DE10215335A1 - Device and method for quantifying bubbles - Google Patents

Abstract

The invention relates to a device for quantifying bubbles, wherein ultrasound sectional images are recorded which overlap and are mutually displaced in parallel, the bubbles being excited to independent, characteristic signals and these signals being measured and methods for this quantification.

Description

The invention relates to devices and methods for the quantification of Bubbles according to the teaching of the claims, especially those through diagnostic ultrasound to independent, characteristic signals can be excited, especially those signals that with the Destruction of the bubbles go hand in hand.

The terms used in the present invention are as follows Definitions:

bladder

Bubble in the sense of the present application are gas-containing in essential spherical phases with a diameter <10 microns, the are either free or encased. In the case of enveloped bubbles, the envelope can surface-active substances (surfactants), proteins, lipids and / or contain natural and / or a synthetic polymer. Bubbles according to the The present application can also, for example, gas-filled microcapsules, gas filled microparticles, gas filled microspheres, microballoons and Microbubbles stabilized by surfactants.

Ultrasound contrast agents

Agents containing bubbles for use in ultrasound diagnostics and / or -therapy.

independent, characteristic signal

An independent, characteristic signal is one of the scatter signal independent, non-linear echo signal of a bubble.

Stimulated Acoustic Emission (SAE)

The bubbles preferred according to the invention can be excited with ultrasound be destroyed. This creates an independent and from the broadcast signal deviating signal from the scattering and movement of the bubbles is independent. This independent, characteristic signal is called stimulated acoustic emission (SAE) (synonym: loss of correlation (LOC)).

Saturation bubble concentration

The saturation bladder concentration is the bladder concentration per milliliter Fluid (tissue or blood) above which there is an overlay of the SAE signals come within a sound field and a quantification of the Bubbles becomes impossible from the sum of the SAE signals too when using arithmetic correction formulas no longer on the number the bubbles can be closed.

SAE layer thickness

Thickness of the layer per ultrasound sectional image, from which the in the sectional image SAE signals shown come from a scan with parallel overlap. The SAE layer thickness is equal to the displacement distance per Ultrasonic cut.

Region of interest (ROI)

Measuring window in the sectional view.

Morphometric Units (MU)

Morphometric units are a measure of the area, expressed in the Number of screen pixels that the displayed SAE signals occupy in the video image. The screen pixels are usually counted automatically Video densitometry. Is the number of screen pixels of a single SAE Known signals can from the morphometric units on the number of bubbles getting closed.

Corrected SAE (SAE _corr. ) Or corrected MU (MU _corr. )

If the size of the bubbles is smaller than the layer thickness of the ultrasound cross-sectional image, SAE signals may appear in the video image. The probability increases with increasing bladder concentration and layer thickness of the ultrasound sectional image. Both parameters have a direct influence on the percentage color area of the SAE signals in the video image. If the bubbles are randomly distributed in the volume examined, the MU can be corrected if SAE signals are superimposed as long as there is no complete color saturation in the ROI:


FP - Screen color pixels that lie within the measurement window (ROI) in the area of the SAE effects shown.
MU - ΣFP within the measurement window (ROI)
GP - screen gray pixels that are within the measurement window (ROI) but outside of the SAE effects shown.

About the quotient from the sum of the color pixels and the sum of the SAE signals


Within the ROI, the corrected MU (MU _corr. ) can be converted into a corrected SAE signal number (SAE _corr. ):

The application of this correction formula implies a random distribution of the bubbles ahead in the volume examined.

2D array

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

3D array

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

transducer

Device for sonography containing piezo elements directions in relation to the transducer ( Fig. 6)
X = across the transducer (2D array)
Y = lateral
Z = axial (direction of sound propagation)

The device according to the invention and the method according to the invention are used independent, characteristic signals from bubbles, preferably at their ultrasound-induced destruction occurs (stimulated acoustic emission, SAE signals).

The principle of stimulated acoustic emission (SAE) is shown in Fig. 1.

Bubbles that can emit independent characteristic signals described for example in the documents EP 0077752, EP 0123235, EP 0212568, EP 0224934, EP 0274961, EP 0398935, EP 0644777, EP 0458745 and WO 01/68150.

In principle, all gas-filled microbubbles that are used as Ultrasound contrast media are suitable to use diagnostic ultrasound to SAE Effects are stimulated. For what is on the market Ultrasound contrast medium Levovist®, the microparticle from galactose and Pamitic acid contains and after adding water a suspension stabilized gas bubbles, SAE effects were also unspecific Enrichment in the liver observed. Ultrasound Med Biol. 1999 Nov; 25 (9): 1341-52.

Particulate ultrasound contrast media containing bubbles in the form are preferred gas-filled microcapsules or gas-filled microparticles, the shell of which Polyalkyl cyanoacrylates, in particular polybutyl cyanoacrylate (EP 0644777). Functionalized polyalkyl cyanoacrylates are particularly preferred according to WO 01/68150, in particular functionalized polybutyl cyanoacrylate described in WO 01/68150.

The SAE signals of various bubbles can also be used according to the US patent 5,425,366 can be detected 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. Examples include:
Spectral Doppler, Power Doppler, Color Doppler and Harmonic (2nd, Sub, Wideband, Pulse Inversion, Ultraharmonic, Color Doppler, Harmonic Power Doppler).

The SAE signal strength is so high that even individual bubbles can be detected. The individual SAE signal is displayed 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 bubble (<10 µm) ( Fig. 2 ). In many ultrasound diagnostic devices currently available, the layer thickness of a single image is approximately 1 mm, depending on the device setting ( FIG. 3). 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.

The device according to the invention and the method according to the invention are not limited to imaging or video. Counting the SAE Signals within an ROI can be determined based on the received signals (radio frequency (RF) data), although the saturation bubble concentration at Counting of the SAE signals at the level of the received signals differs from that Saturation bubble concentration when counting the SAE signals in the can distinguish two-dimensional ultrasound sectional image. The device and the counting of the received signals can be integrated into an ultrasound device become.

• 1. durch die Schichtdicke des Ultraschallschnittbildes (Fig. 3) und
• 2. durch die Größe eines im Ultraschallschnittbild dargestellten SAE-Signales bei Zerstörung einer einzigen Blase (Fig. 2).

In the two-dimensional ultrasound sectional image, the saturation bubble concentration is determined by

• 1. by the layer thickness of the ultrasound sectional image ( Fig. 3) and
• 2. by the size of a SAE signal shown in the ultrasound sectional image when a single bubble is destroyed ( FIG. 2).

A low saturation bubble concentration is required for qualitative imaging be of advantage because even at low bladder concentrations a gapless Contrast in the sectional image is created.

For quantifying bubbles, however, is a small one Saturation bubble concentration is disadvantageous because it is already low Bladder concentrations lead to contrast saturation in the image and themselves Bubble concentrations above the saturation bubble concentration in the picture can no longer be absolutely determined or differentiated.

As long as bubbles are only distributed unbound in the blood, the Control the bladder concentration there by dosing so that the Saturation bubble concentration is not reached.

However, there is a specific or non-specific enrichment the bubbles (for example in tissues or on structures), the Saturation bladder concentration usually exceeded because of that enriching tissues or the enriching structures are so dense, that the bubbles are there in a concentration above the Enrich saturation bladder concentration.

By reducing the dose, the saturation bladder concentration in the Target area may also be undercut here, but the Enriched number of bubbles would be in an unknown ratio to maximum saturation. Therefore, such a measurement would be neither reproducible, nor would it return a value that corresponds to the number of those present there Enrichment factors correlated.

Therefore, a method for determining the number of bubbles is above the Saturation bubble concentration desirable.

• - durch Phagozytose von Blasen in Organen des RES oder in anderen Zellen, die erkrankungsassoziert phagozitieren wie z. B. tumorassoziierte Makrophagen oder Makrophagen in atherosklerotischen Plaques.
• - durch Adhäsion bzw. durch langsames Entlangrollen von Blasen beispielsweise am Gefäßendothel
• - durch Einwandern von Blasen in "Gefäßsackgassen" oder "blind" endenden Blutgefäßen. Dies kann im Übergangsbereich zu Nekrosen, bei stark ödematösen oder entzündlichen Prozessen, Hämatomen oder Gefäßausbuchtungen wie Aneurysmen, gefäßbildenden Tumoren wie Hämangiome oder durch Einspülen in Nekrosen auftreten.

For example, non-specific enrichment of bubbles takes place:

• - by phagocytosis of blisters in organs of the RES or in other cells that phagocitate disease-associated such as. B. tumor-associated macrophages or macrophages in atherosclerotic plaques.
• - by adhesion or by slowly rolling bubbles along the vascular endothelium, for example
• - by immigration of bubbles in "vascular blind alleys" or "blind" ending blood vessels. This can occur in the transition area to necrosis, in highly edematous or inflammatory processes, hematomas or bulges such as aneurysms, vascular tumors such as hemangiomas or by washing into necrosis.

A specific enrichment of bubbles can be made by a specific or electrostatic binding to physiological or pathophysiological Structures can be achieved. The blisters usually wear in these cases specifically binding molecules on their surface.

For the measurement of ultrasound contrast there is the possibility of video images digitize, and the brightness (gray or color values) in definable Measure regions of the image over time. However, this type of measurement solves not the problem of contrast saturation at high bladder concentrations. If at all, quantification is only possible for very small ones Bladder concentrations possible.

3D-Echotech has the need to quantify bubbles tried to satisfy with an ultrasound machine for quantification of Bladder signals in the blood. With this device, the video image of the Ultrasound device digitized, broken down into color and gray values and optionally by average brightness value or number of screen pixels within one defined region of interest (ROI) evaluated. The scope This measuring system is based on the bladder flooding in blood vessels Quantify brightness values within a definable ROI. But also this method is due to the layer thickness (approx. 1-2 mm) of the sound field not suitable to quantify above approx. 3000 particles or bubbles / ml. But even below this concentration there is none with this device reproducible measurement possible because the transducer is guided by hand, and so some areas overlap several times or to different degrees, other areas are not sounded at all. This problem won't either through the possibility of the also developed by Echotech electromagnetic position determination of the transducer solved because this is only accurate in the sub-millimeter range.

The Kretz-Technik company offers 3D ultrasound methods, in which a 2D array (Linear array, sector array, phased array, as in 2D sonography) within of a transducer is automatically pivoted at a certain angle. With this method, however, there is no overlap in the axial direction (Z) evenly. Several neighboring sound fields overlap in the vicinity almost completely, while with increasing distance to the transducer gaps arise, which are then interpolated. As a result, the SAE Signal yield in an examined tissue from the distance to the transducer depends. This device cannot quantify bubbles be carried out reproducibly. This applies in particular if different patients are examined and / or different Examiner perform the exam.

With none of the devices mentioned it is possible to blow bubbles in vivo, ex vivo or quantify in vitro, especially not in concentrations above one Concentration of approx. 3000 bubbles / ml 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.

The object of the present invention is an apparatus and a method for quantifying bubbles to provide a determination the bladder concentration in vivo, ex vivo and in vitro above the Saturation bladder concentration allows. The method should be suitable Quantify bubble quantities reproducibly, their concentration too determine and with respect to the SAE signals shown in the video image Allow spatial resolution in the histological range.

The invention solves the problem by the devices and methods according to of claims.

The invention solves the problem of quantifying bubbles passing through diagnostic ultrasound to independent, characteristic signals can be stimulated, especially those related to the destruction of the Bubbles are accompanied by devices and processes that vary from volumes of Take the object under examination of ultrasound sectional images that overlap and are shifted parallel to each other.

The volumes preferably overlap between 20% and 99.99%.

The volumes particularly preferably overlap between 40% and 99.9% and most preferably between 70% and 99%.

In addition to the parallel overlap, the volumes can also differ Overlap cross direction.

The overlap and shift of the volumes can be done by a Transducer is moved relative to the object under examination. It can either the object under examination versus a fixed transducer be moved or the transducer relative to the fixed Examination subject. The volumes can overlap and shift also done with a transducer that is self-overlapping and parallel records shifted ultrasound sectional images.

• - Dignitätsbestimmung von Tumoren,
• - Staging von Tumoren,
• - Therapieverlaufskontrolle z. B. bei der Chemotherapie
• - Erhöhung der Reproduzierbarkeit und damit der Vergleichbarkeit von Befunden unterschiedlicher Untersucher
• - Vermeidung von den Patienten belastenden Biopsien
• - Reduktion der Schichtdicke, aus der die im Ultraschallbild dargestellten SAE-Signale stammen, wodurch Blasen-Anreicherungsgebiete im Millimeter-Bereich räumlich hochauflösend im Mikrometer-Bereich abbilden und bezüglich der Blasenkonzentration quantifizieren lassen.

The device according to the invention and the method according to the invention for the quantifiability of bubbles can be used for:

• - Dignity determination of tumors,
• - staging of tumors,
• - Therapy progress control z. B. in chemotherapy
• - Increasing the reproducibility and thus the comparability of findings from different examiners
• - Avoidance of biopsies that are stressful for the patient
• - Reduction of the layer thickness from which the SAE signals shown in the ultrasound image originate, as a result of which bubble enrichment areas in the millimeter range can be spatially high-resolution depicted in the micrometer range and quantified with regard to the bubble concentration.

The blisters are and are usually contained in ultrasound contrast media applied in the form of suspensions.

Not only in the areas of clinical diagnostics, but also in the basic medical and biological research and preclinical Research and development open up new ones Examination possibilities.

• - in vivo im Tierversuch
• - ex vivo in entnommenen Organen oder Geweben
• - in vitro z. B. in Zellkulturen

um pathologische oder physiologische Prozesse auf molekularer Ebene abzuklären. Bubbles can be quantified:

• - in vivo in animal experiments
• - ex vivo in harvested organs or tissues
• - In vitro e.g. B. in cell cultures

to clarify pathological or physiological processes at the molecular level.

In drug discovery and development of specific binding systems can are checked with the method according to the invention, which Binding systems work at all, which binding molecules under what conditions exist in which places in which quantity, which Influencing factors determine the binding ability and enrichment which Kinetics with bubbles or, in the case of ultrasound-induced Drug release, how much of an encapsulated drug is released.

The principle of the method according to the invention is particularly simple using the example of complete bubble destruction in each ultrasound sectional image illustrate:

• a) Im ersten Bild wird ein kontrastgesättigtes Ultraschallschnittbild erhalten (Fig. 4).
• b) Die Blasen werden durch die Beschallung zerstört. Es sind keine SAE- Signale mehr zu detektieren.
• c) Nachfolgend wird das Schallfeld zwischen 2 Bildern um eine definierte Verschiebungsstrecke gegen das Gewebe verschoben.
• d) Wurden im 1. Bild alle Blasen zerstört, so können im 2. Bild und allen nachfolgenden Bildern nur die neu hinzugekommenen detektiert werden. Diese sind quantifizierbar (Fig. 5b) und c)).

Fig. 5b) and c) show the inventive method:

• a) A contrast-saturated ultrasound sectional image is obtained in the first image ( FIG. 4).
• b) The bubbles are destroyed by the sonication. SAE signals can no longer be detected.
• c) The sound field between two images is then shifted against the tissue by a defined displacement distance.
• d) If all bubbles were destroyed in the 1st image, only the newly added ones can be detected in the 2nd image and all subsequent images. These can be quantified ( Fig. 5b) and c)).

With a parallel displacement distance of 10 µm against the tissue and the slice images overlap with a layer thickness of 1 mm for example 99%. In the 2nd cross-section only those are new added bubbles are detected, which are in the 10 µm layer.

The distance of the shift between two images is between 5 µm and the scan thickness (approx. 1-2 mm). The optimal route shift depends of the highest bladder concentration / mL tissue or blood in the scanned Volume.

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 the SAE signals are not superimposed in the ultrasound sectional image.

This can happen if the level of bladder accumulation is underestimated. In these cases, the displacement distance per ultrasound sectional image must be reduced in order to ensure absolute quantifiability. Fig. 9-11 show the relationship between the displacement distance and the morphometric units or the number of SAE signals in the sectional image.

The bubble concentration is preferred for such SAE layer thicknesses determined whose sectional images each 20 to 80%, preferably 30 to 70% Have color saturation. In favor of a reduced examination time can the displacement distance can be increased, however, as long as the Saturation bladder concentration is reached. For an optimal compromise There can be a defined between the examination time and the accuracy of the measurement Program that varies the SAE layer thickness. The through that Program-controlled optimal displacement distance can be based on a sufficiently large population of patients can be determined.

If individual contrast-saturated sectional images do occur, it is absolute quantifiability no longer exists. A quantifiability However, the non-contrast-saturated sectional images remain possible. Even if in certain areas of the study area Color saturation is present thanks to the standardized scanning process defined parallel displacement of the sound fields and by the higher spatial resolution of the method according to the invention reproduced Measurement possible. Such a measurement can e.g. B. for a comparison between in different patients or for therapy monitoring suffice for the same patient.

If not all bubbles are destroyed in a cross-section, you can do so before starting the next position also one or more further destruction pulses send.

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

• a) im ersten Bild werden alle Blasen zerstört
• b) in den nachfolgenden Bildern werden die Blasen so angeregt, dass sie ein eigenständiges, charakteristisches Signal geben.
• c) In jedem Bild werden dann die jeweils neu hinzukommenden Signale ermittelt.

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

• a) in the first picture all bubbles are destroyed
• b) in the following pictures the bubbles are excited in such a way that they give an independent, characteristic signal.
• c) The newly added signals are then determined in each image.

• - Subtraktion des jeweiligen Vorwertes oder
• - durch einfache, vorzugsweise automatische Zählung der Signale nach Senden eines Zerstörungspulses.

The determination of the newly added signals can be determined by if the transmission of the signal is not accompanied by the complete destruction of the bubbles

• - Subtraction of the respective pre-value or
• - By simple, preferably automatic counting of the signals after sending a destruction pulse.

The independent, characteristic signals go with the destruction of the If there are bubbles, the number of signals in the overlap area can usually as with SAE signals by simple, preferably automatic counting be determined.

• A) das Gewebe bzw. der Patient gegenüber dem fixierten Schallkopf verschoben wird oder
• B) der Schallkopf gegenüber dem zu untersuchenden fixierten Gewebe verschoben wird oder
• C) ein Schallkopf verwendet wird, der in der Lage ist, zeitlich nacheinander sich überlappende und verschobene Schnittbilder zu erstellen.

The defined parallel shift can be achieved in the

• A) the tissue or the patient is moved relative to the fixed transducer or
• B) the transducer is moved relative to the fixed tissue to be examined or
• C) a transducer is used, which is able to create overlapping and shifted sectional images one after the other in time.

The different displacement options can also be combined become.

• A) Das Untersuchungsobjekt (Patient, Tier, ein exenteriertes oder isoliertes Organ oder eine Zellkulturschale) wird automatisch (z. B. durch Servomotor oder DC-Motor angetrieben), am fixierten Schallkopf vorbeigefahren.
• B) Der Schallkopf wird automatisch mit Hilfe einer speziellen Vorrichtung über dem Untersuchungsobjekt (Patient, Tier, ein exenteriertes oder isoliertes Organ oder eine Zellkulturschale) verschoben. Die Vorrichtung kann ein Rahmen sein, innerhalb dessen der Schallkopf in einer Halterung fixiert ist und durch Motortrieb entlang einer Parallelführung bewegt wird.
  Im einfachsten Fall kann ein Einzelkristall in x- und y-Richtung mit definierten Überlappungsbereichen verschoben werden. Diese Variante bietet sich für Kulturschalen von Zellen an, an deren Oberfläche sich Blasen spezifisch oder in deren Inneren unspezifisch (z. B. Phagozytose) angereichert haben. Hier reicht die Aussage, wie viele Blasen pro Fläche bzw. Volumen sich angereichert haben, aus. Die Daten müssen nicht in Bilder umgerechnet werden.
• C) Mit einem Schallkopf enthaltend ein 3D-Array können durch eine entsprechende Steuerung des Scans benachbarte Schallkeulen innerhalb einer Linie oder Schallkeulen benachbarter Linien in Querrichtung definiert zur Überlappung gebracht werden. Bei dieser Variante ist eine weitere Bewegung von Schallkopf oder Untersuchungsobjekt nicht mehr nötig.
  Der Schallkopf kann auch ein 2D-Array enthalten, welches innerhalb des Schallkopfes parallel mit definierter Überlappung in Querrichtung bewegt wird. Die laterale Auflösung kann innerhalb eines 2D-Schnittbildes gesteigert werden, wenn sich die einzelnen Scan-Linien, aus denen das Schnittbild aufgebaut ist, ebenfalls definiert überlappen. Die einzelnen Schallfelder, die jeweils von einer Gruppe benachbarter Piezoelemente erzeugt werden, werden dabei im Gegensatz zur konventionellen Steuerung stärker und vor allem definiert zur Überlappung gebracht, indem kleine Schrittweiten (bis hinunter zu einem Piezo-Element) gewählt werden (Fig. 7). Wie beim definiert überlappenden Scan in Querrichtung zum Schallkopf erhöht sich die Auflösung um den Faktor, in dem die "Verschiebung" zur Breite der Piezo-Elementgruppe steht.

The following variants of parallel displacement are described as examples:

• A) The examination object (patient, animal, an eccentric or isolated organ or a cell culture dish) is automatically moved past the fixed transducer (e.g. powered by a servo motor or DC motor).
• B) The transducer is automatically moved using a special device over the examination object (patient, animal, an eccentric or isolated organ or a cell culture dish). 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 is suitable for culture dishes of cells on the surface of which bubbles have accumulated specifically or inside which have become non-specific (e.g. phagocytosis). Here it is sufficient to say how many bubbles have accumulated per area or volume. The data does not have to be converted into pictures.
• C) 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 parallel within the transducer with a defined overlap in the transverse 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) ( Fig. 7). As with the defined overlapping scan in the transverse direction to the transducer, the resolution increases by the factor in which the "shift" to the width of the piezo element group is.

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

• - Größe,
• - Hülle,
• - Bindungsmolekülen auf deren Oberfläche,
• - Anregbarkeit durch verschiedene Pulsformen
• - Zerstörbarkeit durch Schall
• - Art des charakteristischen Signals.

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

• - size,
• - cover,
• - binding molecules on their surface,
• - Excitability through different pulse shapes
• - Destructibility by sound
• - type of characteristic signal.

• a) indem bei einer Position hintereinander verschiedene Pulsformen gesendet und/oder verschiedene charakteristische Signale empfangen werden. Kommt es dabei nicht zur vollständigen Zerstörung der Blasen, kann ein Zerstörungspuls gesendet werden, bevor zur nächsten Position gefahren wird oder,
• b) indem das zu untersuchende Gebiet mehrfach mit entsprechender definierter Verschiebung gescannt wird, wobei jeder Scan mit einer anderen für die jeweilige Blasenart notwendige Pulsform durchgeführt wird.

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 scanning the area to be examined 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 can be used, as well as different ones Sound field shifts can be combined as long as the resulting intersection of the sound fields defined and in relation to their spatial expansion can be calculated and as long as the used Bubbles can be detected using characteristic signals.

• a) es werden 2 unterschiedliche Blasenarten injiziert:
  Blasenart A, mit geringem Schalldruck zerstörbar und mit Bindungsmolekül
  Anti-X für in-vivo-Bindungsstelle X auf der Oberfläche und
  Blasenart B, mit hohem Schalldruck zerstörbar und mit Bindungsmolekül
  Anti-Y für in-vivo-Bindungsstelle Y auf der Oberfläche.
• b) Anfahrt der errechneten Position
• c) ein oder mehrere Bilder mit niedrigem Schalldruck, Datenspeicherung
• d) ein oder mehrere Bilder mit hohem Schalldruck, Datenspeicherung
• e) definierte Verschiebung des Schallfeldes
• f) Wiederholung c)-e) bis zum Ende des Scans
• g) Auswertung der Daten für Bindungsmolekül X und Y, deren Anzeige auf dem Monitor und Speicherung

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 distribution of the binding sites in the examination region:

• a) 2 different types of bubbles are injected:
  Bubble type A, destructible with low sound pressure and with binding molecule
  Anti-X for in vivo binding site X on the surface and
  Bubble type B, destructible with high sound pressure and with binding molecule
  Anti-Y for in vivo binding site Y on the surface.
• b) Approach of the calculated position
• c) one or more images with low sound pressure, data storage
• d) one or more images with high sound pressure, data storage
• e) defined displacement of the sound field
• f) repetition c) -e) until the end of the scan
• g) Evaluation of the data for binding molecule X and Y, their display on the monitor and storage

In the old variants mentioned, the saturation bubble concentration is around Factor increased in which the between 2 pictures or sound clubs distance covered to the layer thickness of the entire sound field. at a 10 µm shift and 1 mm layer thickness increases the Saturation bladder concentration by a factor of 100. At the same time it decreases the layer thickness of the tissue from which the newly added bubbles stem from the same factor.

• A) Vergleich der gemessenen MU bei konstant gehaltener SAE-Schichtdicke und Dosis zur intraindividuellen Verlaufskontrolle einer Therapie.
• B) B.
  • 1. Zunächst Ermittlung der Abhängigkeit der gemessenen MU von der SAE-Schichtdicke und/oder Dosis in einem speziellen Messfenster (Leber, Tumoroberfläche, Gefäßendothel etc.) anhand einer möglichst großen Population. Die Population kann aus Individuen bestehen, die sich hinsichtlich der Verteilung und des Anreicherungsgrades der Blasen im Messfenster nicht unterscheiden d. h. die homogen ist oder aber die diesbezüglich gruppiert ist. Diese Abhängigkeiten müssen in der Regel bei verschiedenen Ultraschallgerätesettings (Schallkopf, Detektionsmode. . .) ermittelt werden.
  • 2. Ermittlung von linearen Korrelationsfunktionen im Bereich 20 bis 80%, vorzugsweise im Bereich 30 bis 70% Farbsättigung
  • 3. ggf. Integration der linearen Korrelationsfunktionen in eine Auswertungssoftware des Ultraschallgerätes
  • 4. Zählung der SAE-Signale im entsprechenden Messfenster eines Patienten
  • 5. anhand der linearen Korrelationsfunktionen: Bestimmung der absoluten Blasenanzahl bzw. der Blasenkonzentration im speziellen Messfenster des Patienten, wenn Farbsättigung im Geltungsbereich der linearen Korrelationsfunktion liegt.
• C) C.
  • 1. Zunächst Ermittlung der Abhängigkeit der absolut gezählten SAE- Signale von der SAE-Schichtdicke und/oder Dosis in einem speziellen Messfenster (Leber, Tumoroberfläche, Gefäßendothel etc.) anhand einer möglichst großen Population. Die Population kann aus Individuen bestehen, die sich hinsichtlich der Verteilung und des Anreicherungsgrades der Blasen im Messfenster nicht unterscheiden d. h. die homogen ist oder aber die diesbezüglich gruppiert ist. Diese Abhängigkeiten müssen in der Regel bei verschiedenen Ultraschallgerätesettings (Schallkopf, Detektionsmode etc.) ermittelt werden.
  • 2. Durchführung von Kurvenfits, Ermittlung von die jeweilige Kurve beschreibenden Korrelationsfunktionen
  • 3. ggf. Integration der Korrelationsfunktionen in eine Auswertungssoftware des Ultraschallgerätes
  • 4. Zählung der SAE-Signale im entsprechenden Messfenster eines Patienten
  • 5. anhand der Korrelationsfunktionen: Bestimmung der absoluten Blasenanzahl bzw. der Blasenkonzentration im speziellen Messfenster des Patienten.

A histological resolution can be achieved in this way. Since this principle can also be used in vivo, one can speak of an "in vivo sono-histology" or, in the case of a specific bladder accumulation, of an sonographic "in vivo immuno-histology" 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 SAE layer thickness and dose for the intraindividual follow-up of a therapy.
• B) B.
  • 1. First, determine the dependence of the measured MU on the SAE layer thickness and / or dose in a special measurement window (liver, tumor surface, vascular endothelium, etc.) using the largest possible population. The population can consist of individuals who do not differ in terms of the distribution and the degree of enrichment of the bubbles in the measurement window, ie who are homogeneous or who are grouped in this regard. These dependencies usually have to be determined with various ultrasound device settings (transducer, detection mode ...).
  • 2. Determination of linear correlation functions in the range 20 to 80%, preferably in the range 30 to 70% color saturation
  • 3. If necessary, integration of the linear correlation functions into an evaluation software of the ultrasound device
  • 4. Counting the SAE signals in the corresponding measurement window of 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) C.
  • 1. First determine the dependence of the absolutely counted SAE signals on the 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 terms of the distribution and the degree of enrichment of the bubbles in the measurement window, ie who are homogeneous or who are 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 into an evaluation software of the ultrasound device
  • 4. Counting the SAE 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 figures are briefly described below:

Fig. 1 Principle of stimulated acoustic emission:
1 sound wave of low amplitude
2 high amplitude sound wave
3 scatter signal, bubble remains intact
4 SAE signal destroying the bladder

Fig. 2 size of SAE signals in the sectional view

Fig. 3 layer thickness conventional sectional view

Fig. 4 color saturated sectional view

Fig. 5 Principle of the inventive method
a displacement distance 100 µm
b displacement distance 50 µm
c displacement distance 10 µm
5 bubble
6 SAE signal
7 destroyed bubble

Fig. 6 directional definitions relating to a transducer
23 transducer
24 Transverse direction to the transducer
25 lateral
26 Axial (direction of sound propagation)

Fig. 7 multi-element transducer with defined overlapping sound fields (2D array)
a Image composition with low beam overlap
b Image structure with strong beam overlap
8 bubble
9 SAE signal
10 destroyed bubble
11 piezo element
12 piezo element, which sends an ultrasonic signal together with neighboring piezo elements.

Fig. 8 3-D view of the scanned with different displacement distances agar phantom 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
Section 15 : displacement distance 100 µm

Fig. 9 dependence of the MU on the SAE layer thickness
(Agar Phantom, Example 1)

Fig. 10 dependence of the MU _corr. on the SAE layer thickness
(Agar Phantom, Example 1)

Fig. 11 dependence of the SAE _corr. on the SAE layer thickness
(Agar Phantom, Example 1)

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 = 350 µm
b SAE layer thickness = 20 µm

Fig. 13: Bladder accumulation in the liver, rat 2 (example 2): 1.10 ⁷ blisters / ml.
Ex vivo measurement, 30 min. pi
a SAE layer thickness = 350 µm
b SAE layer thickness = 20 µm

Fig. 14 as a function of the MU of the SAE-layer thickness
Rat 1 (example 2): 1.10 ⁸ bubbles / ml. Ex vivo measurement, 30 min.

Fig. 15 dependence of the MU _corr. 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 _corr. on the SAE layer thickness
Rat 1 (example 2): 1.10 ⁸ bubbles / 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 _corr. on the SAE layer thickness
Rat 2 (example 2): 1.10 ⁷ bubbles / ml. Ex vivo measurement, 30 min.

Fig. 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 Graphic representation
b Sonographic longitudinal 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
20 color signals (MU _FP )
21 gray signals (MU _GP )
22 Sum of MU _FP and MU _GP , or the measured tissue volume

Fig. 21 color signals of the enriched in tumor-specific bubbles (4 mice, Example 3) compared to the isotype control (4 mice, Example 3).

The invention is described in more detail by the following examples:

example 1 Agar phantom containing 30,000 bubbles / ml a) Preparation of the bases

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

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

665 ml of water (Ampuwa, Fresenius) are in a kettle (Braun, Type 3 217 WK 210) cooked and transferred to a 1000 ml beaker. It 1.7 g of benzoic acid (Merck) for preservation and 10 g of agar (Merck) added with stirring. The agar solution is in a microwave (Bosch HMT 702 C, 800 W) briefly boiled again and then to 40 ° C cooled.

Before the bubbles are added to the agar solution, the bubble concentration is adjusted to 6.10 ⁸ bubbles / ml with 0.02% Triton × 100 solution. 33 µl (2.10 ⁷ bubbles) of this are carefully stirred into the agar solution without air bubbles. The agar solution is then poured into a glass bowl (14.19.4 cm) in which it solidifies as a gel (agar phantom).

c) Experimental setup and implementation

An approx. 4 cm wide strip of the agar phantom is placed in water filled plastic basin. 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) is vertical with a tripod attached over the phantom so that it dips into the water.

• 1. per Hand in Querrichtung unter dem Schallkopf mit einer Geschwindigkeit von ca. 2 cm/s verschoben, was einer Verschiebungsstrecke von ca. 350 µm/frame oder
• 2. auf einen Servomotor (Limes 150, OWIS GmbH, Motorcontroller DC 500) gestellt, und automatisch mit unterschiedlichen Geschwindigkeiten unter dem fixierten Schallkopf vorbeigefahren. Die Geschwindigkeiten werden dabei so gewählt, dass unter Berücksichtigung der gegebenen Bildrate des Ultraschallgerätes von 5,8 Bildern/Sekunde und kontinuierlicher Bewegung des Agar-Phantoms folgende Verschiebungsstrecken je Bild resultierten: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 µm/frame. Die dazu erforderlichen Geschwindigkeiten (µm/s) werden wie folgt errechnet:
  
  Geschwindigkeit [µm/s] = Verschiebestrecke/Bild [µm/frame] × Bildrate des Ultraschallgerätes [frame/s]

The pelvis is put together with the phantom

• 1. moved by hand in the transverse direction under the transducer at a speed of approx. 2 cm / s, which is a displacement distance of approx. 350 µm / 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 are selected 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 (µm / s) required for this are calculated as follows:
  
  Speed [µm / s] = displacement distance / image [µm / frame] × frame rate of the ultrasound device [frame / s]

d) quantification of the SAE signals

The resulting video images are video densitometrically with QuantiCon © (Echotech 3D Imaging Systems GmbH) evaluated as follows:

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

In order to separate colored noise signals from SAE signals, a threshold value of 40 is 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 are used for the evaluation. Fig. 8 shows the 3D view of the scanned agar phantom, how it is generated from the 3D data set in Quanticon. Both the number of pixels of the gray values and the number of pixels of the segmented color values of all images in the data set are exported and processed with MS Excel.

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 is 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 is first determined. For this purpose, the sum of the SAE signals is first determined by hand counting in 20 sectional images. The sum of the segmented color pixels is 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 is determined. The about this factor of Mu _corr. The graphic converted to the number of corrected SAE signals (SAEu _corr. ) is shown in FIG. 11.

Table 1 lists the data on which FIGS. 9 to 11 are based: Table 1

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

The points outside this range are also considered linear Taking correlation into account, the correlation coefficients worsen until to 0.971 for MU and 0.981 for the corrected MU / SAE.

e) Result

• 1. All the pictures when moving the phantom by hand and one Displacement distance of approx. 350 µm / frame is complete saturated with SAE signals. The saturation bubble concentration is exceeded and quantification impossible.
• 2. With the automatic shift with overlap of the sound fields, only a few SAE signals are detected with a shift of 10 µm / frame ( FIG. 8). As the speed increases, the number of SAE signal frame increases. Color saturation in the image only occurs from a shift of 100 µm / frame.
  Thus, a bubble concentration of 30,000 bubbles / ml with a layer thickness of 100 µm leads to SAE saturation. From this, a saturation bubble concentration of approx. 3,000 bubbles / ml can be concluded for a layer thickness of 1 mm.

f) discussion

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

Due to the defined overlapping displacement of an object against the In each sectional image, only the newly added bubbles become transducers detected from the non-overlapping volume. So 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 to the displacement distance or SAE layer thickness is available. A 10 µm shift from frame to frame increases at the same time the spatial resolution and the saturation bubble concentration around the factor 100 with a layer thickness of the sound field of 1 mm.

Example 2 Ex vivo quantification of nonspecifically enriched bubbles in the Rat liver a) Making the bubbles

The bubbles are produced according to Example 4 (a)

b) investigation

Two rats (Wistar SCHOE, 220 g, female) are examined. Various doses of the bladder suspension prepared according to a) are injected iv via the tail vein into the rats. For rat 1, a dilution to 1.10 ⁸ bubbles / ml is prepared, for rat 2 to 1.10 ⁷ bubbles / ml in 0.9% NaCl solution containing 0.02% Triton × 100.
Rat 1: 1.10 ⁸ bubbles / kg (1.10 ⁸ bubbles / ml, 220 µl IV)
Rat 2: 1.0 ⁷ bubbles / kg (1.10 ⁷ bubbles / ml, 220 µl IV)
30 minutes after the injection, the rats are killed by ip 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):

• 1. per Hand in Querrichtung unter dem Schallkopf mit einer Geschwindigkeit von ca. 2 cm/s verschoben, was einer Verschiebungsstrecke von ca. 350 µm/frame entspricht oder
• 2. auf einen Servomotor (Limes 150, OWIS GmbH, Motorcontroller DC 500) gestellt, und automatisch mit unterschiedlichen Geschwindigkeiten unter dem fixierten Schallkopf vorbeigefahren. Die Geschwindigkeiten werden dabei so gewählt, dass unter Berücksichtigung der gegebenen Bildrate des Ultraschallgerätes von 5,8 Bildern/Sekunde und kontinuierlicher Bewegung des Gefäßes folgende Verschiebungsstrecken je Bild resultierten:
  Ratte 1 (1.10⁸ Blasen/kg): 15, 20, 30 µm/frame
  Ratte 2 (1.10⁷ Blasen/kg): 20, 40, 80, 120 µm/frame

The right lobe of the liver is removed and placed in a container filled with 0.9% sodium chloride solution.
The vessels with the liver lobes will:

• 1. moved by hand in the transverse direction under the transducer at a speed of approx. 2 cm / s, which corresponds to a displacement distance of approx. 350 µm / 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 are selected 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 is carried out in the same way as in Example 1 Device settings. The liver is placed at a distance of 2 cm on the transducer passed. The same ROI is used for all measurements. The size The ROI (approx. 2.6 mm measured on the scale of the ultrasound device) is so selected that the ROI is completely in the range for all evaluated images of the liver tissue. All measurements in Quanticon are the same Magnification selected.

To determine the ratio of screen pixels to object length [mm] in the ultrasound image a square ROI of 2 mm edge length (determined using the length measurement scale shown in the ultrasound image).

To do this, the image is enlarged accordingly using the zoom function available in Quanticon. The pixel sum in this ROI (MU) is 225. From this, a ratio of Embedding 56.25 pixels / mm ² (225/4) is calculated. The bladder concentration / ml liver is then calculated from the measured volume (area of the ROI X displacement distance / frame).

The following formula is used to convert the raw data (FP, GP, S) into the bladder concentration G in the tissue:


C - bladder concentration in [bubbles / ml] or [SAE _corr. / Ml]
S - SAE layer thickness, or the shift between 2 images in [mm]
FP - Screen pixels that are 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.
Σsae - 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

• 1. Alle Bilder, die beim Verschieben der Leber per Hand und einer Verschiebungsstrecke von ca. 350 µm/frame entstanden, sind vollständig mit SAE-Signalen gesättigt (Fig. 12a, Fig. 13a). Die Sättigungsblasenkonzentration ist überschritten und eine Quantifizierung unmöglich. 2. Bei der automatischen Verschiebung mit Überlappung der Schallfelder werden bei Ratte 1 (Dosis: 1.10⁸ Blasen/kg) bei SAE-Schichtdicken von 20 bis 120 µm/frame SAE-Signale in einer quantifizierbaren Anzahl detektiert (Fig. 12b: SAE-Schichtdicke), bei Ratte 2 (Dosis: 1.10⁷ Blasen/kg) bei SAE-Schichtdicken von 10 bis 30 µm/frame (Fig. 13b: SAE-Schichtdicke). Mit zunehmender Geschwindigkeit nimmt auch die Anzahl der SAE- Signalelframe zu.
• 2. Bei Ratte 1 errechnet sich ein linearer Korrelationskoeffizient von 1,000 für die Abhängigkeit der unkorrigierten MU von der SAE-Schichtdicke (Fig. 14). Der lineare Korrelationskoeffizient für die Abhängigkeit der korrigierten MU (Mu_corr.) bzw. korrigierten SAE (SAE_corr.) von der SAE-Schichtdicke zwischen 15 µm und 30 µm liegt jeweils bei 0,995 (Fig. 15+16).
• 3. Bei Ratte 3 errechnet sich ein linearer Korrelationskoeffizient von 0,984 für die Abhängigkeit der unkorrigierten MU von der SAE-Schichtdicke (Fig. 17). Der lineare Korrelationskoeffizient für die Abhängigkeit der korrigierten MU (Mu_corr.) bzw. korrigierten SAE (SAE_corr.) von der SAE-Schichtdicke zwischen 20 µm und 120 µm liegt jeweils bei 0,995 (Fig. 18+19).
• 4. Die Blasenanzahl steht in einem linearen Verhältnis zur SAE-Schichtdicke.

Table 2 compares the bladder concentrations of the rats. The tables also contain the mean values of the percentage of color saturation for the respective SAE layer thickness. Table 2

• 1. All images that were created when the liver was shifted by hand and a shift distance of approximately 350 μm / frame are completely saturated with SAE signals ( FIG. 12a, FIG. 13a). The saturation bubble concentration has been exceeded and quantification is impossible. 2. In the case of the automatic shift with overlapping of the sound fields, a quantifiable number of SAE signals are detected in rat 1 (dose: 1.10 ⁸ bubbles / kg) with SAE layer thicknesses from 20 to 120 μm / frame ( FIG. 12b: SAE layer thickness ), in rat 2 (dose: 1.10 ⁷ bubbles / kg) with SAE layer thicknesses of 10 to 30 µm / frame ( Fig. 13b: SAE layer thickness). As the speed increases, the number of SAE signal frame increases.
• 2. In rat 1, a linear correlation coefficient of 1,000 is 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 (Mu _corr. ) Or corrected SAE (SAE _corr. ) On the SAE layer thickness between 15 µm and 30 µm is in each case 0.995 ( Fig. 15 + 16).
• 3. In rat 3, a linear correlation coefficient of 0.984 is calculated for the dependence of the uncorrected MU on the SAE layer thickness ( FIG. 17). The linear correlation coefficient for the dependence of the corrected MU (Mu _corr. ) Or corrected SAE (SAE _corr. ) On the SAE layer thickness between 20 µm and 120 µm is in each case 0.995 ( Fig. 18 + 19).
• 4. The number of bubbles is in a linear relationship to the SAE layer thickness.

c) Possible uses

The method according to the invention is also in removed organs, in which bubbles have accumulated non-specifically, applicable. There is a close one Correlation between the number of SAE signals shown in the cross section and the corresponding SAE layer thickness. The bladder concentration in the Livers of the examined rats differ significantly depending on the dose applied.

Example 3 In vivo quantification of specifically enriched USKM in the F9 Teratoma of the mouse

With the method according to Example 1, the enrichment of specific Bubbles with anti-CD105 antibodies coupled to their surface in tumors of F9 tumor-bearing mice (swiss-nude) after i. v. Injection in vivo examined. Bubbles serve as control substance, on the surface of which IgG2a- Antibodies (isotype control) are coupled.

a) Production of the specific bubbles

The production and characterization of the specific bubbles follows Example 4-a. .e. Antibody coupling takes place according to Example 4-f.

b) Preparation of the isotype control

The production and characterization of the isotype control bubbles follows Example 4-a. .e. The isotype coupling takes place according to example 4-g.

c) tumor inoculation

F9 tumor cells grown subconfluently in culture are trypsinized, centrifuged and resuspended in PBS with Ca ²⁺ / Mg ²⁺ . After staining with trypan blue and calculating the cell concentration, the cell suspension is adjusted to a concentration of 6.10 ⁷ cells / ml. The cell suspension is chilled on ice until use. From this cell suspension, 10 female nude mice (Swiss nude, 18-20 g body weight) each 50 µl per animal subcutaneously (= 3 × 10E6 cells / animal) inoculated into the left flank region using a Hamilton syringe and 27 G cannula. The growth of the tumors (length and width) is checked by measurement with a caliper, and the volume of the tumor is approximately determined using the following formula:

L × W ^2/2

L = length [mm]
B = width [mm]

The animals are 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

4 mice are injected with an ultrasound contrast agent containing specific bubbles (produced and characterized according to Example 4-a.. E & antibody coupling according to Example 4-f) iv while awake in a dose of 1 × 10 ⁹ bubbles / kg body weight (corresponds to 200 µl of the suspension according to Example 4-g each 20 g mouse). The specific bubbles are gas-filled microcapsules, the shell of which consists of functionalized polybutyl cyanoacrylate and to whose surface anti-CD105 antibodies are coupled. As a control substance, an additional ultrasound contrast agent containing unspecific bubbles (produced and characterized according to Example 4-a.. E & isotype coupling according to Example 4-g) is injected into 4 further mice in the same way. The non-specific bubbles are gas-filled microcapsules, the shell of which also consists of functionalized polybutyl cyanoacrylate, but to the surface of which IgG _2a (isotype control) is coupled. The tumors of all animals are 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 examination, the mice are mixed with a 1: 1 volume mixture a dilution (1 + 9) of Rompun® 2% (20 mg xylazine / ml) and one Dilution (1 + 4) of Ketavet® 100 mg / ml (100 mg ketamine base / ml) in each physiological NaCl solution anesthetized. From this anesthetic mixture 200 µl each 20 g mouse i. p. injected.

The mouse is placed on a carrier attached to the moving part of a servo motor (Limes 150, OWIS GmbH, Motorcontroller DC 500) is attached with adhesive tape fixed. The servo motor is adjusted vertically with a tripod so that the carrier with the mouse in a water basin (cut, square plastic bottle) enough. Before each scan, the pool is fresh and at 37 ° C tempered water filled. A transducer (L 10-5, ATL UM9) comes with Coupling gel provided and with a tripod horizontally on the side wall of the Water basin put on. The mouse becomes more visual and sonographic Control so far into the pelvis that the tumor has not yet entered it Sound field device. If air bubbles adhere to the skin, they will removed by using a round curved wire, the one with liquid soap is gently stroked over the skin. After that, the mouse turns some Retracted millimeters. Before the scan, the necessary device settings are made on Ultrasound device set (color Doppler mode, MI: 1.1, persistence: 0, priority: maximum, depth of penetration: 3 cm, focus: 2 cm). The scan runs with a constant SAE layer thickness of 285 µm / frame driven. The tumor lies in the Area of focus. After the scan, the image is "frozen" so that the Transducer sends no signal. The mouse moves to the starting position scaled back. The transducer is turned on again and the animal is again with the same device settings and the same SAE layer thickness scanned. The second scan is needed to identify color signals that not caused by SAE signals and serves as a control value. Such signals can come from flowing blood or larger air bubbles are caused.

• a) Fixierung des Tieres auf einem Kunststoffträger mit einer Aussparung von mindestens der Tumorgröße, so dass der Tumor in der Aussparung liegt
• b) Messung der Atembewegung des Tieres (z. B. mit Dehnungsmessstreifen, Staudruck-Messverfahren.) und Verwendung als Triggersignal zur Steuerung sowohl des Ultraschallgerätes, als auch des Servomotors.

With the anesthetic used here, the animal's breathing frequency is usually low enough to be able to carry out a scan of approx. 3 cm at 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 can be avoided as follows:

• a) fixation of the animal on a plastic carrier with a recess of at least the size of the tumor so that the tumor lies in the recess
• b) Measurement of the animal's breathing movement (e.g. with strain gauges, dynamic pressure measurement method) and use as a trigger signal to control both the ultrasound device and the servo motor.

evaluation

A sectional image is generated in the scan direction ( FIG. 20b) and this is 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 is evaluated with Quanticon (3D-Echotech). Only the color signals in the tumor (MU _FP : 20) and not the gray signals (MU _GP : 21) are 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.

g) Result

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

h) Possible uses

The result shows that the method according to the invention also for Quantification of specifically enriched bubbles in the living organism suitable is. It is even then with the method according to the invention significant difference detectable, if not with maximum spatial Resolution is scanned and there are some areas of some cross-sectional images too SAE saturation is coming. The number of signals at a specific Bladder accumulation can result from residual signals from the blood vessel system and / or be distinguished from signals of an unspecific bladder accumulation.

Example 4 Production of ultrasound contrast media (a) Preparation of the microcapsule suspension

In a cylindrical steel reactor (internal dimensions: height = 37 cm, diameter = 29 cm) are 7 l of an aqueous 1% (m / m) octoxynol solution in a pH of 2.5 submitted and with a mixer (dissolver disc: Diameter = 60 mm, agitator shaft: length = 30 cm, immersion angle = 10 °) at high shear rate (4000 rpm) mixed and tempered (temperature of the Solution = 32 ° C) so that self-gassing (i.e. one by dispersing conditional air entry into the medium with strong foam formation). 100 g Butyl cyanoacrylic acid are added quickly (<3 minutes) and dispersed (Immediately before the addition, the speed of rotation of the mixer is raised to 8000 rpm elevated). It is polymerized for 60 minutes with self-fumigation, whereby gas-filled microcapsules (bubbles in the sense of the application underlying definition). In a separating funnel, it will Flotat separated in the gravitational field of the earth for 3 days, the shelter drained and the flotate with 3 l of an aqueous 0.02% octoxynol solution resuspended. The microcapsule suspension thus obtained has one Polymer content of 9.46 mg / ml, a density of 0.943 g / ml and a pH from 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) is determined using a particle counter from Particle Sizing Systems, type AccuSizer 770 (measuring medium: aqueous 0.02% (mlm) Triton X100 solution). The volume-weighted particle size distribution ranges from 0.8 to 10 µm with a maximum at 1.8 µm and the microcapsule concentration is 7.2.10 ⁹ (± 1.10 ⁸ ) particles per mL.

(c) Functionalization of the gas-filled microcapsules by partial Seitenkettenhydrolyse

2500 g of a microcapsule suspension according to 4 (a) are mixed with stirring with 501 g of sodium hydroxide solution with a concentration of 8.10 ^-2 mol / l. A pH of 12 is established in the reaction mixture. The mixture is stirred at room temperature for 20 minutes. The pH is 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) is 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 ranges from 0.8 to 10 µm with a maximum at 1.8 µm and the microcapsule concentration is 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) is purified by flotation at least once against 0.02% Triton-X100 solution (pH = 4.5, adjusted with hydrochloric acid). 7.5.10 ⁸ particles of the cleaned suspension are placed in a scintillation vial with 15 ml sodium acetate buffer and stirred on a magnetic stirrer using a stick stirrer. (Sodium acetate buffer = solution A: 1.36 g NaCH3COOO.3H ₂ OM = 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 water, 400 ml of solution A are introduced and adjusted to a pH of 4.5 with solution B.)

300 µg streptavidin (company SIGMA) are in 300 µl sterile filtered water solved. The solution becomes a sodium acetate buffer - microcapsule suspension given. The pH should be 4.5 ± 0.2. Then be quick 750 µg EDC (1 ethyl-3- (3-dimethylaminopropyl) carboiimide hydrochloride; company SIGMA) dissolved in 750 µl sterile filtered water and in the buffer solution given.

It is now stirred for an hour with occasional pH control and stirred overnight (approx. 17 h) at 4 degrees Celsius.

Finally, a solution of 188 ul 1 M ethanolamine solution (company SIGMA) in 3.75 ml of water.

After flotation (24 h), the batch is drained down to a volume of 1 ml was concentrated. Then 10 ml Hepes buffer / Triton solution 0.01% (30 ml 5 M NaCL solution + 10 ml 1 M Hepes solution (Gibco 15630-056) ad 1000 ml Triton solution 0.01% pH 7.4) added, floated again and the shelter again to a volume drained from 1 ml. The concentrated suspension is now in one Siliconized Eppendorf reaction vessel transferred and stored in the refrigerator. The coupling success is in the FACS (flow cytometer from Becton Dickinson) with FITC biotin. With the help of a saturation series Quantified 200,000 streptavidin molecules per microcapsule.

(f) Binding of biotinylated anti-CD105 antibodies to functionalized gas-filled microbubbles

The suspension of gas-filled microbubbles according to Example 4a-d) to which Streptavidin according to Example 4e) was bound for 10 minutes Room temperature with biotinylated anti-CD105 antibody (advice 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 is made up to 1e8 bubbles / ml with PBS buffer, containing 0.02% Triton × 100, diluted. The CD105 coupling and thinning is freshly made for each animal.

(g) Binding of biotinylated IgG2a to functionalized gas-filled microcapsules

The suspension of gas-filled microbubbles according to Example 4a-d) to which streptavidin according to Example 4e) was bound is 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) in a ratio of 0.5 µg antibody to 1e6 microbubbles.

After incubation, the suspension is made up to 1e8 bubbles / ml with PBS buffer, containing 0.02% Triton × 100, diluted. Isotype coupling and dilution is freshly made for each animal.

Claims (22)

1. A device for quantifying bubbles, characterized in that ultrasound cross-sectional images are taken, which overlap and are mutually displaced in parallel, the bubbles stimulating independent, characteristic signals and these signals being measured. 2. Device according to claim 1, characterized in that the Sending the independent, characteristic signal with the Destruction of the bladder goes hand in hand. 3. Device according to one of claims 1 or 2, characterized characterized that the volumes vary between 20% and 99.99% overlap. 4. Device according to one of claims 1 to 3, characterized characterized that the volumes vary between 40% and 99.9% overlap. 5. Device according to one of claims 1 to 4, characterized characterized that the volumes vary between 70% and 99% overlap. 6. Device according to one of claims 1 to 5, characterized characterized in that the volumes are parallel and transverse overlap and are shifted. 7. Device according to one of claims 1 to 6, characterized characterized in that the device includes a transducer which is moved relative to the object under examination. 8. The device according to claim 7, characterized in that the Examination object moved relative to a fixed transducer becomes. 9. The device according to claim 7, characterized in that the Transducer moved relative to a fixed examination object becomes. 10. The device according to claim 7, characterized in that the Transducer defines shifted ultrasound sectional images. 11. Method for quantifying bubbles, characterized in that Ultrasound sectional images of the bubbles in the examination object are recorded, with the ultrasound radiation Bubbles destroyed by scattering and movement of the Bubble independent and different from the irradiation signal independent characteristic signals are detected and measured become. 12. The method according to claim 11, characterized in that the independent, characteristic signal with the destruction of the bladder goes hand in hand and the determination of the number of bubbles above the Saturation bubble concentration is, preferably the Bladder concentration is greater than 3000 bubbles per milliliter. 13. The method according to any one of claims 11 or 12, characterized characterized that the volumes vary between 20% and 99.99% overlap. 14. The method according to any one of claims 11 to 13, characterized characterized that the volumes vary between 40% and 99.9% overlap. 15. The method according to any one of claims 11 to 14, characterized characterized that the volumes vary between 70% and 99% overlap. 16. The method according to any one of claims 11 to 15, characterized characterized in that the volumes are parallel and transverse overlap and are shifted. 17. The method according to any one of claims 11 to 16, characterized characterized that a transducer opposite the Examination object is moved. 18. The method according to claim 17, characterized in that the Examination object moved relative to a fixed transducer becomes. 19. The method according to claim 17, characterized in that the Transducer moved relative to a fixed examination object becomes. 20. The method according to claim 17, characterized in that the Transducer defines shifted ultrasound sectional images. 21. The method according to at least one of claims 11-20, wherein the Bubbles are quantified in vivo, ex vivo or in vitro. 22. The method according to at least one of claims 11-21, wherein overlapping and shifted parallel to each other Ultrasound sectional images are recorded.