WO2003082117A1 - Device and method for quantifying bodies by means of ultrasound - Google Patents

Abstract

The problem with quantifying bodies, particularly bubbles, in ultrasonic diagnostics is that the concentration of bodies, particularly bubbles, is often so high that the obtained image data is saturated concerning the representation thereof, rendering quantification impossible. In order to solve said problem, the bodies contained in cross-sectional layers of an object that is to be analyzed are stimulated by means of ultrasound so as to emit characteristic signals, whereupon said signals are recorded, sets of data are formed from the signals, the sets of data are transformed into a representation of the arrangement of the bodies in the object that is to be analyzed, and the number of bodies is detected therefrom. At least two sets of ultrasonic signals are recorded from overlapping cross-sectional layers in the object that is to be analyzed.

Description

Device and method for quantifying bodies using ultrasound

The invention relates to a device and a method for the quantification of bodies contained in an examination object, which can be excited to independent, characteristic signals by diagnostic ultrasound, in particular those signals which are associated with the destruction of a body, preferably of bubbles. Furthermore, the invention also relates to a use of the device for ultrasound diagnosis and / or therapy on humans or animals and a use of the device and a method for the in-vivo and ex-vivo mapping of physiologically high or low-regulable molecular markers in Organs and tissues from postembryonic ontogenesis and from pathologically high or down-adjustable molecular markers in organs and tissues during pathogenesis and for the characterization of cell cultures by in-mapping of up and down-adjustable molecular markers of cells.

Ultrasound diagnostics has found a permanent place in everyday clinical practice. Details can be shown better in ultrasound recordings by injection of contrast media, since stronger ultrasound signals can be generated in this way. All very small bodies (micro-bodies) that deliver a detectable signal that can be distinguished from the tissue signal, in particular gas-filled very small (micro) bubbles, are suitable as contrast agents. These bodies are fed to the examination object and can be destroyed in the ultrasound field, for example in the case of bubbles, under certain conditions. These bubbles emit independent, characteristic ultrasound signals. If the bubbles are destroyed by the ultrasound field, the independent, characteristic signals are referred to as "stimulated acoustic emission (SAE)".

Bubbles that can emit independent characteristic signals are described, for example, in EP 0 398 935 B1, EP 0 644 777 B1, EP 0 458 745 A1 and WO 01/68150 A1. The principle of stimulated acoustic emission (SAE) is shown schematically in the following figures. They show in detail:

1: the principle of "stimulated acoustic emission" using an ultrasound-bladder,

2: an ultrasound sectional image from which the size of the SAE signals can be seen, FIG. 3: a conventionally recorded ultrasound sectional image.

1 shows a first sound wave with low amplitude 1 and a second sound wave with high amplitude 2. When the sound wave with low amplitude 1 is irradiated, a bladder 3 that is sonicated remains intact. A scatter signal is generated. In contrast to this, the bladder 4 is destroyed when a sound wave with high amplitude is irradiated, a SAE signal being generated.

SAE signals of various bubbles can be detected according to US 5,425,366 A, US 6,186,951 B1 and DE 198 13 174 A1 using color Doppler mode.

SAE signals can also be displayed independently of the movement of the bubbles with other ultrasound device modes, which were originally developed primarily to represent movement. The spectral Doppler, power Doppler, color Doppler and harmony method (2nd, sub, wideband, pulse inversion, ultraharmonic, color Doppler, harmonic power Doppler method) are mentioned as examples.

The SAE signal strength is so high that even individual bubbles can be detected. The individual SAE signal is shown on the monitor considerably larger (diameter approx. 0.5 - 1 mm depending on the device, transducer and settings) than it corresponds to the actual size of the undestroyed bladder (<10 μm). 2 shows the size of SAE signals in the sectional view. In many ultrasound diagnostic devices currently available, the layer thickness of a single image is approximately 1 mm, depending on the device setting. This is illustrated in Figure 3. The layer thickness of a conventional sectional image can be seen there. As a rule, a bladder concentration of approx. 2,000-3,000 blisters / ml tissue / blood leads to a complete SAE contrast in the sonographic cross-section. In the two-dimensional ultrasound sectional image, the saturation bubble concentration is determined by the layer thickness of the ultrasound sectional image (shown by way of example in FIG. 3) and by the size of an SAE signal shown in the ultrasonic sectional image when a single bubble is destroyed (several individual bubbles are shown in FIG. 2).

A low saturation concentration / saturation bladder concentration can be advantageous for qualitative imaging, since in particular, even at low bladder concentrations, a complete contrast is created in the sectional image.

On the other hand, a low saturation concentration / saturation bubble concentration is disadvantageous for the quantification of bodies, since even small concentrations of bodies, in particular bubbles, lead to contrast saturation in the image and their concentrations above the saturation concentration in the image are no longer absolute determine or no longer distinguish bodies, especially bubbles.

As long as bodies, in particular bladders, are only distributed unbound in the blood, their concentration can be controlled there by the dosage so that the saturation concentration / saturation bubble concentration is not reached. However, if there is a specific or non-specific accumulation of the body, for example in cells, tissues, organs or other enriching structures, the saturation concentration / saturation bladder concentration is usually exceeded because the enriching tissue or the enriching structures are so dense, that the bodies accumulate there in a concentration above the saturation concentration / saturation bubble concentration.

By reducing the dose, the saturation concentration / saturation bladder concentration in the target area could also be undercut here, but the enriched number of bodies, in particular bubbles, would be in an unknown ratio to the maximum saturation. Such a measurement would therefore neither be reproducible nor would it provide a value that correlates with the number of enrichment factors present there. Therefore, a method for determining the number of bodies (number of bubbles) above the saturation concentration / saturation bubble concentration is desirable.

Bodies, in particular bladders, can accumulate non-specifically or specifically on or in cells, tissues, organs or other structures, for example. This enrichment mechanism is often referred to in the literature as passive or active targeting (Lanza, G., Wickline, S .: "Targeted ultrasonic contrast agents for molecular imaging and therapy". Prague. Cardiovasc. Dis., 2001, 44: 13- 31; Dayton, PA, Ferrara, KW: "Targeted imaging using ultrasound". J. Magn. Reson. Imaging, 2002, 16: 362-377).

Unspecific enrichment of bodies, especially bubbles, takes place for example:

a) by phagocytosis of bodies / bladders in organs of the reticuloendothe- lial system (RES) or in other cells which e.g. disease-related, such as in tumor-associated macrophages or macrophages in atherosclerotic processes, or tumor cells themselves or cells that are stimulated to phagocytosis, e.g. Endothelial cells of the

Liver with certain diseases of the liver; b) by adhesion or by slowly rolling bodies / blisters along, for example on the vascular endothelium; c) by immigration of bodies / blisters in or by fenestrations, eg of the endothelium, in "vascular cul-de-sacs" or "blind" blood vessels; this can occur in the transition area to necrosis, in the case of severely edematous or inflammatory processes, hemorrhages, hematomas or bulges of the vascular system, such as aneurysms, vascular tumors, such as hemangiomas, or by washing into necrosis; d) by transporting phagocytized or adherent bodies / bladders, which are phagocytosed, for example, by monocytes or adhered to cells of the blood, such as leukocytes, through endothelial barriers, such as the blood-brain barrier, to pathological areas, for example. A specific enrichment of bodies, in particular bubbles, can be achieved by a covalent or non-covalent, including an electrostatic bond, hydrogen bond or Van der Waais bond, to physiological or pathophysiological structures. As a rule, the bodies, in particular bubbles, carry target-specific binding molecules on their surface in these cases.

To measure ultrasound contrast, there is the option of digitizing video images and measuring the brightness (gray or color values) in definable image regions over time. However, this type of measurement does not solve the problem of contrast saturation at high concentrations of bodies / bubbles. If at all, quantification is only possible with a very low concentration of the body, especially blisters.

3D-Echotech, Halbergmoos, Germany, has also attempted to satisfy the need for the quantifiability of bubbles with an ultrasound device for the quantification of bubble signals in the blood. With this device, the video image of the ultrasound device is digitized, broken down into color and gray values and optionally evaluated according to the average brightness value or the number of screen pixels within a defined measurement window (ROI). The field of application of this measuring system is to quantify the blistering in blood vessels based on the brightness values within a definable ROI. However, this method is also unsuitable for quantifying the bubbles above a concentration of approx. 3,000 particles or bubbles / ml. Even below this concentration, a reproducible measurement is not possible with this device.

Kretztechnik, Zipf, Austria, offers a 3D (three-dimensional) ultrasound method, in which a 2D (two-dimensional) array (linear array, sector array, phased array, as in 2D sonography) within of a transducer is automatically pivoted at a certain angle. However, bubbles cannot be quantified reproducibly with this device. This applies in particular when different patients are examined and / or different examiners carry out the examination. None of the devices mentioned makes it possible to quantify bodies, in particular bubbles, in vivo, ex vivo or in vitro, in particular not in concentrations above a concentration of approximately 3,000 bubbles / ml of tissue or blood. Below this concentration, spatial resolution is only possible in the millimeter range. No other measurement methods to solve this problem are known to date.

A disadvantage of the known methods and devices is that it is not possible to display small structures, since the sound lobes emanating from a transducer have minimum dimensions that limit the resolution. For example, the thickness of the measuring layer detected by the sound waves is approximately 1 mm, so that finer structures can no longer be resolved.

For the above reasons it is not possible to use ultrasound technology e.g. to use for the detection of molecular markers:

Key biological technologies such as genomics / bioinformatics and proteomics are used to identify new molecular markers (Yao, T .: "Bioinformatics for the genomic sciences and towards Systems biology. Japanese activities in the post-genome era". Prog. Biophys. Mol. Biol., 2002, Jul.-Aug., 80 (1-2): 23-42; Wu, W., Hu, W., Kavanagh, JJ: "Proteomics in cancer research". Int. J. Gynecol Cancer, 2002, Sep.-Oct, 12 (5): 409-423) With these technologies, the sequences of, for example, human genes are analyzed using special bioinformatics tools in order to obtain proteins belonging to pharmacologically interesting protein families, in particular receptors and enzymes These are used in wϊro processes, for example, using freshly isolated (primary) cells and (immortalized) cell cultures (from diseased or healthy tissue), but these cell cultures are disadvantageous because the expression spectrum differs from molecular ones Markers can change, for example, through the insulation process. Such investigated cell cultures only reflect the expression pattern of molecular markers at the exact time when they were isolated. Furthermore, the cell cultures are generally immortalized cells (which can be expanded in vitro indefinitely by special manipulation) and cannot reflect the real Vo ratios. Another very common method of identifying molecular markers is by immunohistochemical examination of tissues. Tissues of special interest, for example tumors, are histologically processed after their removal and immunohistologically specifically examined for the expression of one or more molecular markers. Only after histological identification of such markers can they be imaged later in vivo by using appropriate antibodies which are coupled to corresponding signal generators. Such images are generally only possible in terms of quality and mostly only if there is a very strong accumulation of the antibody-carrying signal transmitter (Fonsatti, E., Jekunen, AP, Kairemo, KJA et al .: “Endoglin is a suitable target for efficient imaging of solid tumors: In vivo evidence in a canine mammary carcinoma model ". Clinical Cancer Research, Vol. 6, May 2000: 2037-2043).

Many diagnostic methods for the detection of molecular markers are currently being tested in vivo. Although the detection of known molecular markers is possible using known diagnostic methods, these methods generally lack high spatial resolution. This is the case, for example, with PET (positron emission tomography), NIRF (near infrared fluorescence imaging) and scintigraphy. In other cases, high concentrations of signal transmitters are required, for example in MRI (magnetic resonance imaging) and CT (computer tomography). There is also literature on the use of ultrasound contrast media for passive targeting (without antibody conjugate, e.g. without specific binding to molecular markers) and for active targeting (with antibody conjugate, e.g. with specific binding to molecular markers). However, none of the diagnostic methods described to date meet the criteria that can be achieved using the device known from DE 102 15 335 A, namely spatial resolution in the micrometer range and highly sensitive quantification of signal transmitters in the target area.

The present invention is therefore based on the object of finding a method and a device for the quantification of bodies in an examination object, with which the concentration of the bodies can be determined in vivo, ex vivo and in vitro above the saturation bubble concentration is possible. With the drive and the device should be able to reproducibly quantify body quantities and determine their concentration. In addition, it should be possible, with respect to the independent, characteristic signals shown in the video image, for example the SAE signals, to allow spatial resolution, preferably in the histological range, in order, for example, to be able to identify molecular markers in a target-specific manner.

The object is achieved by the device for quantifying bodies contained in an examination object according to claim 1, their use for ultrasound diagnosis and / or ultrasound therapy on humans or animals according to claim 10, their use for in-vivo and ex-v / Vo mapping of molecular markers in organs and tissues and in w ^' fro mapping of cells for the characterization of cell cultures according to claim 11 and the method for quantifying bubbles contained in a test object according to claim 12 solved. Preferred embodiments of the invention are specified in the subclaims.

definitions:

In addition, reference is made to the figures explained below. It shows in detail:

Fig. 4: Color-saturated sectional image

5: Principle of the method according to the invention: a: displacement distance: 100 μm b: displacement distance: 50 μm c: displacement distance: 10 μm

Fig. 6: Directions in relation to the transducer

Fig. 7: Multi-element transducer with defined overlapping sound fields (2D array) a: Image construction with a low beam overlap b: Image construction with a strong beam overlap 8: 3D view of the agar phantom scanned with different displacement distances from example 1: a: SAE layer thickness = 10 μm to 100 μm,

Sectional image 13: displacement distance 10 μm b: SAE layer thickness = 50 μm to 100 μm

Sectional image 14: displacement distance 50 μm c: SAE layer thickness = 100 μm

Sectional image 15: displacement distance 100 μm

9: Dependence of the MU on the SAE layer thickness (agar phantom, example 1)

Fig. 10: Dependence of the MU _CO rr. on the SAE layer thickness (agar phantom, example 1)

Fig. 11: Dependence of the SAE _CO rr. on the SAE layer thickness (agar phantom, example 1)

a) body:

Bodies in the sense of the present application are solid particles and / or bubbles with a diameter <10 μm. Solid particles contain at least one surface-active substance (e.g. surfactant), at least one carbohydrate, protein, lipid and / or natural and / or synthetic polymer.

b) bubble:

Bubbles in the sense of the present application are gas-containing phases with a diameter <10 μm, which are either free or coated. The phases can be substantially spherical. In the case of enveloped bubbles, the envelope can contain at least one surface-active substance (eg surfactant), at least one protein, lipid and / or natural and / or synthetic polymer. Bubbles according to the present application can also, for example, gas-filled microcapsules, gas-filled microparticles, gas-filled microspheres, microballoons and surfactant-stabilized microbubbles.

c) Ultrasound contrast media:

Ultrasound contrast media are agents that contain bodies, in particular bladders, for use in ultrasound diagnostics and / or therapy.

d) Independent, characteristic signal:

An independent, characteristic signal (CS signal, CS effect) is an echo signal of a body, in particular a bladder, which is independent of the scatter signal. This also includes all non-linear signals such as 2nd harmonic, subharmonic or ultra-harmonic, as well as in particular SAE signals, such as those which arise when bubbles are destroyed in the ultrasound field.

e) Stimulated Acoustic Emission (SAE):

Bubbles preferred according to the invention can be destroyed under certain conditions when excited with ultrasound. This creates an independent signal that deviates from the transmission signal and is independent of the scattering and movement of the bubbles. This independent, characteristic signal is called stimulated acoustic emission (SAE) (synonym: Loss of Correlation (LOC)).

f) Saturation Concentration / Saturation Bubble Concentration:

The saturation concentration / saturation bubble concentration is the concentration of bodies / bubbles per milliliter of object under examination, e.g. tissue or blood, above which the independent, characteristic signals, in particular the SAE signals, are superimposed within a sound field and above which one It is impossible to quantify the bubbles because the number of bodies / bubbles can no longer be inferred from the sum of the CS signals, even when using arithmetic correction formulas. g) CS / SAE layer thickness:

In the event that the bubbles are destroyed, this is the thickness of the layer per ultrasound sectional image (cut layer) from which the CS signals shown in the sectional image (in this case SAE signals) are used in a scan with parallel overlap come. In the event that the bodies / bubbles are not destroyed during the sonication with the CS signals, it is the thickness of the layer per ultrasound sectional image from which the CS signals shown in the sectional image originate in a scan with a parallel overlap of bubbles that are added each time the ultrasound field is shifted. The CS / SAE layer thickness is equal to the displacement distance per ultrasound sectional image. This layer thickness is preferably significantly less than the cut layer. The cut layer is created in the examination object by irradiation of a sound lobe (sound field). This is done in a conventional manner in that piezo crystal elements emit sound pulses which are coupled and focused into the medium to be sonicated by suitable means. By moving the sound lobe protruding into the examination object, for example along the surface of the examination object, a cut layer can be gradually moved away from the sound lobe and the corresponding image can be generated by the sound signals arriving at the receiver. In both cases, the layer thickness is defined by the feed between two images.

h) Region of interest (ROI):

This is the measurement window in the sectional view.

i) Morphometric Units (MU):

Morphometric units are a measure of the area, corresponding to a section layer, expressed in the number of screen pixels that the CS signals represented in the video image occupy. The screen pixels are usually counted automatically by video densitometry. If the number of screen pixels of an individual CS signal is known, the number of bodies / number of bubbles can be concluded from the morphometric units. j) Corrected SAE (SAE _CO rr.) or corrected MU (MU _corr .):

If the bodies / bubbles are smaller than the thickness of a layer for an ultrasound cross-sectional image, CS signals can be superimposed in the video image. The probability of such a superimposition increases with increasing concentration of bodies / bubbles and layer thickness of the ultrasound sectional image, because in this case a larger number of bodies / bubbles is detected. Both parameters have a direct influence on the percentage color area of the CS signals in the video image. If the bodies / bubbles are randomly distributed in the examined volume, the value of MU can be corrected if CS signals are superimposed as long as there is no complete color saturation in the ROI:

ΣFP

MUcorr = ∑FPx 1 + -

∑FP + ∑GP

FP: color screen pixels that lie within the measurement window (ROI) in the area of the CS effects shown, MU: FP within the measurement window (ROI),

GP: Screen gray pixels that are within the measurement window (ROI) but outside the CS effects shown.

About the quotient of the sum of the color pixels and the sum of the CS signals

(Fsae = within the ROI, the corrected MU (MU _cor r.) Can be converted into a

the number of CS signals (SAE _cor r.) are converted:

". "MUcorr

SAEcorr =

fsae

The application of this correction formula requires a random distribution of the body / bubbles in the volume examined. k) 2D array:

A 2D array consists of a one-dimensional row of piezo elements.

I) 3D array:

A 3D array consists of several adjacent rows of piezo elements.

m) transducer (ultrasonic transducer):

Device for sonography, containing piezo elements.

n) Directions in relation to the transducer:

6, a transducer 23 is shown schematically. The directions in relation to the transducer are defined as follows:

x = transverse to the transducer (reference number 24: 2D array) y = lateral (reference number 25) z = axial (direction of sound propagation) (reference number 26)

o) Molecular markers:

Intra-, inter- and / or extracellular molecular structures, for example receptors, ligands, enzymes or their structural components.

In order to be able to quantify bodies, preferably bubbles, in an examination object, in particular those bubbles are used which can be excited to produce independent, characteristic signals, and above all bubbles which are destroyed during the sonication. The device according to the invention and the method according to the invention serve to quantify bodies, preferably bubbles, contained in an examination object. The device comprises at least one transducer for emitting an ultrasound field, which excites the bodies contained in a layer in the examination object, preferably bubbles, for emitting characteristic ultrasound signals. At least one of the transducers is also used to receive the characteristic signals. Furthermore, at least one data processing system for determining the number of bodies, preferably bubbles, is provided in the layer from the characteristic signals. Furthermore, means are provided in the manner according to the invention with which the ultrasound field after excitation of the bodies, preferably bubbles, can be displaced in a first layer in the examination object such that bodies / bubbles can be excited in a second layer after the displacement, the first layer and the overlap second layer and are shifted parallel and defined against each other. Ultrasound signal data records are thus obtained from different cut layers in the examination object, which contain information about the presence of bodies / bubbles in the individual cut layers, in particular about the spatial arrangement of the bubbles in the cut layers. In relation to a transducer with piezocrystals arranged in rows, the layers are preferably shifted against one another in such a way that they overlap in the transverse direction (x direction) to the rows of piezocrystals.

The transducer can either be designed to emit ultrasound signals as well as to receive the signals originating from the bodies, preferably bubbles. Alternatively, the transducer can also be designed exclusively for emitting the ultrasound signals. In this case, a further receiving head is required to detect the incoming ultrasound signals from the bodies, preferably bubbles.

To carry out the method according to the invention for quantifying bodies, preferably bubbles, contained in the examination object, bodies / bubbles contained in a layer are excited with an ultrasound field to produce characteristic ultrasound signals. The characteristic ultrasound signals emitted by the bodies / bubbles are received, and the number of bodies / bubbles is determined from the received ultrasound signals, in each case by the Displacement of the ultrasound field after excitation of bodies / bubbles in a first layer in the examination object bodies / bubbles in a second layer overlapping the first layer and excited in a defined and defined manner against this shifted layer.

The device according to the invention and the method according to the invention are not limited to embodiments using imaging or the video image. The CS signals within a region of interest (ROI) can also already be counted on the basis of the received signals (radio frequency (RF) data), although the concentration of saturation bubbles when the CS signals are counted at the level of the received signals from the Can distinguish saturation bubble concentration when counting the CS signals in the two-dimensional ultrasound image. The device according to the invention and the counting of the received signals can be integrated into an ultrasound device.

In contrast to known devices and methods, it is possible with the device and the method according to the invention to also determine concentrations of bodies, preferably bubbles, which are above a limit which is in the range from 1000 to 3000 bodies (bubbles Vml:

With known radiological and cardiological devices and methods, which preferably operate in the frequency range from 2.5 to 12 MHz, saturation can already be observed at a concentration above about 1000 bodies (bubbles) / ml, so that quantification becomes impossible. This is due to the fact that the spatial resolution in this case is not sufficiently high: the spatial resolution of ultrasonic transducers is determined by their frequency. The spatial resolution increases with higher frequency. At the same time, however, the sound pressure and thus the depth of penetration of the ultrasonic wave into the examined tissue are reduced. For medical diagnostics, for which greater penetration depths are required, ultrasonic transducers in the frequency range from approximately 2.5 MHz (tissue penetration depth of approximately 30 cm) to 12 MHz (tissue penetration depth of approximately 7 cm) are used. The spatial resolution in the x direction or x and y direction (in a plane perpendicular to the sound lobe) when using a single-crystal ultrasound transducer with a round cross section is in the mm range in these cases. Hence the thickness of the Sound field at the narrowest point in the sound beam emitted by the transducer in the mm range. For this reason, the smallest volume that can be resolved by such an ultrasound device is approximately 1 mm ³ . If a body, preferably a bubble, in this volume is destroyed by ultrasound, the exact spatial position of the corresponding CS signal within this volume cannot be determined exactly. Consequently, this signal is shown in the image in a size that is also in the millimeter range. If there are at least two bodies, preferably bubbles, in this volume, these can no longer be discriminated by the ultrasound device due to the lack of spatial resolution. Concentrations on bodies, preferably bubbles of more than about 1000 bodies (bubbles) / ml, therefore lead to saturation. This is also the reason why the above-mentioned device from SD-Echotech is not suitable for quantifying the bodies, in particular bubbles, since the thickness of the sound field is in the range of approx. 1-2 mm. Another reason why this device is unsuitable for the quantification of bodies, in particular bubbles, is that the transducer is guided by hand, so that some areas of the examination object overlap several times or to different degrees, other areas even not be sounded. This problem is also not solved by the possibility of electromagnetic position determination of the transducer, which was also developed by 3D-Echotech, since this is only accurate in the sub-millimeter range.

For the first time, the present invention enables a significantly higher spatial resolution than with known methods and devices. At the same time, calculable volumes are used. This makes it possible for the first time to quantify bodies, preferably bubbles, in an examination volume even if the saturation concentration / saturation bubble concentration would normally be exceeded.

The Kretztechnik device is also unsuitable for quantification, since the overlap of the sound fields in the axial direction (z) is not uniform. In the close range, several adjacent sound fields overlap almost completely, while gaps arise with increasing distance from the transducer, which are then interpolated. As a result, the signal yield in an examined tissue also depends on the distance to the transducer. The aforementioned object is only achieved by the present invention.

The principle of the method according to the invention can be illustrated particularly easily using the example of complete bubble destruction in each ultrasound sectional image:

4, 5 b) and c) the mode of operation of the invention can be shown:

a) A contrast-saturated ultrasound sectional image is obtained in the first image (Fig.

4). b) The bubbles are destroyed by the sonication (Fig. 5a). SAE signals can then no longer be detected. Independent bubbles emitting characteristic signals are denoted by 6, undestroyed bubbles in the non-excited cut layer by 5. c) Subsequently, the sound field between two images is shifted against the tissue by a defined displacement distance so that the sonicated volumes of the examination object overlap, whereby the longitudinal axes of both volumes are parallel to each other. The longitudinal axis is defined as a line that runs in the direction of the direction of sound propagation (direction z in FIG. 6; from top to bottom in FIG. 5b) through the center of the sound field (see also FIG. 7). Accordingly, in the sense of the present invention, a parallel displacement of the two layers which overlap and from which ultrasound sectional images are obtained means that the

Longitudinal axes of the two layers are parallel to each other. d) If all bubbles were destroyed in the first image, only the newly added ones can be detected in the second image and all subsequent images. With a parallel shift of the sound field against the examination object of, for example, 10 μm, this means that the bubbles newly added in the second image come from a volume with a layer thickness of 10 μm. Since the displacement is carried out in parallel in the aforementioned sense, the layer thickness of the volume in which the bubbles which have not yet been destroyed are located is continuous, regardless of the shape of the sound field same, namely 10 μm. These bubbles which have not yet been destroyed can be quantified (FIGS. 5 b) and c)). Destroyed bubbles are labeled 7.

The principle of the method according to the invention can also be used for such bodies, preferably bubbles, which can be excited with diagnostic ultrasound to form an independent and characteristic signal independent of destruction. Such a signal can be, for example, a harmonic or subharmonic signal.

The procedure here is analogous to that of an SAE signal:

In the case of bubbles:

a) in the first image / a first layer all bubbles are destroyed, b) in the subsequent images / layers the bubbles are excited so that they give an independent, characteristic signal, c) as long as the cumulative scan distance is less than or equal to the layer thickness of the sound field, the signals that are added are determined in each additional image / layer, d) the cumulative scan distance is greater than the layer thickness of the sound field, or if signal saturation is reached in an image / layer, the bubbles in the sound field can be destroyed.

In the case of solid particles:

a) in the first image / a first layer, no independent, characteristic signals of solid particles can be detected after excitation; This can be achieved, for example, by starting the scan at a position that is free of particles, or by moving the transducer out of an area in which independent, characteristic signals of solid particles can be detected after excitation, before the scan begins. is moved to an adjacent area, in which this is not the case, b) in the subsequent images / layers, the solid particles become like this stimulated that they produce an independent, characteristic signal, c) as long as the cumulative scan distance is less than or equal to the layer thickness of the sound field, the newly added signals are determined in each image / layer.

If the transmission of the signal does not result in the complete destruction of the body, preferably the bubbles, the determination of the newly added signals can be determined by subtracting the respective pre-value or by simple, preferably automatic counting of the signals after sending a destruction pulse.

If the independent, characteristic signals are accompanied by the destruction of the body, preferably the bubbles, the number of signals in the overlap area can generally be determined by simple, preferably automatic, counting, as in the case of SAE signals.

With a parallel displacement distance of 10 μm against the tissue and a layer thickness of the sound field of 1 mm, for example, the sectional images (cut layers) overlap by 99%. In the second sectional image, only the newly added bodies, preferably bubbles, that are in the 10 μm layer are detected.

The distance of the shift between two images is at least 5 μm, preferably at least 10 μm and particularly preferably at least 20 μm. In a very particularly preferred embodiment, the displacement distance is at least 50 μm. The shift distance is smaller than the scan thickness (approx. 1 - 2 mm). The optimal distance shift depends on the highest body concentration / ml, preferably bladder concentration / ml in the scanned volume. It is advantageous to set an overlap of the volumes between 20% and 99.99%, preferably between 40% and 99.9% and particularly preferably between 70% and 99%. If the displacement distance is chosen too large or the ultrasound sectional images do not overlap at all (FIG. 5a), absolute quantification is also not possible with the method according to the invention, since there is an uncorrectable superposition of the CS signals in the ultrasonic sectional image.

The displacement distance can be chosen to be too large, for example, or the ultrasound sectional images may not overlap if the degree of accumulation of the body, preferably bubbles, is underestimated. In these cases, the displacement distance per ultrasound sectional image must be reduced in order to ensure absolute quantifiability. 9 to 11 show the relationship between the displacement distance and the morphometric units (MU) or the number of independent, characteristic signals in the sectional image.

The concentration of bodies, preferably bubbles, is preferably determined at those CS / SAE layer thicknesses whose sectional images each have 20 to 80%, preferably 30 to 70%, color saturation. In favor of a reduced examination time, however, the displacement distance can be increased as long as the saturation bubble concentration is not reached in any sectional image. A defined program that varies the CS / SAE layer thickness can ensure an optimal compromise between the examination time and the accuracy of the measurement. The optimal displacement distance controlled by the program can be determined, for example, on the basis of a sufficiently large population of patients (examination objects).

However, if individual contrast-saturated sectional images occur accidentally, the absolute quantifiability is no longer possible. However, quantification using the non-contrast-saturated sectional images remains possible.

Even if there is color saturation in certain areas of the examination area, the standardized scan process with a defined parallel shift of the sound fields and the higher spatial resolution of the method according to the invention enable a reproduced measurement. Such a measurement can be sufficient, for example, for a comparison between different patients or for a therapy course check for the same patient. If not all bubbles are destroyed in a sectional view, you can also send one or more further destruction pulses before moving to the next position.

The overlap and displacement of the volumes of the cut layers can be achieved by displacing the transducer and the examination object against one another (relative to one another).

The defined parallel shift can be achieved by

I. the tissue or the examination object, for example a patient, is shifted relative to a fixed transducer or

II. The transducer is moved relative to the fixed examination object to be examined, for example a tissue, or III. a transducer is used, which is able to create overlapping and shifted sectional images one after the other, i.e. the volumes are shifted and overlapped with a transducer, which itself takes overlapping and parallel shifted ultrasound images; For this purpose, the transducer can be controlled in such a way that overlapping and in particular transversely shifted ultrasound sectional images or sound lobes

Cut layer are included.

The different displacement options can also be combined.

The following variants of parallel displacement are described as examples:

I. The examination object (patient, animal, an eccentric or isolated organ or a cell culture dish) is automatically moved past the fixed transducer (e.g. driven by a servo motor or DC motor).

The transducer is automatically moved over / opposite the examination object (patient, animal, an eccentric or isolated organ or a cell culture dish) with the help of a special device. The device can be a frame within which the transducer is fixed in a holder and is moved by a motor drive along a parallel guide.

In the simplest case, a single crystal can be shifted in the x and y directions with defined overlap areas. This variant offers e.g. for culture dishes of cells on the surface of which bodies, in particular bladders, have accumulated specifically or inside which have become nonspecific, for example by phagocytosis. Here it is sufficient to state how many bodies, in particular bubbles, per area or volume have accumulated. The data does not have to be converted into pictures.

In addition, by means of a corresponding control of the scan, adjacent sound lobes can be brought to overlap within a line in the transverse direction in a defined manner with a transducer containing a 2D array.

III. With a transducer containing a 3D array, by correspondingly controlling the scan, adjacent sound lobes within a line or sound lobes of adjacent lines can be brought to overlap in a defined manner in the transverse direction. With this variant, further movement of the transducer or examination object is no longer necessary.

The transducer can also contain a 2D array, which is moved within the transducer in parallel with a defined overlap in the transverse direction (x direction). The lateral resolution can be increased within a 2D sectional image if the individual scan lines from which the sectional image is constructed also overlap in a defined manner. In contrast to conventional control, the individual sound fields, which are each generated by a group of neighboring piezo elements, are overlapped more strongly and above all by selecting small increments (down to a piezo element).

This is shown, for example, in FIG. 7: A small area 12 of piezo elements, comprising a few array elements, is excited to oscillate by an array of individual piezo elements 11 (FIG. 7 a). As a result, a sound wave in the form of a sound lobe 9 is emitted into the examination object. Undestroyed bubbles 8 located within the sound lobe are destroyed and thereby emit independent, characteristic signals 9. The destroyed bubbles are identified by the reference number 10. Subsequently, a newly formed group of piezo elements of the array is excited to vibrate, which differs from the group used in the first excitation by only one piezo element by which the group is now offset (FIG. 7b). Due to the overlap of the resulting sound lobe with the previously emitted sound lobe, only the newly detected bubbles are excited into independent, characteristic signals.

As with the defined overlapping scan in the transverse direction to the transducer, the resolution increases by the factor in which the displacement is related to the width of the piezo element group.

To move the transducer and / or the examination object, at least one motor drive is therefore provided for displacing the examination object or the at least one transducer relative to one another.

If the transducer is to be moved relative to the examination object, it must be moved in a defined way. For this purpose, a movable holder for the transducer is provided. The examination object is fixed in a holding device. Of course, it is not absolutely necessary to hold the examination object with the holding device by clamping, clamping or another similar fixation. It is also sufficient that the object is held practically motionless by the holding device. For example, a patient can be placed on a couch on which the patient rests during the examination. Such holding devices can be used for holding the examination object both when it is not moved during the examination, but only the transducer, and when the examination object is moved and the transducer is not. The above-mentioned holding devices can also be used for the examination object in the event that both the transducer and the examination object are moved against one another at the same time. To quantify the bodies, preferably bubbles, an image evaluation system for data processing and image display is provided in a preferred embodiment, with which the quantification can be carried out by video densitometry. For this purpose, the data sets formed from the ultrasound signal sets can be reshaped in a known manner in such a way that a video image of the individual cut layers is created, each cut layer containing the information from the areas which do not overlap with a previously excited cut layer (new cut layer area (CS / SAE-layer)). The bodies represented in the individual video images, in particular bubbles, can then be counted manually or obtained by video-densitometric determination of the color image points (color pixels) and division of the value obtained therefrom by the average number of color pixels per bubble representation.

After the number of bodies, preferably bubbles, has been determined using the method described above, it may be necessary to make a corrected determination of the number using the following formula:

V FP

MUcorr = ∑FPx 1 + ∑FP

∑ + ∑

in which

FP are the color pixels representing the body, in particular bubble signals, in a locally resolved screen representation within the overlap layer and GP are screen gray pixels in a locally resolved screen representation, which are not the pixels representing the body, in particular bubbles.

The device according to the invention is particularly suitable for ultrasound diagnosis and / or therapy on humans or animals. Not only in the areas of clinical diagnostics, but also in basic medical and biological research and pre-clinical research and development, new possibilities of investigation open up:

The bodies, preferably bubbles, can be tested in vivo in animal experiments, ex vivo in harvested organs or tissues and in vitro e.g. be quantified in cell cultures in order to clarify pathological or physiological processes at the molecular level.

In the process of finding active substances and developing specific binding systems, the method according to the invention can be used to test which binding systems work at all, which binding molecules are present under which conditions at which locations and in what quantity, which influencing variables determine the binding capacity and accumulation, what kinetics the body, preferably bubbles, or , in the case of ultrasound-induced active ingredient release, what amounts of an encapsulated active ingredient are released.

The device according to the invention and the method according to the invention can be used, for example, for the / nv / Vo mapping of physiologically high or low regulatable molecular markers in organs and tissues from postembryonic ontogenesis (ontogenesis: total development of an individual from the unfertilized / fertilized egg cell to to natural death: this comprises four phases: 1. embryonic development, 2. postembryonic development, 3. time of sexual maturity and reproduction and 4. time of aging).

For this purpose, both passively and actively accumulative bodies, preferably bubbles, can be used: For this purpose, the bodies can be provided with binding molecules specific for the markers that are located on the surface (actively accumulable bodies / bubbles). In an alternative embodiment of the invention, passively accumulative bodies can also be used, i.e. Bodies / bubbles without such specific binding molecules.

With this method, molecular markers can be localized in vivo on different cells in humans and animals and their influence on ontogenesis can be determined. Furthermore, a time-controlled mapping of known and newly identified molecular markers in the whole organism can be carried out in vivo. This enables a previously unknown function of the molecular marker in ontogenesis to be determined in vivo.

Of particular interest for gerontology is the identification of molecular markers in the 4th phase of ontogeny (time of aging) and their influence on aging processes. For this purpose, the spatially high-resolution sonography according to the invention can be used to investigate the signs of aging from which these molecular markers originate and which they can lead to, furthermore what basis these receptors represent for age-related changes or diseases of the whole organism and whether there are any findings for development be derived from medications that alleviate age-related complaints or even help to avoid them.

In a further interesting application of the device according to the invention and the method according to the invention, a / n-wVo mapping of pathologically high or low regulatable molecular markers in organs and tissues during pathogenesis can also be carried out using passively and actively accumulative bodies, in particular bladders ,

With this method, disease-associated molecular markers can be localized in vivo on different cells in humans and animals and therefore their influence on the pathogenesis of diseases in vivo can be determined.

Furthermore, a time-controlled mapping of known and newly identified disease-associated molecular markers in the whole organism can be carried out in vivo during the pathogenesis. As a result, a function of the molecular markers in pathogenesis in vivo which has not yet been known can be determined.

Furthermore, new therapeutic drugs and / or treatment strategies can be derived from this knowledge by the fact that the determined surface states of cells (status of the cells with regard to the ac- tivierung / the rest state) regulated and these states are tracked with the inventive method.

Finally, a highly sensitive therapy course control in vivo is also possible by tracking the surface conditions of cells.

The above methods can also be performed ex vivo on harvested organs and tissues.

The in v / Vo mapping of the molecular markers in the organs and tissues from postembryonic ontogeny and during pathogenesis can of course be applied to various animal species and / or animal models, in particular in gerontology or for disease models. The mapping can generally also be used for all animal species, for example agricultural livestock, domestic and domestic animals and wild animals, and for humans. Since mapping can be used in vivo, there is often no need for time-consuming and financially expensive conventional in-house processes, for example immunohistochemistry, which often have the additional disadvantage of having to identify and identify molecular markers at exactly one point in time to characterize.

Furthermore, the device according to the invention and the method according to the invention can also be used for the characterization of cell cultures by ro-mapping of molecular markers of cells of different origin that can be regulated up and down, for example under different cultivation conditions or for characterizing new cell lines or for re-characterizing existing ones Cell lines after different passages or immediately before their application, especially when implanting tumor cells in mice, rats and other animal models. In this case, plant cells can also be characterized in addition to animal and human cells.

In this case too, the bodies, preferably bubbles, can be provided with binding molecules specific for the markers. For the identification of molecular markers in the respective areas of application, reference is expressly made to the following exemplary publications, which are hereby incorporated by reference into the disclosure content of the present application, the issues described therein being able to be investigated using the device and the method according to the present invention:

1. Phelps, M .: "Positron emission tomography provides molecular imaging of biological processes". Proc. Natl. Acad. Soc, USA 2000, 97: 9226-9233.

2. Weissleder, R., "Molecular imaging: exploring the next frontier". Radiology, 1999, 212: 609-614.

3. Bremer, C, Tung, C.H., Weissleder, R., "In Vivo Molecular Target Assessment of Matrix Metalloproteinase Inhibition". Nat. Med., 2001, 7: 743-748.

4. Anderson, SA, Rader, RK, Westlin, WF, et al., "Magnetic Resonance Contrast Enhancement of Neovasculature with Alpha _v ß ₃ -Targeted Nanoparticles". Magn. Reson. Med., 2000, 44: 433-439.

5. Sipkins, DA, Cheresh, DA, Kazemi, M.R., Nevin, L.M., Bednarski, M.D., Li, K.C., "Detection of Tumor Angiogenesis In Vivo by alphaVbeta3-Targeted Magnetic Resonance Imaging". Nat. Med., 1998, 4: 623-626.

6. Bredow, S., Lewin, M., Hofmann, B., Marecos, E., Weissleder, R., "Imaging of tumor neovasculature by targeting the TGF- [beta] binding receptor endoglin".

Eur. J. Cancer, 2000, 36: 675-681.

7. Sipkins, DA, Gijbels, K., Trapper, F.D., Bednarski, M., Li, K.C.P., Steinman, L., "ICAM-1 expression in autoimmune encephalitis visualized using magnetic resonance imaging". J. Neuroimmunol., 2000, 104: 1-9. 8. Lindner, J.R., Song, J., Christiansen, J., Klibanov, A.L., Xu, F., Ley, K., "Ultrasound assessment of inflammation and renal tissue injury with microbubbles targeted to P-selectin". Circulation, 2001, 104: 2107-2112.

10. Klibanov, AL, Hughes, MS, Marsh, JN, et al., "Targeting of ultrasound contrast material: An in vitro feasibility study". Acta Radiol. Suppl., 1997, 38: 113-120. 11. Demos, SM, Dagar, S., Klegerman, M., Nagaraj, A., Mcpherson, DD, Onyuksel, H., "In vitro targeting of acoustically reflective immunoliposomes to fibrin under various flow conditions". J. Drug Target, 1998, 5: 507-518.

12. Villanueva, F.S., Jankowski, R.J., Klibanov, S., et al., "Microbubbles Targeted to Intercellular Adhesion Molecule-1 Bind to Activated Coronary Artery Endothelial Cells". Circulation, 1998, 98: 1-5.

The device and the method for quantifying bodies, preferably bubbles, therefore allow or support, for example:

- a dignity determination of tumors,

- staging of tumors,

- a therapy progress check e.g. in chemotherapy,

- an increase in reproducibility and thus the comparability of findings from different examiners,

- avoidance of biopsies, which are stressful for the patient,

a reduction in the layer thickness from which the CS signals shown in the ultrasound image originate, as a result of which bubble enrichment regions in the millimeter range can be spatially high-resolution depicted in the micrometer range and quantified with regard to the bubble concentration.

The bodies, in particular bubbles, are usually contained in ultrasound contrast media and are preferably applied in the form of suspensions.

In principle, all bodies, in particular (micro) bubbles, which are suitable for use as ultrasound contrast media, can be excited with diagnostic ultrasound to produce CS effects. For the commercially available ultrasound contrast agent Levovist ^® (Schering AG, Berlin), which contains microparticles from galactose and palmitic acid and which results in a suspension of stabilized gas bubbles after the addition of water, CS effects were also observed after unspecific accumulation in the liver (Blomley, MJ et al .: "Stimulated acoustic emission to image a late liver and spleen-specific phase of Levovist in normal volunteers and patients with and without liver disease". Ultrasound Med. Biol., 1999, Nov., 25 (9): 1341- Particulate ultrasound contrast media containing bubbles in the form of gas-filled microcapsules or gas-filled microparticles, the shell of which consists of polyalkyl cyanoacrylates, in particular polybutyl cyanoacrylate, are preferred according to the invention. These are described in EP 0 644 777 B1, in particular in the examples: Thereafter, cyanoacrylic acid alkyl esters, for example butyl cyanoacrylic acid, are dispersed in acidic aqueous solution which additionally contains a surfactant. The resulting microparticles can be obtained by centrifugation. Bubbles with functionalized polyalkyl cyanoacrylates, which are produced according to the process described in WO 01/68150 A1, are particularly preferred. The method described there is therefore expressly included in the disclosure content of the present application. The polymers described in WO 01/68150 A1 are used to produce the bubbles preferred according to the invention. Therefore, these starting materials for producing the bubbles are expressly included in the disclosure content of the present application.

The method according to the invention can also be used for ultrasound contrast media which contain different types of body, preferably types of bubbles.

The bodies can differ in at least one of the following characteristics:

- type of solid material,

- size,

- binding molecules on their surface, - excitability through different pulse shapes,

- destructibility by sound,

- type of characteristic signal

- type of gas in the body / bladder,

- type of shell of a body / bladder, e.g. Material thickness, elasticity ,.

For example, such bubble mixtures or their distribution patterns in the examination object can also be quantified with high spatial resolution using the method according to the invention, a) by sending different pulse shapes in succession at a position and / or receiving different characteristic signals; if the bubbles are not completely destroyed, a destruction pulse can be sent before moving to the next position; or, b) by the area to be examined being scanned several times with a correspondingly defined displacement, each scan being carried out with a different pulse shape required for the respective bubble type.

Different pulse shapes as well as different sound field shifts can be combined with each other, as long as the resulting overlap of the sound fields is defined and can be calculated with regard to their spatial expansion and as long as the bubbles used can be detected using characteristic signals.

If the bubbles differ simultaneously in the binding molecule on the surface and in their destructibility by sound, the following can be used to determine the in-vivo, ex-vivo and in-v / fro distribution of the binding sites in the examination region:

a) Two different types of bubbles are injected: i. Bubble type A, destructible with low sound pressure and with binding molecule

Anti-X for in vivo binding site X on the surface, and ii. Bubble type B, destructible with high sound pressure and with anti-Y binding molecule for in vivo binding site Y on the surface, b) Approaching the calculated position, c) Taking one or more images with low sound pressure, data storage, d) Taking one or several images with high sound pressure, data storage, e) defined shift of the sound field, f) repetition of steps c) - e) until the end of the scan, g) evaluation of the data for the binding molecules X and Y, their display on the Monitor and storage. In all the variants mentioned, the saturation bubble concentration is increased by the factor in which the distance covered between two images or sound lobes is related to the layer thickness of the entire sound field. With a 10 μm shift and a layer thickness of 1 mm, the saturation bubble concentration increases by a factor of 100. At the same time, the layer thickness of the tissue from which the newly added bubbles originate is reduced by the same factor.

Histological resolution can be achieved in this way. Since this principle can also be used in vivo, one can speak of an “in wVo sono histology” or, in the case of a specific bladder accumulation, a sonographic “/ π-Vo immunohistology” with regard to the signals shown.

The measured MU can be evaluated for diagnosis as follows, for example:

A. Comparison of the measured MU with a constant CS / SAE layer thickness and dose for the intraindividual follow-up of a therapy.

B. 1) First determine the dependence of the measured MU on the CS / SAE

Layer thickness and / or dose in a special measurement window (liver, tumor surface, vascular endothelium, etc.) based on the largest possible population. The population can consist of individuals who do not differ in the distribution and the degree of enrichment of the bubbles in the measurement window, i.e. which is homogeneous or which is grouped in this regard. These dependencies generally have to be determined with various ultrasound device settings (transducer, detection mode, etc.).

2) Determination of linear correlation functions in the range from 20 to 80%, preferably in the range from 30 to 70% color saturation.

3) If necessary Integration of the linear correlation functions in an evaluation software of the ultrasound device.

4) Counting the CS signals in the corresponding measurement window of an examination object, for example a patient. 5) Using the linear correlation functions: Determination of the absolute number of bubbles or the bubble concentration in the special measurement window of the patient if color saturation is within the scope of the linear correlation function.

C. 1) First, determine the dependence of the absolutely counted CS signals on the CS / SAE layer thickness and / or dose in a special measurement window (liver, tumor surface, vascular endothelium, etc.) based on the largest possible population. The population can consist of individuals who do not differ with regard to the distribution and the degree of enrichment of the bubbles in the measurement window, i.e. which is homogeneous or which is grouped in this regard. These dependencies usually have to be determined with various ultrasound device settings (transducer, detection mode, etc.). 2) Execution of curve fits, determination of correlation functions describing the respective curve.

3) If necessary Integration of the correlation functions in an evaluation software of the ultrasound device.

4) Counting the CS signals in the corresponding measurement window of a patient. 5) Using the correlation functions: Determination of the absolute number of bubbles or the bubble concentration in the special measurement window of the patient.

The following examples are intended to illustrate the invention, without the scope of the invention being restricted thereby. For this purpose, reference is also made to the following figures. They show in detail:

Fig. 12: bladder accumulation in the liver, rat 1 (example 2): 1 * 10 ⁸ bubbles / ml; Ex vivo measurement, 30 min. pi a: SAE layer thickness = approx. 1 mm b: SAE layer thickness = 20 μm

Fig. 13: bladder accumulation in the liver, rat 2 (example 2): 1 ^* 10 ⁷ bubbles / ml; Ex vivo measurement, 30 min. pi a: SAE layer thickness = approx. 1 mm b: SAE layer thickness = 20 μm

Fig. 14: Dependence of the MU on the SAE layer thickness

Rat 1 (Example 2): 1 * 10 ⁸ bubbles / ml; Ex vivo measurement, 30 min.

Fig. 15: Dependence of the MU _co "on the SAE layer thickness

Rat 1 (Example 2): 1 * 10 ⁸ bubbles / ml; Ex vivo measurement, 30 min.

Fig. 16: Dependence of the SAE _CO r _r on the SAE layer thickness rat 1 (example 2): 1 * 10 ⁸ biases / ml; Ex vivo measurement, 30 min.

Fig. 17: Dependence of the MU on the SAE layer thickness

Rat 2 (Example 2): 1 * 10 ⁷ bubbles / ml; Ex vivo measurement, 30 min.

Fig. 18: Dependence of the MU _corr on the SAE layer thickness

Rat 2 (Example 2): 1 * 10 ⁷ bubbles / ml; Ex vivo measurement, 30 min.

Fig. 19: Dependence of the SAE _∞ rr on the SAE layer thickness

Rat 2 (Example 2): 1 ^* 10 ⁷ bubbles / ml; Ex vivo measurement, 30 min.

20: Enrichment of specific bubbles, on the surface of which anti-CD-105 antibodies are coupled, in the F9 teratoma of a mouse (Example 3) a: Graphical representation b: Longitudinal sonographic section of the tumor in the scan direction area between lines 16 and 17: tumor on first scan

Area between lines 18 and 19: tumor in the second scan

21: Color signals of the specific bubbles enriched in the tumor (four mice, example 3) in comparison to the isotype control (four mice, example 3). Example 1 :

Agar phantom containing 30,000 bubbles / ml:

(a) Making the Bubbles:

The bubbles are made according to Example 4 (a).

(b) Preparation of the agar phantom containing 30,000 bubbles / ml:

® 665 ml of water (Ampuwa, Fresenius) were in a kettle (Braun, type 3

217 WK 210) boiled and transferred to a 1000 ml beaker. 1.7 g of benzoic acid (Merck) for preservation and 10 g of agar (Merck) were added with stirring. The agar solution was boiled again briefly in a microwave (Bosch HMT 702 C, 800 W) and then cooled to 40 ° C.

Before the bubbles were added to the agar solution, the bubble concentration was adjusted to 6 * 10 ⁸ bubbles / ml with 0.02% (m / m) Triton X 100 solution. Thereof were carefully stirred without introduction of air bubbles into the solution Agarlö- 33 ul ^(* 10 ⁷ bubbles 2). The agar solution was then placed in a glass bowl

(14 cm * 19 cm * 4 cm), in which this solidified as a gel (agar phantom).

(c) Experimental setup and implementation:

An approximately 4 cm wide strip of the agar phantom was placed in a plastic basin filled with water. A transducer (L 10-5, ATL UM9, color Doppler mode, MI: 1, 1, persistence: 0, priority: maximum, size of the color box: maximum, depth of penetration: 3 cm, focus: 2 cm) was with a Tripod mounted vertically above the phantom so that it dipped into the water.

The pelvis was made together with the phantom 1. moved by hand in the transverse direction under the transducer at a speed of approx. 2 cm / s, which corresponded to a displacement distance of approx. 1 mm / frame, or

2. placed on a servo motor (Limes 150, OWIS GmbH, motor controller DC 500) and automatically passed under the fixed transducer at different speeds. The speeds were chosen so that taking into account the given frame rate of the ultrasound device of 5.8 images / second and continuous movement of the agar phantom, the following displacement distances per image resulted: 10, 20, 30, 40, 50, 60, 70, 80 , 90, 100 μm / frame. The speeds required for this

(μm / s) were calculated as follows:

Speed [μm / s] = displacement distance / image [μm / frame] x frame rate of the ultrasound device [frame / s]

(d) Quantification of the SAE signals:

® The resulting video images were evaluated video quantitatively using QuantiCon (Echotech 3D Imaging Systems GmbH) as follows:

During the scan, the video images were digitized at a sample rate corresponding to the frame rate given by the ultrasound device (here 5.8 frames / second) (QuantiCon). For this purpose, a trigger delay of 172 ms (1000 ms / 5.8) was previously entered in QuantiCon.

In order to separate colored noise signals from SAE signals, a threshold value of 40 was first defined for the color values before the evaluation of the 3D data set. Only the "segmented color values" (40-255) then remaining above this threshold value were used for evaluation. FIG. 8 shows the 3D view of the scanned agar phantom as it was generated from the 3D data set in Quanticon the number of pixels of the gray values as well as the number of pixels of the segmented color values of all images in the data set were exported and further processed with MS-Excel ^® (Microsoft). The graphic of the segmented color values (MU) created in MS Excel is shown in FIG. 9 as a function of the SAE layer thickness. Each individual color value was corrected in accordance with the correction formula defined under “corrected MU”. The resulting dependence on the SAE layer thickness is shown in FIG. 10 (MU _corr .).

In order to convert the sum of the segmented color values into individual SAE signals or individual bubbles, the conversion factor F _sae was first determined. For this purpose, the sum of the SAE signals was first determined by counting by hand in 20 sectional images. The sum of the segmented color pixels was then divided by the sum of the SAE effects counted by hand in the corresponding images. An average of 44 color pixels per SAE signal was determined. The graphic converted from MU _cor r .. to the number of corrected SAE signals (SAE _CO rr.) By this factor is shown in FIG. 11.

The data on which FIGS. 9 to 11 are based are listed in Table 1: Table 1:

The curve range between 30 and 70 μm SAE layer thickness (~ 37 - 81% color saturation gung) can be described with a linear correlation function. Correlation coefficients are 0.990 for the uncorrected MU and 0.996 for the corrected MU / SAE.

If the points outside this range are also taken into account in the linear correlation, the correlation coefficients worsen down to 0.971 for MU and 0.981 for the corrected MU / SAE.

(e) Result:

(a) All images that were created when the phantom was moved by hand and a displacement distance of approx. 1 mm / frame are completely saturated with SAE signals. The saturation bubble concentration has been exceeded and quantification is impossible. (b) In the automatic shift with overlapping of the sound fields, only a few SAE signals were detected with a shift of 10 μm / frame (FIG. 8). With increasing speed, the number of SAE signals / frame also increased. Only after a shift of 100 μm / frame did the image become saturated in color.

Thus, a bubble concentration of 30,000 bubbles / ml with a layer thickness of 100 μm leads to SAE saturation. This results in a saturation bubble concentration of approx. 3,000 bubbles / ml for a layer thickness of 1 mm.

(f) Discussion:

In contrast to conventional scanning methods, the method according to the invention enables bubble quantification even with bubble concentrations greater than 3,000 bubbles / ml.

Due to the defined overlapping displacement of an object against the transducer, only the newly added bubbles from the non-overlapping volume are detected in each sectional image. In this way, the SAE layer thickness can be reduced to the respective displacement distance per image. The saturation bubble concentration increases in the ratio in which the actual layer thickness of the sound field relates to the displacement distance or to the SAE layer thickness. A 10 μm shift from image to image simultaneously increases the spatial resolution and the saturation bubble concentration by a factor of 100 with a layer thickness of the sound field of 1 mm.

Example 2:

Ex vivo quantification of nonspecifically enriched blisters in the liver of rats

(a) Making the Bubbles:

The bubbles were made according to Example 4 (a).

(b) Investigation:

Two two rats (Wistar SCHOE, 220g, female) were examined. The rats were injected iv with various doses of the bladder suspension prepared according to a) via the tail vein. For rat 1 a dilution to 1 * 10 ⁸ bubbles / ml, for rat 2 to 1 * 10 ⁷ bubbles / ml in 0.9% (m / m) NaCl solution containing 0.02% (m / m ) Triton X 100.

Rat 1: 1 * 10 ⁸ bubbles / kg (1 * 10 ⁸ bubbles / ml, 220 μl iv) Rat 2: 1 * 10 ⁷ bubbles / kg (1 * 10 ⁷ bubbles / ml, 220 μl iv)

30 minutes after injection, the rats were injected i.p. Injection of 1 ml of a 1: 1 volume mixture of Rompun 2% (20 mg xylazine / ml) and Ketavet 100 mg / ml (100 mg ketamine base / ml) killed:

The right lobe of the liver was removed and placed in a vessel filled with 0.9% (m / m) sodium chloride solution.

The vessels with the liver lobes were:

1. moved by hand in the transverse direction under the transducer at a speed of approx. 2 cm / s, which corresponded to a displacement distance of approx. 1 mm / frame or 2. placed on a servo motor (Limes 150, OWIS GmbH, motor controller DC 500) , and automatically passed at different speeds under the fixed transducer; the speeds were chosen so that, taking into account the given frame rate of the ultrasound device of 5.8 images / second and continuous movement of the vessel, the following displacement distances per image resulted:

Rat 1 (1 ^* 10 ⁸ bubbles / kg): 15, 20, 30 μm / frame Rat 2 (1 ^* 10 ⁷ bubbles / kg): 20, 40, 80, 120 μm / frame The ultrasound examination was carried out analogously to Example 1 with the same device settings. The liver was moved past the transducer at a distance of 2 cm. The same ROI was used for all measurements. The size of the ROI (approx. 2 mm * 6 mm, measured on the scale of the ultrasound device) was chosen so that the ROI was completely in the area of the liver tissue for all evaluated images. The same magnification was selected in Quanticon ^® for all measurements.

To determine the ratio of screen pixel to object length [mm], a square ROI of 2 mm edge length (determined using the length measurement scale shown in the ultrasound image) was measured in the ultrasound image. For this purpose, the image was enlarged accordingly using the zoom function available in Quanticon ^® . The pixel sum in this ROI (MU) was 225. From this, a ratio of Embedding 56.25 pixels / mm ² (225/4) could be calculated. The bladder concentration / ml liver was then calculated from the measured volume (area of the ROI x displacement distance / frame). The following formula was used to convert the raw data (FP, GP, S) into the bladder concentration C in the tissue:

C: bubble concentration in [bubbles / ml] or [SAE _cor r / ml] S: SAE layer thickness, or the shift between two images in [mm]

FP: Screen pixels that lie within the measurement window (ROI) in the area of the SAE effects shown. GP: Screen pixels that lie within the measurement window (ROI) outside of the SAE effects shown. ∑sae: Sum of the SAE signals shown in an ultrasound image

F _f : sum of all screen pixels in mm ² (measured using the length scale shown in the ultrasound image)

(c) Result:

The bladder concentrations of the rats are compared with one another in Table 2. Tab. 2 also contains the mean values of the percentage color saturation for the respective SAE layer thickness.

Tab. 2

1. All images that were created when the liver was shifted by hand and a shift distance of approximately 1 mm / frame were completely saturated with SAE signals (FIG. 12a, FIG. 13a). The saturation bubble concentration was exceeded and quantification was impossible.

2. During the automatic shift with overlapping of the sound fields, SAE signals in a quantifiable number were detected in rat 1 (dose: 1 * 10 ⁸ bubbles / kg) at SAE layer thicknesses of 20 to 120 μm / frame (FIG. 12b: SAE -Layer thickness: 20 μm), in rat 2 (dose: 1 ^* 10 ⁷ bubbles / kg) in SAE layer thicknesses from 10 to 30 μm / frame (FIG. 13b: SAE layer thickness: 20 μm). With increasing speed, the number of SAE signals / frame also increased.

3. In rat 1, a linear correlation coefficient of 1,000 was calculated for the dependence of the uncorrected MU on the SAE layer thickness (FIG. 14). The linear correlation coefficient for the dependence of the corrected MU (MUc _o rr.) Or corrected SAE (SAE _cor r.) On the SAE layer thickness between 15 μm and 30 μm was in each case 0.995 (FIGS. 15 and 16).

4. In rat 2, a linear correlation coefficient of 0.984 was calculated for the dependence of the uncorrected MU on the SAE layer thickness (FIG. 7). The linear correlation coefficient for the dependence of the corrected MU (MUc _o rr.) Or corrected SAE (SAE _cor r.) On the SAE layer thickness between 20 μm and 120 μm was in each case 0.995 (FIGS. 18 and 19).

5. The number of bubbles is in a linear relationship to the SAE layer thickness.

(d) Discussion:

The method according to the invention can also be used in removed organs in which bubbles accumulate non-specifically. There is a close correlation between the number of SAE signals shown in the sectional view and the corresponding SAE layer thickness. The bladder concentration in the livers of the rats examined differs significantly depending on the dose applied.

Example 3:

In v / Vo quantification of specifically enriched USKM in the F9 teratoma of the mouse:

With the method according to Example 1, the accumulation of specific bubbles, to the surface of which anti-CD 105 antibodies were coupled, in tumors of F9 tumor-bearing mice (swiss-nude) after iv injection in vivo was examined. As Control substance served bubbles, on the surface of which IgG2a antibodies (isotype control) were coupled.

(a) Production of specific bubbles:

The specific bubbles were prepared and characterized according to Example 4 (a) to (e). The antibody coupling was carried out according to Example 4 (f).

(b) Preparation of the isotype control:

The isotype control bubbles were prepared and characterized according to Example 4 (a) to (e). The isotype coupling was carried out according to Example 4 (g).

(c) Tumor Inoculation:

F9 tumor cells grown subconfluently in culture were trypsinized, centrifuged and resuspended in PBS with Ca ²⁺ / Mg ²⁺ . After staining with trypan blue and calculation of the cell concentration, the cell suspension was adjusted to a concentration of 6 * 10 ⁷ cells / ml. The cell suspension was chilled on ice until use. From this cell suspension, ten female nude mice (Swiss.nude, 18-20g KGW) each 50 μl per animal were subcutaneously (= 3 * 10 ⁶ cells / animal) inoculated into the left flank region using a Hamilton syringe and 27G cannula. The growth of the tumors (length and width) was checked by measurement with a caliper, and the volume of the tumor was approximately determined using the following formula:

L x W ^2/2

L: length [mm] W: width [mm]

The animals were examined 12 to 14 days after tumor inoculation. In the present study, the tumor sizes at that time were between 230 and 1600 mm ³ . (d) Application of the specific bubbles and the isotype control

Four mice were given an ultrasound contrast agent, containing specific bubbles (produced and characterized according to Example 4 (a) to (e) and antibody coupling according to Example 4 (f)), in the waking state iv in a dose of 1 * 10 ⁹ bubbles / kg body weight injected (corresponded to 200 μl of the suspension according to Example 4 (g) each 20 g mouse). The specific bubbles were gas-filled microcapsules, the shell of which consisted of functionalized polybutyl cyanoacrylate and to whose surface anti-CD105 antibodies were coupled. As a control substance, four further mice were injected in the same way with an ultrasound contrast agent containing non-specific bubbles (produced and characterized according to Example 4 (a) to (e) and isotype coupling according to Example 4 (g)). The non-specific bubbles were gas-filled microcapsules, the shell of which also consisted of functionalized polybutyl cyanoacrylate, but to the surface of which IgG _2a (isotype control) was coupled.

The tumors of all animals were examined 60 minutes after application of the respective ultrasound contrast agent with regard to the degree of enrichment using the method according to Example 1.

(e) Ultrasound examination:

For the study, the mice were treated with a 1: 1 volume mixture of a dilution (1 + 9) of Rompun ^® 2% (20 mg xylazine / ml) and a dilution (1 + 4) of Ketavet ^® 100 mg / ml (100 mg Ketamine base / ml) anesthetized in physiological NaCl solution. 200 μl each of 20 g of mouse were injected ip from this anesthetic mixture.

The mouse was attached to a carrier that was attached to the moving part of a servo motor (Limes 150, OWIS GmbH, motor controller DC 500) with adhesive tape. The servo motor was adjusted vertically with a tripod so that the carrier reached into a water basin (cut, square plastic bottle) with the mouse. Before each scan, the pool was washed with fresh water at a temperature of 37 ° C crowded. A transducer (L 10-5, ATL UM9) was provided with coupling gel and placed horizontally on the side wall of the pool with a tripod. The mouse was moved so far into the pelvis under visual and sonographic control that the tumor had not yet reached the sound field. If air bubbles adhered to the skin, they were removed by gently stroking the skin with a round wire that had been smeared with liquid soap. The mouse was then moved back a few millimeters. Before the scan, the necessary device settings were set on the ultrasound device (color Doppler mode, MI: 1, 1, persistence: 0, priority: maximum, depth of penetration: 3 cm, focus: 2 cm). The scan was run with a constant SAE layer thickness of 285 μm / frame. The tumor was in the focus area. After the scan, the image was "frozen" so that the transducer sent no signal. The mouse was moved back to the starting position. The transducer was switched on again and the animal was scanned again with the same device settings and the same SAE layer thickness a second scan was needed to identify color signals that were not caused by SAE signals and served as a control value, such signals could have been caused by flowing blood or larger air bubbles.

With the anesthetic used here, the animal's respiratory rate was generally low enough to be able to perform a scan of approx. 3 cm with the SAE layer thickness used here within one pause without movement artifacts (Fig. 20b). With longer examination times (e.g. with a lower SAE layer thickness) or with a higher breathing frequency, breathing artifacts could be avoided as follows:

a) fixation of the animal on a plastic carrier with a recess of at least the tumor size so that the tumor was in the recess, b) measurement of the animal's breathing movement (e.g. with strain gauges, dynamic pressure measurement methods, etc.) and use as a trigger signal for control tion of both the ultrasound device and the servo motor. (f) Evaluation:

A sectional image was generated in the scan direction (FIG. 20b) and this was superimposed on the same scale with the corresponding graphic of the measured values (FIG. 20a). The sum of the colored screen pixels within the tumor was evaluated with Quanticon® (3D-Echotech). Only the color signals in the tumor (MUFP: 20) and not the gray signals (MUGP: 21) were taken into account for the evaluation. Lines 16 and 17 drawn in FIGS. 20a / b delimit the tumor area during the first scan. The area of the second scan is shown between lines 18-19.

In addition, the following means in detail: Reference number 20: color signals (MUFP), reference number 21: gray signals (MUGP), reference number 22: sum of MUFP and MU _G p, or the measured tissue volume.

(g) Result:

For the isotype control, significantly fewer color signals (MU) were measured in the tumor (FIG. 21).

(h) Discussion:

The result shows that the method according to the invention is also suitable for the quantification of specifically enriched bubbles in the living organism. With the method according to the invention, a significant difference can be detected even if scanning is not carried out with maximum spatial resolution and SAE saturation occurs in some areas of some sectional images. The number of signals for a specific bladder accumulation can be distinguished from residual signals of the blood vessel system and / or signals of an unspecific bladder accumulation. Example 4: Preparation of the ultrasound contrast media:

(a) Preparation of the microcapsule suspension:

7 l of an aqueous 1% strength (m / m) octoxynol solution at a pH of 2.5 were placed in a cylindrical steel reactor (internal dimensions: height = 37 cm, diameter = 29 cm) and mixed with a mixer (dissolver disc: Diameter = 60 mm, stirrer shaft: length = 30 cm, immersion angle = 10 °) with high shear rate (4000 rpm) mixed and tempered (temperature of the solution = 32 ° C), so that self-gassing (i.e. an air entry into the Medium with strong foaming) took place. 100 g of butyl cyanoacrylic ester were added rapidly (<3 minutes) and dispersed (immediately before the addition, the speed of rotation of the mixer was increased to 8000 rpm). The polymerization was carried out for 60 minutes with self-gassing, during which gas-filled microcapsules (bubbles in the sense of the definition on which the application is based) formed. The flotate in the gravitational field of the earth was separated in a separating funnel for three days, the shelter was drained and the flotate was resuspended with 3 l of an aqueous 0.02% (m / m) octoxynol solution. The microcapsule suspension thus obtained had a polymer content of 9.46 mg / ml, a density of 0.943 g / ml and a pH of 3.5.

(b) Particle size and concentration of the gas-filled microcapsules:

The particle size distribution of the microcapsule suspension according to Example 4 (a) was determined using a particle counter from Particle Sizing Systems, type AccuSizer 770 (measuring medium: aqueous 0.02% (m / m) Triton X100 solution). The volume-weighted particle size distribution ranged from 0.8 to 10 μm with a maximum at 1.8 μm, and the microcapsule concentration was 7.2 * 10 ⁹ (± 1 * 10 ⁸ ) particles per ml.

(c) Functionalization of the gas-filled microcapsules by partial side chain hydrolysis:

2,500 g of a microcapsule suspension according to Example 4 (a) were mixed with stirring with 501 g of sodium hydroxide solution with a concentration of 8 * 10 ^"2 mol / l. In the reaction mixture the pH was 12. The mixture was stirred at room temperature for 20 minutes. The pH was then adjusted to pH 3.5 with 1 N hydrochloric acid.

(d) Particle size and concentration of the gas-filled microcapsules:

The particle size distribution of the microcapsule suspension according to Example 4 (c) was determined using a particle counter from Particle Sizing Systems, type AccuSizer 770 (measuring medium: aqueous 0.02% (m / m) Triton X100 solution). The volume-weighted particle size distribution ranged from 0.8 to 10 μm with a maximum at 1.8 μm, and the microcapsule concentration was 6.0 * 10 ⁹ (± 1 * 10 ⁸ ) particles per ml.

(e) Binding of streptavidin to functionalized gas-filled microcapsules:

The microcapsule suspension according to Example 4 (c) was purified by flotation at least five times against 0.02% (m / m) Triton-X100 solution (pH = 4.5, adjusted with hydrochloric acid). 7.5 * 10 ⁸ particles of the cleaned suspension were placed in a scintillation vial with 15 ml of sodium acetate buffer and stirred on a magnetic stirrer using a stick stirrer. (Sodium acetate buffer = solution A: 1.36 g NaCH ₃ COO * 3 H ₂ O, M = 136.08 g / mol, Riedl-de Haen ad 500 ml water, solution B: 571 μl glacial acetic acid, M = 60, 05 g / mol, ad 500 ml of water, 400 ml of solution A were introduced and adjusted to a pH of 4.5 with solution B.)

300 μg streptavidin (company SIGMA) were dissolved in 300 μl sterile-filtered water. The solution was added to the sodium acetate buffer microcapsule suspension. The pH should be 4.5 ± 0.2. Then 750 μg EDC (1 ethyl 3- (3-dimethylaminopropyl) carboximide hydrochloride (SIGMA) were quickly dissolved in 750 μl sterile-filtered water and added to the buffer solution.

The mixture was then stirred for an hour with occasional pH control. The mixture was stirred further at 4 ° C. overnight (approx. 17 h).

Finally, a solution of 188 μl of 1 M ethanolamine solution (SIGMA) in 3.75 ml of water was added to the mixture. After flotation (24 h), the mixture was concentrated to a volume of 1 ml by draining off the shelter. 10 ml of Hepes buffer / Triton solution 0.01% (m / m) (30 ml of 5 M NaCl solution + 10 ml of 1 M Hepes solution (Gibco 15630-056) ad 1000 ml of Triton solution, 0 , 01% (m / m), pH 7.4), floated again and the shelter drained again to a volume of 1 ml. The concentrated suspension was then transferred to a siliconized Eppendorf reaction vessel and stored in the refrigerator.

The coupling success was checked in the FACS (flow cytometer from Becton Dickinson) with FITC-Biotin. With the help of a saturation series, 200,000 streptavidin molecules per microcapsule were quantified.

(f) Binding of biotinylated anti-CD 105 antibodies to functionalized gas-filled microbubbles:

The suspension of gas-filled microbubbles according to Example 4 (a) to (d) to which streptavidin according to Example 4 (e) had been bound was treated with biotinylated anti-CD105 antibody (Rat Anti-Mouse CD105 / Endoglin, Biotin Conjugate, Southern Biotechnology Associates, Inc., Cat. No. 1860-08) in a ratio of 0.5 μg antibody to 1e6 microbubbles. After incubation, the suspension was diluted to 1e8 bubbles / ml with PBS buffer containing 0.02% (m / m) Triton x 100. The CD105 coupling and dilution was freshly made for each animal.

(g) Binding of biotinylated IgG _2a to functionalized gas-filled microcapsules:

The suspension of gas-filled microbubbles according to Example 4 (a) to (d), to which streptavidin according to Example 4 (e) had been bound, was treated for 10 minutes at room temperature with biotinylated IgG _2a isotype control (Rat IgG _2a isotype control, biotin Conjugate, Southern Biotechnology Associates, Inc., Cat. No. 0117-08) at 0.5 µg antibody to 1e6 microbubbles. After incubation, the suspension was diluted to 1e8 bubbles / ml with PBS buffer containing 0.02% (m / m) Triton x 100. Isotype coupling and dilution were freshly made for each animal.

Claims

claims

1. Device for quantifying bodies contained in an examination object, comprising a) at least one transducer for emitting an ultrasound field, with which the bodies contained in a layer in the examination object can be excited to emit characteristic ultrasound signals, and for receiving the characteristic signals, b) at least one data processing system for determining the number of bodies in the layer from the characteristic signals, and c) means with which the ultrasound field can be displaced in a first layer in the examination object after excitation of the bodies in such a way that bodies after the displacement in one second layer can be excited, the first layer and the second layer overlapping and being shifted parallel and defined against each other.

2. Device according to claim 1, characterized in that the bodies represent bubbles.

3. Device according to claim 1 or 2, characterized in that the ultrasonic field is displaceable by at least 5 microns.

4. Device according to one of claims 1 to 3, characterized in that at least one transducer has a two-dimensional or a three-dimensional array of piezo crystals.

5. The device according to claim 4, characterized in that means are provided with which the examination object and the two-dimensional array are displaceable against each other.

6. The device according to claim 4, characterized in that a control is provided for the piezo crystals in the three-dimensional array, with which adjacent sound fields originating from piezo crystals in a row of piezo crystals in the array or sound fields originating from adjacent rows of piezo crystals in the array can be brought to overlap ,

7. The device according to claim 4, characterized in that means are provided with which the object under examination and at least one transducer are displaceable against each other.

8. Device according to one of the preceding claims, characterized in that at least one motor drive is provided for a displacement of the object under examination and the at least one transducer against each other.

9. Device according to one of the preceding claims, characterized in that a holding device with which the examination object can be clamped, and a movable holder for the at least one transducer are provided.

10. Device according to one of the preceding claims, characterized in that an image evaluation system for data processing and image display is provided with which the quantification of the body can be carried out by video densitometry.

11. The device according to claim 10, characterized in that the body represents a bubble.

12. Use of the device according to any one of claims 1-11 for ultrasound diagnosis and / or ultrasound therapy on humans or animals.

13. Use of the device according to one of claims 1-11 for the in vivo and ex-wVo mapping of physiologically high or low regulatable molecular markers in organs and tissues from the postembryonic on- togenesis, for the in-vivo and ex-v / Vo-mapping of pathologically high or down-adjustable molecular markers in organs and tissues during pathogenesis and for the characterization of cell cultures by in iz / fro-mapping of high and down-adjustable molecular Marking the cells, the bodies having specific binding molecules for the markers.

14. Use of the device according to claim 13, characterized in that the bodies represent bubbles.

15. Method for quantifying bodies contained in an examination object, in which a) bodies contained in a layer in the examination object are excited with an ultrasound field to produce characteristic ultrasound signals, b) the ultrasound signals are received, c) the number of bodies is determined from the received ultrasound signals and d) after an excitation of bodies in a first layer, the ultrasound field is shifted in such a way that bubbles are excited in a second layer after the shift, the first layer and the second layer overlapping and being shifted in parallel and in a defined manner relative to one another.

16. The method according to claim 15, characterized in that the bodies represent bubbles.

17. The method according to claim 16, characterized in that the bubbles are destroyed during the excitation and are thereby excited to emit the characteristic signals.

18. The method according to any one of claims 15 - 17, characterized in that the ultrasonic field is shifted by at least 5 microns.

19. The method according to any one of claims 15-18, characterized in that the examination object and at least one transducer emitting the ultrasound field are displaced relative to one another.

20. The method according to claim 19, characterized in that the examination object is displaced relative to at least one fixed transducer or that the at least one transducer is displaced relative to a fixed examination object.

21. The method according to any one of claims 15 - 20 for ultrasound diagnosis and / or therapy on humans or animals.

22. The method according to any one of claims 15-20 for carrying out an in-vivo and ex-wVo mapping of physiologically high or down-adjustable molecular markers in organs and tissues from postembryonic ontogenesis, an in-vivo and ex-v / Vo-mapping of pathologically up or down-regulable molecular markers in organs and tissues during the pathogenesis and for the characterization of cell cultures by in w ^' / ro-mapping of up and down-regulatable molecular markers of the cells, whereby bubbles are used for the markers have specific binding molecules.