 the angel of the plane wave relative to the transmitter surface. In the example the angle 6 is selected at 16°.

Figure 12 schematically shows a method 200 according to the invention.

The method comprises providing 201 a plurality of transmitters arranged to a transmitter surface. The method comprises selecting 202 a first set of transmitters of the plurality of transmitters for transmitting a first plane wave into the medium. The method comprises selecting 203 a second set of transmitters of the plurality of transmitters for transmitting a second plane wave into the medium. The method comprises transmitting 204 a first plane wave and a second plane wave for creating an interference sheet in the medium. The method may be considered comprising the steps described above. The steps may be performed in a different order, repeatedly, and/or in parallel if the dependency between the steps allow this.

It will also be clear that the above description and drawings are included to illustrate some embodiments of the invention, and not to limit the scope of protection. Starting from this disclosure, many more embodiments will be evident to a skilled person without departing from the scope of the invention as set forth in the appended claims. These embodiments are within the scope of protection and the essence of this invention and are obvious combinations of prior art techniques and the disclosure of this patent. Devices functionally forming separate devices may be integrated in a single physical device.

The term “substantially” herein, such as in “substantially all emission” or in “substantially consists”, will be understood by the person skilled in the art. The term “substantially” may also include embodiments with “entirely”, “completely”, “all”, etc. Hence, in embodiments the adjective substantially may also be removed. Where applicable, the term “substantially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. The term “comprise” also includes embodiments wherein the term “comprises” means “consists of”.

The term "functionally" will be understood by, and be clear to, a person skilled in the art. The term “substantially” as well as “functionally” may also include embodiments with “entirely”, “completely”, “all”, etc. Hence, in embodiments the adjective functionally may also be removed. When used, for instance in “functionally

SUBSTITUTE SHEET (RULE 26) parallel”, a skilled person will understand that the adjective “functionally” includes the term substantially as explained above. Functionally in particular is to be understood to include a configuration of features that allows these features to function as if the adjective “functionally” was not present. The term “functionally” is intended to cover variations in the feature to which it refers, and which variations are such that in the functional use of the feature, possibly in combination with other features it relates to in the invention, that combination of features is able to operate or function. For instance, if an antenna is functionally coupled or functionally connected to a communication device, received electromagnetic signals that are receives by the antenna can be used by the communication device. The word “functionally” as for instance used in “functionally parallel” is used to cover exactly parallel, but also the embodiments that are covered by the word “substantially” explained above. For instance, “functionally parallel” relates to embodiments that in operation function as if the parts are for instance parallel. This covers embodiments for which it is clear to a skilled person that it operates within its intended field of use as if it were parallel.

Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. Thus, these terms are not necessarily intended to indicate temporal or other prioritization of such elements.

The devices or apparatus herein are amongst others described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation or devices in operation.

It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims. In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. Use of the verb "to comprise" and “to include”, and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. Also, the use of introductory phrases such as “at least one” and “one or more” in the claims should not be construed to imply that the introduction of another claim element by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim element to inventions containing only one such element, even when the same

SUBSTITUTE SHEET (RULE 26) claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an." The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.

The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In the device or apparatus claims enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

The invention further applies to an apparatus or device comprising one or more of the characterising features described in the description and/or shown in the attached drawings. The invention further pertains to a method or process comprising one or more of the characterising features described in the description and/or shown in the attached drawings.

It will be appreciated that the invention also applies to computer programs, particularly computer programs on or in a carrier, adapted to put the invention into practice. The program may be in the form of a source code, a code intermediate source and an object code such as in a partially compiled form, or in any other form suitable for use in the implementation of the method according to the invention. It will also be appreciated that such a program may have many different architectural designs. For example, a program code implementing the functionality of the method or system according to the invention may be sub-divided into one or more sub-routines. Many different ways of distributing the functionality among these sub-routines will be apparent to the skilled person. The sub-routines may be stored together in one executable file to form a self-contained program. Such an executable file may comprise computerexecutable instructions, for example, processor instructions and/or interpreter instructions (e.g. Java interpreter instructions). Alternatively, one or more or all of the sub-routines may be stored in at least one external library file and linked with a main program either statically or dynamically, e.g. at run-time. The main program contains at least one call to at least one of the sub-routines. The sub-routines may also comprise function calls to each other. An embodiment relating to a computer program product comprises computer-executable instructions corresponding to each processing stage of at least one of the methods set forth herein. These instructions may be sub-divided into sub-routines and/or stored in one or more files that may be linked statically or dynamically. Another embodiment relating to a computer program product comprises

SUBSTITUTE SHEET (RULE 26) computer-executable instructions corresponding to each means of at least one of the systems and/or products set forth herein. These instructions may be sub-divided into sub-routines and/or stored in one or more files that may be linked statically or dynamically.

The carrier of a computer program may be any entity or device capable of carrying the program. For example, the carrier may include a data storage, such as a ROM, for example, a CD ROM or a semiconductor ROM, or a magnetic recording medium, for example, a hard disk. Furthermore, the carrier may be a transmissible carrier such as an electric or optical signal, which may be conveyed via electric or optical cable or by radio or other means. When the program is embodied in such a signal, the carrier may be constituted by such a cable or other device or means. Alternatively, the carrier may be an integrated circuit in which the program is embedded, the integrated circuit being adapted to perform, or used in the performance of, the relevant method.

The various aspects discussed in this patent can be combined in order to provide additional advantages. The mere fact that certain measures are recited in mutually different claims does not indicate that a combination of these measures cannot be used to advantage. Furthermore, some of the features can form the basis for one or more divisional applications.

SUBSTITUTE SHEET (RULE 26)

Claims

1 . Method (200) for imaging a medium, comprising:

- providing (201 ) a plurality of transmitters arranged to a transmitter surface;

- selecting (202) a first set of transmitters of the plurality of transmitters for transmitting a first plane wave into the medium;

- selecting (203) a second set of transmitters of the plurality of transmitters for transmitting a second plane wave into the medium; and

- transmitting (204) a first plane wave and a second plane wave for creating an interference sheet in the medium; wherein the wave amplitude inside the interference sheet is above a threshold, and outside the interference sheet below the threshold; and wherein transmitting the first plane wave and the second plane wave comprises timing the transmission of the first plane wave relative to the transmission of the second plane wave for positioning the interference sheet.

2. Method according to the preceding claim, wherein timing comprises delaying the transmission of the first plane wave relative to the transmission of the second plane wave for positioning the interference sheet.

3. Method according to any of the preceding claims, wherein positioning the interference sheet comprises a lateral shift of the interference sheet relative to the transmitter surface.

4. Method according to any of the preceding claims, wherein timing comprises timing with discrete time steps having a discrete step size; and wherein preferably the discrete step size is based on a clock frequency provided to at least the first set of transmitters and the second set of transmitters.

5. Method according to any of the preceding claims, further comprising the steps of:

- transmitting a first plane wave; and

- transmitting a second plane wave.

6. Method according to the preceding claim, comprising:

- providing a plurality of receivers, wherein at least a set of receivers from the plurality of receivers is arranged for receiving echoes from the first plane wave and the second plane wave;

- after transmitting the first plane wave, receiving a first set of echoes from the first plane wave with the set of receivers;

- after transmitting the second plane wave, receiving a second set of echoes

SUBSTITUTE SHEET (RULE 26) from the second plane wave with the set of receivers; and

- after transmitting the first plane wave and the second plane wave, receiving a third set of echoes from the first plane wave and the second plane wave with the set of receivers.

7. Method according to the preceding claim, comprising deducing image information related to the interference sheet based on subtracting the first set of echoes and the second set of echoes from the third set of echoes.

8. Method according to any of the preceding claims, wherein selecting the first set of transmitters and selecting the second set of transmitters comprises selecting the first set of transmitters and selecting the second set of transmitters such that these transmitters are adjacent.

9. Method according to the preceding claim, wherein transmitting the first plane wave and the second plane wave comprises transmitting the first plane wave and the second plane wave with the adjacent sets of transmitters such that the interference sheet originates from a separation line separating the adjacent sets of transmitters and into the medium.

10. Method according to the preceding claim, wherein the separation line is a straight line or curved line.

11 . Method according to any of the preceding claims, wherein a mesh comprises vertices and edges; wherein the plurality of transmitters are arranged to respective vertices; wherein the edges have a length; wherein the length of the edges is selected such that the mesh forms a regular or repeating pattern; wherein the mesh forms the transmitter surface; wherein preferably the vertices form a grid, more preferably a square grid, a rectangular grid, a triangular grid, or a hexagonal grid; and wherein selecting the first set of transmitters and selecting the second set of transmitters comprises selecting the first set of transmitters and selecting the second set of transmitters such that these sets boarder each other at one edge, two edges, three edges, four edges or more edges apart from each other.

12. Method according to the preceding claim, wherein an area is defined between the first set of transmitters and the second set of transmitters; and wherein the area is straight.

SUBSTITUTE SHEET (RULE 26)

13. Method according to any of the preceding claims 11 -12, wherein selecting the first set of transmitters and selecting the second set of transmitters comprises selecting the first set of transmitters and selecting the second set of transmitters such that these sets boarder each other one edge apart from each other for transmitting the first plane wave, the second plane wave, and the first and the second plane wave; wherein the method comprises:

- selecting a third set of transmitters of the plurality of transmitters for transmitting a third plane wave into the medium;

- transmitting a third plane wave; and

- transmitting a first plane wave and a third plane wave for creating a second interference sheet in the medium; wherein the wave amplitude inside the second interference sheet is above the threshold, and outside the second interference sheet below the threshold; wherein the third set of transmitters is one edge shifted compared to the second set of transmitters, such that the third set of transmitters and the second set of transmitters boarder each other two edges apart from each other; and wherein at least at the boundary between the transmitter surface and the medium, preferably a part of the interference sheet, more preferably over the whole interference sheet, the interference sheet and the second interference sheet are shifted from each other over half an edge length.

14. Method according to any of the preceding claims 11 -13, wherein the method comprises:

- selecting a fourth set of transmitters of the plurality of transmitters for transmitting a fourth plane wave into the medium;

- selecting a fifth set of transmitters of the plurality of transmitters for transmitting a fifth plane wave into the medium;

- transmitting a fourth plane wave;

- transmitting a fifth plane wave; and

- transmitting a fourth plane wave and a fifth plane wave for creating a third interference sheet in the medium; wherein the wave amplitude at the third interference sheet is above the threshold, and outside the third interference sheet below the threshold; and wherein the set of transmitters formed by the first set of transmitters and the second set of transmitters do not overlap with the set of transmitters formed by the

SUBSTITUTE SHEET (RULE 26) fourth set of transmitters and the fifth set of transmitters, or wherein the interference sheet and the third interference sheet intersect, preferably are perpendicular.

15. Method according to any of the preceding claims 11-14, wherein the transmitter surface is rectangular; wherein selecting the first set of transmitters and selecting the second set of transmitters comprises selecting the first set of transmitters in a first rectangle and selecting the second set of transmitters in a second rectangle; wherein the first plane wave has a first normal vector; wherein the second plane wave has a second normal vector; wherein the first cross-propagation angle is defined as the angle between the first and second normal vectors; wherein the interference sheet has an interference depth reaching into the medium; wherein the method comprises:

- selecting a sixth rectangular set of transmitters of the plurality of transmitters for transmitting a sixth plane wave into the medium;

- selecting a seventh rectangular set of transmitters of the plurality of transmitters for transmitting a seventh plane wave into the medium;

- transmitting a sixth plane wave;

- transmitting a seventh plane wave; and

- transmitting a sixth plane wave and a seventh plane wave for creating a fourth interference sheet in the medium; wherein the wave amplitude at the fourth interference sheet is above the threshold, and outside the fourth interference sheet below the threshold; wherein the sixth plane wave has a sixth normal vector; wherein the seventh plane wave has a seventh normal vector; wherein the second cross-propagation angle is defined as the angle between the sixth and seventh normal vectors; wherein the fourth interference sheet has a fourth interference depth reaching into the medium; and wherein the second cross-propagation angle is adapted such that the fourth interference depth is substantially equal to the interference depth.

16. Method according to the preceding claim, wherein the sixth rectangular set of transmitters and the seventh rectangular set of transmitters are located closer to the

SUBSTITUTE SHEET (RULE 26) perimeter of the transmitter surface compared to the first rectangular set of transmitters and the second rectangular set of transmitters.

17. Method according to any of the preceding claims 15-16, wherein the first rectangular set of transmitters, the second rectangular set of transmitters, the sixth rectangular set of transmitters and the seventh rectangular set of transmitters all have a width; wherein the width of the sixth rectangular set of transmitters is smaller compared to the width of the first rectangular set of transmitters; wherein the width of the seventh rectangular set of transmitters is smaller compared to the width of the second rectangular set of transmitters; and wherein preferably the width of the sixth rectangular set of transmitters and the width of the seventh rectangular set of transmitters are both smaller compared to the width of the first rectangular set of transmitters as well as the width of the second rectangular set of transmitters.

18. Method according to claim 11 and claim 3, wherein the lateral shift is based on the discrete time step size, a propagation speed of the plane waves in the medium, and an angle of the plane wave relative to the transmitter surface; and wherein one discrete time step results in a lateral shift of less than the edge length.

19. Method according to the preceding claim, wherein every integer multiple of the one discrete time step is unequal to the edge length.

20. Method according to any of the preceding claims, wherein the medium comprises a medium boundary, typically adjacent to or in contact with the transmitter surface; and wherein the interference sheet originates adjacent to the medium boundary and propagates substantially away, preferably away, from the medium boundary, or wherein the interference sheet originates at a distance from the medium boundary and propagates substantially away, preferably away, from the medium boundary.

21 . Method according to any of the preceding claims, wherein the transmitter surface is a 2D transmitter surface.

22. Method according to the preceding claim, wherein the 2D transmitter surface is a

2D flat transmitter surface or a 2D curved transmitter surface.

SUBSTITUTE SHEET (RULE 26)

23. Method according any of the preceding claims, wherein the first plane wave has a first normal vector; wherein the second plane wave has a second normal vector; wherein a first cross-propagation angle is defined as the angle between the first and second normal vectors; and wherein the cross-propagation angle is less than 150 degrees, preferably in a range from 120-150 degrees, more preferably 130-150 degrees, most preferably 140- 150 degrees.

24. Method according the preceding claim, wherein the interference sheet is substantially flat; wherein the interference sheet defines a sheet normal vector normal to the interference sheet; wherein a first angle is between the first normal vector and the sheet normal vector; wherein a second angle is between the second normal vector and the sheet normal vector; and wherein the first angle is minus the second angle.

25. Method according any of the preceding claims, wherein the medium comprises a contrast agent; and wherein the threshold is one of a buckling threshold, a collapse threshold, and a cavitation threshold of the contrast agent.

26. Method according the preceding claim, wherein the contrast agent is an engineered harmonic gas vesicle or a microbubble.

27. Method according any of the preceding claims, comprising providing a plurality of transducers, wherein each transducer comprises one transmitter of the plurality of transmitters and one receiver of the plurality of receivers.

28. Method according any of the preceding claims, wherein the plurality of transmitters and the plurality of receivers are arranged for transmitting and receiving ultrasound, respectively.

29. Method according the preceding claim, wherein the threshold is a pressure threshold.

30. Method according the preceding claim, wherein the interference sheet is substantially flat; wherein the interference sheet defines a sheet normal vector normal to the interference sheet;

SUBSTITUTE SHEET (RULE 26) wherein the transmitter surface is substantially flat; and wherein the sheet normal vector is substantially parallel to the transmitter surface.

31 . Method according any of the preceding claims, wherein the interference sheet is flat or substantially flat.

32. Method according any of the preceding claims, wherein the first set of transmitters and the second set of transmitters are different.

32. Imaging device (100) for imaging a medium, comprising:

- a transmitter surface (110);

- a plurality of transmitters (120) arranged to the transmitter surface;

- a controller arranged for: selecting a first set of transmitters of the plurality of transmitters for transmitting a first plane wave into the medium; selecting a second set of transmitters of the plurality of transmitters for transmitting a second plane wave into the medium; and providing instructions for transmitting a first plane wave and a second plane wave for creating an interference sheet in the medium; wherein the wave amplitude inside the interference sheet is above a threshold, and outside the interference sheet below the threshold; and wherein transmitting the first plane wave and the second plane wave comprises timing the transmission of the first plane wave relative to the transmission of the second plane wave for positioning the interference sheet.

33. Imaging device according to the preceding claim, wherein the controller is further arranged for: providing instructions for transmitting a first plane wave; and providing instructions for transmitting a second plane wave.

34. Imaging device according to the preceding claim, comprising:

- a plurality of receivers, wherein at least a set of receivers from the plurality of receivers is arranged for receiving echoes from the first plane wave and the second plane wave; wherein the controller is arranged for:

- after transmitting the first plane wave, receiving a first set of echoes of the first plane wave from the set of receivers;

- after transmitting the second plane wave, receiving a second set of echoes of the second plane wave from the set of receivers; and

SUBSTITUTE SHEET (RULE 26) - after transmitting the first plane wave and the second plane wave, receiving a third set of echoes of the first plane wave and the second plane wave from the set of receivers.

35. Imaging device according to the preceding claim, wherein the controller is arranged for deducing image information related to the interference sheet based on subtracting the first set of echoes and the second set of echoes from the third set of echoes.

36. Imaging device according to any of the preceding claims 32-35, comprising a plurality of transducers, wherein each transducer comprises one transmitter of the plurality of transmitters and one receiver of the plurality of receivers.

37. Imaging device according to any of the preceding claims 32-36, wherein the plurality of transmitters and the plurality of receivers are arranged for transmitting and receiving ultrasound, respectively.

38. Computer-readable storage product (1000) comprising a computer readable medium (1010) comprising instructions which, when executed by a computer, cause the computer to carry out the steps of the controller of any of the claims 32-37.

39. Method for imaging a medium, comprising:

- providing a plurality of transmitters arranged to a transmitter surface;

- selecting a first set of transmitters of the plurality of transmitters for transmitting a first plane wave into the medium;

- selecting a second set of transmitters of the plurality of transmitters for transmitting a second plane wave into the medium;

- transmitting a first plane wave;

- transmitting a second plane wave; and

- transmitting a first plane wave and a second plane wave for creating an interference sheet in the medium; wherein the wave amplitude inside the interference sheet is above a threshold, and outside the interference sheet below the threshold; wherein a mesh comprises vertices and edges; wherein the plurality of transmitters are arranged to respective vertices; wherein the edges have a length; wherein the length of the edges is selected such that the mesh forms a regular or repeating pattern; wherein the mesh forms the transmitter surface; wherein preferably the vertices form a grid, more preferably a square grid, a

SUBSTITUTE SHEET (RULE 26) rectangular grid, a triangular grid, or a hexagonal grid; and wherein selecting the first set of transmitters and selecting the second set of transmitters comprises selecting the first set of transmitters and selecting the second set of transmitters such that these sets boarder each other at one edge, two edges, three edges, four edges or more edges apart from each other; wherein selecting the first set of transmitters and selecting the second set of transmitters comprises selecting the first set of transmitters and selecting the second set of transmitters such that these sets boarder each other one edge apart from each other for transmitting the first plane wave, the second plane wave, and the first and the second plane wave; wherein the method further comprises:

- transmitting a third plane wave; and

40. Method according to the preceding claim, wherein an area is defined between the first set of transmitters and the second set of transmitters; and wherein the area is straight.

41 Method according to any of the preceding claims 39-40 combined with any of the features of claims 1-32.

SUBSTITUTE SHEET (RULE 26)