U.S. patent application number 13/626826 was filed with the patent office on 2013-04-04 for overlay image contrast enhancement.
The applicant listed for this patent is Desmond Hirson, James Mehi, Stanley Poon, Christopher A. White. Invention is credited to Desmond Hirson, James Mehi, Stanley Poon, Christopher A. White.
Application Number | 20130083981 13/626826 |
Document ID | / |
Family ID | 38049147 |
Filed Date | 2013-04-04 |
United States Patent
Application |
20130083981 |
Kind Code |
A1 |
White; Christopher A. ; et
al. |
April 4, 2013 |
OVERLAY IMAGE CONTRAST ENHANCEMENT
Abstract
A method of creating an image difference overlay comprises
identifying a loop of reference images of a subject and identifying
a loop of data images of the subject. The loop of image data can be
identified after an event, such as the administration of contrast
agent to the subject. A reference loop image frame is compared to
one or more data loop image frames and the reference loop frame is
associated with a data loop image frame which closely resembles the
data loop image frame. Each of the associated frames can then be
processed and used to create an image difference overlay frame.
Inventors: |
White; Christopher A.;
(Toronto, CA) ; Hirson; Desmond; (Thornhill,
CA) ; Poon; Stanley; (Thornhill, CA) ; Mehi;
James; (Thornhill, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
White; Christopher A.
Hirson; Desmond
Poon; Stanley
Mehi; James |
Toronto
Thornhill
Thornhill
Thornhill |
|
CA
CA
CA
CA |
|
|
Family ID: |
38049147 |
Appl. No.: |
13/626826 |
Filed: |
September 25, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11595047 |
Nov 10, 2006 |
8275449 |
|
|
13626826 |
|
|
|
|
60735399 |
Nov 11, 2005 |
|
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Current U.S.
Class: |
382/128 |
Current CPC
Class: |
G06T 2207/10132
20130101; A61B 8/483 20130101; G06T 5/50 20130101; G06T 2207/30084
20130101; G06T 2207/30004 20130101; A61B 6/507 20130101; A61B 8/481
20130101; G06T 2207/30048 20130101; G06T 5/009 20130101; A61B 6/00
20130101; A61B 6/508 20130101 |
Class at
Publication: |
382/128 |
International
Class: |
G06T 5/50 20060101
G06T005/50 |
Claims
1-58. (canceled)
59. A method of producing an enhanced medical image of a subject,
comprising: producing a reference set of medical images of the
subject or a portion thereof using a medical imaging modality,
wherein the reference set of medical images comprises a plurality
of reference set images; creating an area of increased image
contrast in the subject; producing a data set of medical images of
the subject or a portion thereof using the medical imaging
modality, wherein the data set of medical images comprises a
plurality of data set images, each data set image comprising data
received from the area of increased image contrast; comparing at
least one data set image to a plurality of reference set images;
determining the relative similarity between the data set image and
each compared reference set image; selecting a data-reference image
pair, wherein the data-reference image pair comprises a data set
image and a reference set image, and wherein the data-reference
image pair is based on the reference image's determined relative
similarity to the data image; and processing the data-reference
image pair to create the enhanced medical image by removing the
areas of substantial similarity between the data image and the
reference image of the data-reference image pair.
60. A system for producing an enhanced medical image of a subject,
comprising: a medical imaging modality for producing a reference
set of medical images of the subject or a portion thereof, wherein
the reference set of medical images comprises a plurality of
reference set images and wherein the reference medical images
comprise data received from the subject's tissue and for producing
a data set of medical images, wherein the data set of medical
images comprises a plurality of data set images, and wherein the
data set of medical images comprises contrast data received from a
medical imaging contrast agent administered to the subject and data
received from the subject's tissue; and a processor for comparing
at least one data set image to a plurality of reference set images
to identify a data-reference image pair, wherein the data-reference
image pair is identified based on the similarity of tissue data of
the reference image relative to the tissue data of the data image
and for subtracting the reference image from the data image of the
data-reference image pair to create the enhanced medical image.
61. The system of claim 60, further comprising a computer readable
medium having computer readable program code for comparing at least
one data set image to a plurality of reference set images to
identify a data-reference image pair, wherein the data-reference
image pair is identified based on the similarity of tissue data of
the reference image relative to the tissue data of the data image
and for subtracting the reference image from the data image of the
data-reference image pair to create the enhanced medical image.
62. A system for producing an enhanced medical image of a subject,
comprising: means for creating an area of increased image contrast
in the subject; a medical imaging modality for producing a
reference set of medical images of the subject or a portion
thereof, wherein the reference set of medical images comprises a
plurality of reference set images and for producing a data set of
medical images of the subject or a portion thereof, wherein the
data set of medical images comprises a plurality of data set
images, each data set image comprising data received from the area
of increased contrast; and a processor for comparing at least one
data set image to a plurality of reference set images to identify a
data-reference image pair, wherein the data-reference image pair
comprises the reference set image that has the smallest difference
error as to the data set image versus the other compared reference
set images and for subtracting the reference image of the
data-reference image pair from the data image of the data-reference
image pair to create the enhanced medical image.
63. The system of claim 62, further comprising a computer readable
medium having computer readable program code for comparing at least
one data set image to a plurality of reference set images to
identify a data-reference image pair, wherein the data-reference
image pair is identified based on the similarity of tissue data of
the reference image relative to the tissue data of the data image
and for subtracting the reference image from the data image of the
data-reference image pair to create the enhanced medical image.
Description
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] This application is a division of U.S. patent application
Ser. No. 11/595,047, filed on Nov. 10, 2006, now U.S. Pat. No.
8,275,449, which claims the benefit of U.S. Provisional Patent
Application No. 60/735,399 filed Nov. 11, 2005, both of which are
fully incorporated herein and made a part hereof in their
entireties.
BACKGROUND
[0002] Sufficient contrast differences between tissues in acquired
images of patients and small animals are important for medical
diagnosis and biomedical research. To increase the signal intensity
difference between tissues in such acquired images contrast agents
specific for that imaging modality are often used. A comparison
between a single post-contrast agent administrated image with a
single pre-contrast agent image, as selected by the operator, can
be made to identify specific tissue volumes that have undergone
contrast enhancement. Identification of which tissues undergo
contrast enhancement, and how much enhancement, is an important
indicator for many biomedical research and diagnostic applications.
For in vivo imaging, however, respiration, cardiac, and
non-specific motion of patients and small animals causes
post-contrast injection images to be dissimilar when compared to a
pre-single injection image making the comparison difficult.
SUMMARY OF THE INVENTION
[0003] A method of creating an image difference overlay, or
enhanced medical image, comprises identifying a set of reference
images, or frames, of a subject and identifying a set of data
images, or frames, of the subject. A set of images, also referred
to as a "loop" or "cine clip" can comprise one or more images
acquired sequentially. The set of data images can be identified
after an event, such as the administration of contrast agent to the
subject, or after removal of a contrast agent already in the
subject. A single reference image can be compared to one or more
data set images and the reference set frame is associated with a
data set image which closely resembles the reference set image.
Each of the associated images can then be processed and used to
create an enhanced medical image, also referred to throughout as an
image difference overlay, or contrast overlay. In one non-limiting
example, the contrast agent used is a microbubble contrast agent,
and the image sets are acquired using ultrasound.
[0004] Additional advantages of the invention will be set forth in
part in the description which follows, and in part will be obvious
from the description, or may be learned by practice of the
invention. The advantages of the invention will be realized and
attained by means of the elements and combinations particularly
pointed out in the appended claims. It is to be understood that
both the foregoing general description and the following detailed
description are exemplary and explanatory only and are not
restrictive of the invention, as claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate (one) several
embodiment(s) of the invention and together with the description,
serve to explain the principles of the invention.
[0006] FIG. 1 is a flow diagram in block form of an exemplary
embodiment of a method creating a contrast overlay.
[0007] FIG. 2 is a flow diagram in block form showing the creation
of a persisted contrast overlay.
[0008] FIG. 3 is a block diagram illustrating an exemplary
computing operating environment.
[0009] FIG. 4 is a block diagram illustrating an exemplary
ultrasound imaging system.
[0010] FIG. 5 is a flow diagram of an exemplary image processing
block 108 and 110 of FIG. 1.
[0011] FIG. 6A shows a pre-contrast agent injection reference loop
ultrasound image. FIG. 6B shows a post contrast agent injection
data loop ultrasound image. FIG. 6C shows a contrast overlay. FIG.
6D shows the data loop image of FIG. 6B with the contrast overlay
of FIG. 6C blended in.
[0012] FIGS. 7A and 7B shows results of a bolus injection of
microbubbles in healthy kidney at 40 MHz. FIG. 7A shows kidney
before injection and 7C shows the kidney after injection.
Background-subtracted contrast enhancement after injection is shown
in FIG. 7D. FIG. 7B shows pixel intensity averaged over a ROI
encompassing the kidney for bolus injections of 10.sup.5, 10.sup.6,
10.sup.7, and 10.sup.8 micro bubbles (herein referred to as
MB).
[0013] FIGS. 8A, 8B, 8C, and 8D show ultrasound images before MB
destruction in a kidney. The top row shows post-ischemic in FIG. 8A
and healthy contra lateral kidney in FIG. 8B before a destructive
pulse. The bottom row shows background-subtracted images of
post-ischemic in FIG. 8C and healthy contra lateral kidney FIG. 8D
before a destructive pulse.
[0014] FIGS. 9A and 9B show background-subtracted mean pixel
intensity of ROI around kidney showing contrast enhancement in a
post-ischemic kidney in FIG. 9A and in a healthy contra lateral
kidney in FIG. 9B.
[0015] FIG. 10 shows pixel intensity within ROI averaged over 100
frames before a destructive pulse and after MB destruction. Error
bars show standard deviation over 100 frames.
[0016] FIGS. 11A, 11B, 11C, and 11D show ultrasound images before
micro bubble destruction in hind leg. The top row shows 3-hour
TNF-alpha treated in FIG. 11A and healthy untreated hind leg in
FIG. 11B before a destructive pulse. The bottom row shows
background-subtracted images of 3-hour TNF-alpha treated in FIG.
11C and healthy untreated hind leg in FIG. 11D before a destructive
pulse.
[0017] FIG. 12 shows pixel intensity within an ROI averaged over
100 frames before a destructive pulse and after MB destruction in
inflamed and non-inflamed (untreated) hind leg. Error bars show
standard deviation over 100 frames.
[0018] FIG. 13 shows a contrast region measurement created using
exemplary methods of creating an enhanced medical image.
[0019] FIG. 14 shows a contrast region intensity curve vs time
graph created using data obtained from an enhanced medical image
created using the exemplary methods described herein.
[0020] FIG. 15 shows a baseline image prior to DEPO.TM. bolus
injection. Infarct regions in the anterolateral and posterolateral
walls are illustrated as is the area of viable myocardium.
[0021] FIG. 16 shows an image of initial bolus injection as the
left ventricular cavity is filled with DEPO.TM. contrast agent.
[0022] FIG. 17 shows an image of the left ventricle 10 minutes
following injection of DEPO.TM.. The DEPO.TM. contrast agent has
perfused into the viable myocardial microcirculation and has
cleared the left ventricular cavity.
[0023] FIG. 18 shows a processed image showing the myocardial
infarction using the DEPO.TM. data; the overlay indicates regions
of well perfused myocardium and areas without the overlay indicate
areas of myocardial infarction and tissue damage.
[0024] FIG. 19 shows a polar coordinate graph of processed images
demonstrating the location of the region of infarction. Strong
image intensity (y-axis) indicates viable myocardium and conversely
low signal intensity illustrates damaged myocardial tissue.
DETAILED DESCRIPTION
[0025] The present invention may be understood more readily by
reference to the following detailed description of preferred
embodiments of the invention and the Examples included therein and
to the Figures and their previous and following description.
[0026] Before the present compounds, compositions, articles,
devices, and/or methods are disclosed and described, it is to be
understood that this invention is not limited to specific synthetic
methods, specific computer or imaging system architecture or
modality or to particular contrast agent or administration
protocols, as such may, of course, vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular aspects only and is not intended to be
limiting.
[0027] As used throughout, the singular forms "a," "an" and "the"
include plural referents unless the context clearly dictates
otherwise. Thus, for example, reference to "a processor" can
include two or more such processors unless the context indicates
otherwise.
[0028] Ranges may be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another embodiment includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint.
[0029] "Optional" or "optionally" means that the subsequently
described event or circumstance may or may not occur, and that the
description includes instances where said event or circumstance
occurs and instances where it does not.
[0030] By a "subject" is meant an individual. The term subject
includes small or laboratory animals as well as primates, including
humans. A laboratory animal includes, but is not limited to, a
rodent such as a mouse or a rat. The term laboratory animal is also
used interchangeably with animal, small animal, small laboratory
animal, or subject, which includes mice, rats, cats, dogs, fish,
rabbits, guinea pigs, rodents, etc. The term laboratory animal does
not denote a particular age or sex. Thus, adult and newborn
animals, as well as fetuses (including embryos), whether male or
female, are included. The term subject can also include other forms
such as a collection of isolated cells either in vivo or ex vivo as
might be collected on a Petri dish.
[0031] Provided herein are systems and methods for creating an
enhanced medical image, also referred to herein as an image
difference overlay or as a contrast overlay. Such terms are used
interchangeable throughout the description, examples and in the
claims. An enhanced medical image, or contrast overlay can be used
to highlight differences in intensity between a reference image and
a data image. A "reference image" is also referred to throughout as
a "reference set image" and a "data image" is also referred to as a
"data set image." Such terms are used interchangeably
throughout.
[0032] In one aspect, the methods and systems described in this
document illustrate an improved method for the identification and
quantification of small brightness differences in a medically
acquired image, or a medical image, as a result of infusion of
contrast agent specific for that imaging modality.
[0033] When a contrast agent for a given imaging modality is used,
multiple pre-injection images (reference set images) can be used as
a reference set. In this way, the accuracy and simplicity of
matching post-injection images (data set images) with similar
pre-injection images from a set of multiple pre-injection images is
increased. The results include reduced effort on the part of the
operator, and improved accuracy in the results. As used herein, a
pre-injection image, selected from a set of multiple pre-injection
images, matched with a post-injection image, selected from a set of
multiple post-injection images can be referred to as a contrast
overlay or an enhanced medical image.
[0034] The process for image processing uses two or more sets of
images: a reference set, and a post-injection or post increased
contrast data set. The data set typically refers to a series of
sequential images composed as a image loop, or cine clip. The
position of the animal can be made as static as possible to prevent
false positives. The methods described herein have inherent
stability for small changes in image positioning due to respiration
and cardiac motion, or due to other biodynamic cycle motions of a
subject. The reference set or sets and data set or sets can then be
compared with each other to associate the images(s) which are most
similar. Each image of these associated loops can then be processed
with filters to remove local image features and image noise. They
can then be subtracted and a difference map generated to form a
contrast overlay.
[0035] The described system and methods for processing the data
enables more accurate results to be obtained without the need for
complicated ECG or respiration gating. The systems and methods can
also be used to image embryos in vivo where an accurate ECG signal
specific to that embryo is not acquired. Heart beats and the
respiration motion of a subject typically disturbs or moves the
image disallowing direct image subtraction to highlight differences
in intensity. Image subtraction is typically only suitable for
comparing images where only the features of interest have changed.
If the two images are dissimilar, have shifted or undergone some
other transformation, the subtraction process will result in
significant false results. The disclosed systems can also be used
for perfusion imaging. Perfusion imaging can be performed by
injecting contrast agent into the subject, allowing the agent to
circulate and then destroying them in a destruction event. The time
taken for the re-perfusion can be used to establish flow or
perfusion into an organ or portion thereof.
[0036] An exemplary method for creating an image difference overlay
comprises identifying a set of reference images of a subject or
portion thereof. The method further comprises identifying a set of
data images of the subject or the portion thereof. A data image can
be compared to a plurality of images of the reference image loop.
At least one data image can be associated with at least one
reference image. An image subtraction on the associated images can
be performed to produce the contrast overlay or enhanced medical
image.
[0037] In one aspect, the data image is associated with the at
least one reference loop image based on similarity. For example,
the similarity can be determined by summing the absolute difference
of pixel intensity levels and associating the data set image with
the reference set image or images which yield the smallest absolute
difference value. Other methods can be used to determine
similarity.
[0038] The identification of a set of reference images and data
images can comprise acquiring images using an imaging modality. The
imaging modality used can be any medical imaging modality. For
example, the imaging modality can selected from the group
consisting of ultrasound, computed tomography (CT), optical
coherence tomography (OCT), radiography (or X-Ray including
fluorescence), optical detection (either with or without a
magnifying lens or microscope), thermography, nuclear medical
imaging, positron emission tomography, bioluminescent imaging,
biofluorescent imaging, and magnetic resonance imaging (MRI).
[0039] The methods described herein can further comprise
administering a contrast agent to the subject. The administered
contrast agent is typically complementary to the imaging modality
used. For example, a micro bubble (MB) contrast agent can be used
with ultrasound imaging modalities. Contrast agents complementary
to other imaging modalities are known to those skilled in the
art.
[0040] A data set image can comprise data based on signals received
from one or more contrast agent(s) located in the subject or a
portion thereof. Image subtraction on the associated reference set
images can be used to highlight the portion of the image
corresponding to the location of the administered contrast agent in
the subject.
[0041] In one aspect, the contrast agent can be administered to the
subject subsequent to acquiring images of the reference set and the
images of the data set can be acquired subsequent to the
administration of the contrast agent.
[0042] In another aspect, the images of the data set can be
acquired subsequent to the administration of the contrast agent and
the images of the reference set are acquired subsequent to
acquiring the images of the data set. The administered contrast
agent can be substantially cleared from the subject prior to
acquiring the images of the reference set. For example, the micro
bubble contrast agent can be substantially cleared by destroying
the contrast agent with ultrasound. Micro bubble contrast agent can
also be destroyed via chemical interactions with other administered
drugs and can also be destroyed by altering the oxygenation level
of the blood. For example, increasing the percentage oxygen
breathed to the subject can be used to increase the oxygen level in
the blood. Methods to destroy or remove contrast agent for other
modalities can include chemical methods or waiting until the
contrast agent is expelled from the subject. Contrast agent
expulsion from the subject can include being filtered by the liver
and or kidneys or via the contrast agent breaking down or binding
with other molecules and organelles already present in the subject
including cells, oxygen, and carbon dioxide.
[0043] A system for creating a contrast overlay can comprises a
processing unit or processor for identifying a set of reference
images of a subject or portion thereof. The same or another
processor or processing unit of the system can identify a set of
data images of the subject or the portion thereof and can compare a
reference image to a plurality of images of the data image set.
Moreover, the same or another processing unit can associate at
least one reference image with at least one data image and can
performing an image subtraction on the associated images to produce
the contrast overlay. The processing unit can comprise software or
a computer readable medium having computer readable code for
comparing reference set images to data set images and for
subtracting reference set images from data set images.
[0044] Further provided herein is a computer-readable medium having
computer readable program code for creating a contrast overlay. The
computer readable medium comprises program code for inputting an
identified loop of reference images of a subject or portion
thereof. The computer-readable medium further comprises program
code for inputting an identified set of data images of the subject
or the portion thereof and program code for comparing a data image
to a plurality of images of the reference image set and matching at
least one data image with at least one reference image. The
computer-readable medium also can comprise program code for
performing an image subtraction on the matched images to produce
the contrast overlay.
[0045] An exemplary method of creating a contrast overlay comprises
identifying a set of reference images of a subject and identifying
a set of data images of the subject. The set of data images, which
includes a plurality of data set images, can be identified after an
event, such as the administration of contrast agent to the subject
or the removal of contrast agent from the subject either by
destruction or by being expelled from the subject. A data set image
is compared to one or more reference set images, which comprise a
reference set, and the data set image is associated with one or
more reference set images which closely resembles the data set
image. The associated images can then be processed and used to
create a contrast overlay image. In one non-limiting example, the
contrast agent used is a micro bubble contrast agent, and the
images loop images are identified using ultrasound.
[0046] Also, provided herein is a method for creating an image
difference overlay (also called herein a "contrast overlay" or
"enhanced medical image") comprising the steps of identifying a set
of reference images of a subject; identifying a set of data images
of the subject; comparing a data set image to each reference set
image wherein the data set image is associated with one or more
reference set images which most closely resembles the data set
image; performing optional image processing on each of the
associated images; performing image subtraction of the associated
data image with an associated reference image wherein an contrast
overlay frame is created.
[0047] The reference set and data set can comprise a plurality of
reference set and data set ultrasound images. The data set can
comprise "after" ultrasound images. After images are images of the
same subject, or portion thereof, as the reference images acquired
before some event, such as the administration of a contrast agent
in the subject or after the destruction of all or a portion of an
administered contrast agent.
[0048] To obtain an ultrasound image with or without a contrast
agent, high frequency ultrasound can be used. The methods described
herein are not limited to ultrasound and ultrasound contrast agents
however. Any imaging modality can be used with a complementary
contrast agent. Moreover, if CT, Micro-CT, MRI, OTC,
Bioluminescence, Biofluorescence, or another imaging modality is
used, a complementary contrast agent can be selected as would be
clear to one skilled in the art.
[0049] If ultrasound is used, the ultrasound can be transmitted
into the subject at a frequency of about 15 megahertz (MHz) or
greater. Lower frequency or clinical frequency ultrasound, however,
can also be used. Thus, ultrasound can be transmitted into the
subject at a frequency of less than 15 MHz.
[0050] Optionally, the ultrasound is transmitted into the subject
at a frequency of between about and between 15 MHz and about 80
MHz. Thus, the ultrasound can be transmitted into the subject at a
frequency of about and between 20 MHz, 30 MHz, 40 MHz, 50 MHz, 60
MHz, 70 MHz, 80 MHz, or higher. For example, the ultrasound can be
transmitted into the subject at a frequency of about 100 MHz or
higher.
[0051] If high frequency ultrasound is desired, the ultrasound for
use with the disclosed methods can be applied, transmitted and
received using an ultrasonic scanning device that can supply an
ultrasonic signal of at least 15 MHz to the highest practical
frequency. Any system capable of operating at such frequencies can
be used. One such device is the VisualSonics.TM. (Toronto, CA) UBM
system model VEVO.TM. 660. Another device is the VisualSonics.TM.
(Toronto, CA) model VEVO.TM. 770. Another device is the
VisualSonics.TM. (Toronto, CA) model VEVO.TM. 770B. Another such
system can have the following components as described in U.S.
patent application Ser. No. 10/683,890, US patent application
publication 20040122319, which is incorporated herein by reference.
If clinical frequencies of less than 15 MHz are used, any
ultrasound system capable of operating at clinical frequencies can
be used.
[0052] Other devices capable of transmitting and receiving
ultrasound at the desired frequencies can also be used. For
example, ultrasound systems using arrayed transducers can be used.
One such exemplary array system, which is incorporated herein by
reference for its teaching of a high frequency array ultrasound
system, is described in U.S. non-provisional application titled
"HIGH FREQUENCY ARRAY ULTRASOUND SYSTEM" by James Mehl, Ronald E.
Daigle, Laurence C. Brasfield, Brian Starkoski, Jerrold Wen, Kai
Wen Liu, Lauren S. Pflugrath, F. Stuart Foster, and Desmond Hirson,
and filed Nov. 2, 2006 and assigned attorney docket number
22126.0023U2
[0053] In one aspect, the ultrasound images can be high-resolution,
high frequency ultrasound images wherein a contrast agent was
injected into the subject.
[0054] Thus, the methods can comprise the administration of a
contrast agent to a subject. If ultrasound is the imaging modality
used, the contrast agent can be a microbubble contrast agent. A
contrast agent such as a microbubble population for ultrasound
imaging is only exemplary. Contrast agents for CT, Micro-CT, MRI,
Optical Coherence, bioluminescence, bioflorescence, or other
imaging modalities can also be used.
[0055] A microbubble contrast agent typically comprises a plurality
of microbubbles. Non-limiting examples of commercial microbubble
contrast agents include, but are not limited to, Definity.TM.,
Sonovue.TM., Levovist.TM., Optison.TM., MicroMarker.TM., and
MicroMarker.TM. Depo.TM.. Moreover, microbubbles can be obtained
from Targeson (Charlottesville, Va.) or ImaRx (Tucson, Ariz.),
Bracco (Amsterdam, Netherlands), and VisualSonics Inc. (Toronto,
CA). Ultrasound contrast agents are typically gas filed bubbles
with diameters ranging from 1 to 4 microns. The bubble size
distribution can range form sub-micron size to up to 10 micron in
size. An exemplary bubble produced by ImaRx bubble has an average
size of 0.9 microns and an exemplary bubble from Targeson has a
range of 2 to 4 microns in size. Another exemplary bubble product
is the Depo.TM. MicroMarker.TM. product made by Bracco for
VisualSonics. The Depo.TM. product comprises a larger bubble
population that is designed to lodge in the small vasculature of a
subject. The term microbubble as used herein is not intended to be
limited to bubbles of 1 micron or larger. Nano-sized bubbles are
also included in the term microbubble.
[0056] Thus, the methods and systems described herein are not
limited to any particular contrast agent. Microbubble commercial
contrast agents can be used, but one skilled in the art can also
produce microbubbles that would be effective with the disclosed
systems and methods. Combinations of microbubble populations can
also be used. Such microbubbles can be targeted, untargeted or
lodging microbubbles.
[0057] A typical microbubble contrast agent comprises a thin
flexible or rigid shell composed of albumin, lipid or polymer
confining a gas such as nitrogen or a perfluorocarbon. Other
examples of representative gases include air, oxygen, carbon
dioxide, hydrogen, nitrous oxide, inert gases, sulpher fluorides,
hydrocarbons, and halogenated hydrocarbons. Liposomes or other
microbubbles can also be designed to encapsulate gas or a substance
capable of forming gas as described in U.S. Pat. No. 5,316,771. In
another embodiment, gas or a composition capable of producing gas
can be trapped in a virus, bacteria, or cell to form a microbubble
contrast agent. The described ultrasound contrast agents improve
contrast by acting as sound wave reflectors due to acoustic
differences between the agents and surrounding liquid or by
resonating.
[0058] A wide variety of materials can be used in preparing
microbubble membrane or shell. Any compound or composition that
aids in the formation and maintenance of the bubble membrane or
shell by forming a layer at the interface between the gas and
liquid phases can be used. Sonication can be used for the formation
of microbubbles, i.e., through an ultrasound transmitting septum or
by penetrating a septum with an ultrasound probe including an
ultrasonically vibrating hypodermic needle. Optionally, larger
volumes of microbubbles can be prepared by direct probe-type
sonicator action on the aqueous medium in Which microbubbles are
formed in the presence of gas (or gas mixtures) or another
high-speed mixing technique, such as blending or milling/mixing.
Other techniques such as gas injection (e.g. venturi gas
injection), mechanical formation such as through a mechanical high
shear 15 valve (or double syringe needle) and two syringes, or an
aspirator assembly on a syringe, or simple shaking, may be used.
Microbubbles can also be formed through the use of a liquid osmotic
agent emulsion supersaturated with a modifier gas at elevated
pressure introduced into in a surfactant solution.
[0059] Thus, the administered Microbubbles can comprise one or more
gasses. For example, the gas can be a fluorine containing
hydrocarbon gas. Optionally, the gas is selected from the group
consisting of decafluorobutane, octafluorobutane, perfluorohexane,
and dodecofluoropentane. The gas can also be sulfur hexafluoride or
nitrogen. The microbubbles are not limited to these gases, however,
and other gases used for ultrasound contrast agents can also be
used.
[0060] Non-limiting gases that can be used alone or in combination
include, for example, air; nitrogen; oxygen; carbon dioxide;
hydrogen; an inert gas such as helium, argon, xenon or krypton; a
sulphur fluoride such as sulphur hexafluoride, disulphur
decafluoride or trifluoromethylsulphur pentafluoride; selenium
hexafluoride; an optionally halogenated silane such as methylsilane
or dimethylsilane; a low molecular weight hydrocarbon (e.g.
containing up to 7 carbon atoms), for example an alkane such as
methane, ethane, a propane, a butane or a pentane, a cycloalkane
such as cyclopropane, cyclobutane or cyclopentane, an alkene such
as ethylene, propene, propadiene or a butene, or an alkyne such as
acetylene or propyne; an ether such as dimethyl ether; a ketone; an
ester; a halogenated low molecular weight hydrocarbon (e.g.
containing up to 7 carbon atoms); or a mixture of any of the
foregoing. At least some of the halogen atoms in halogenated gases
can be fluorine atoms; thus halogenated hydrocarbon gases may, for
example, be selected from bromochlorodifluoromethane,
chlorodifluoromethane, dichlorodifluoromethane,
bromotrifluoromethane, chlorotrifluoromethane,
chloropentafluoroethane, dichlorotetrafluoroethane,
chlorotrifluoroethylene, fluoroethylene, ethylfluoride,
1,1-difluoroethane and perfluorocarbons. Representative
perfluorocarbons include: perfluoroalkanes such as
perfluoromethane, perfluoroethane, perfluoropropanes,
perfluorobutanes (e.g., perfluoro-n-butane, optionally in admixture
with other isomers such as perfluoro-isobutane), perfluoropentanes,
perfluorohexanes or perfluoroheptanes; perfluoroalkenes such as
perfluoropropene, perfluorobutenes (e.g. perfluorobut-2-ene),
perfluorobutadiene, perfluoropentenes (e.g., perfluoropent-1-ene)
or perfluoro-4-methylpent-2-ene; perfluoroalkynes such as
perfluorobut-2-yne; and perfluorocycloalkanes such as
perfluorocyclobutane, perfluoromethylcyclobutane,
perfluorodimethylcyclobutanes, perfluorotrimethylcyclobutanes,
perfluorocyclopentane, perfluoromethylcyclopentane,
perfluorodimethylcyclopentanes, perfluorocyclohexane,
perfluoromethylcyclohexane or perfluorocycloheptane. Other
halogenated gases include methyl chloride, fluorinated (e.g.,
perfluorinated) ketones such as perfluoroacetone and fluorinated
(e.g., perfluorinated) ethers such as perfluorodiethyl ether. The
use of perfluorinated gases, for example sulphur hexafluoride and
perfluorocarbons such as perfluoropropane, perfluorobutanes,
perfluoropentanes and perfluorohexanes, can be particularly
advantageous in view of the recognized high stability in the
bloodstream of microbubbles containing such gases. Other gases with
physicochemical characteristics which cause them to form highly
stable microbubbles in the bloodstream may likewise be useful.
[0061] Thus, in one aspect, one or more gasses can be enclosed in a
shell to form a microbubble. The shell can comprise a lipid.
Optionally, the shell is a lipid monolayer and the gas is
decafluorobutane.
[0062] A contrast agent can be modified to achieve a desired volume
percentage by a filtering process, such as by microfiltration using
a porous membrane. Contrast agents can also be modified by allowing
larger bubbles to separate in solution relative to smaller bubbles.
For example, contrast agents can be modified by allowing larger
bubbles to float higher in solution relative to smaller bubbles. A
population of microbubbles of an appropriate size to achieve a
desired size distribution can subsequently be selected. Other means
are available in the art for separating microbubble sizes and can
be adapted to select a microbubble population of bubbles such as by
centrifugation. Thus, microbubble populations can be produced with
a proportion of bubbles large enough to lodge in the
microvasculature of a subject. Microbubble populations can also be
selected for a smaller size, including a nanometer size to increase
bubble resonance. For example, a smaller population can be selected
as described in U.S. patent application Ser. No. 11/040,999, U.S.
publication number 20060078501, which is incorporated herein by
reference.
[0063] The number of microbubbles of differing sizes in a
population can be determined, for example, using an optical
decorrelation method. The diameter of microbubbles making up given
population can also be determined and the number percentage of
microbubbles at different sizes can also be determined. For optical
decorrelation methods a Malvin.TM. Zetasizer.TM. or similar
apparatus may be used.
[0064] A plurality of microbubbles can be in a physiologically
acceptable composition for administration to the subject. Such
physiologically acceptable compositions can comprise buffers,
diluents, therapeutic or pharmacologic agents, preservatives and
others compositions known in the art. Thus, an administered
physiologically acceptable composition can comprise a plurality of
microbubbles in combination with one or more additional components.
Such additional components, can be selected by one skilled in the
art based factors including, but not limited to the type of
microbubble used and the desired imaging protocol. Factors related
to imaging protocol that can direct selection of a suitable
additional component, can include, but are not limited to,
administration factors (i.e., for example, location), imaging
factors (i.e., for example, duration, delay between administration
and imaging, tissue or organ imaged, etc.) and subject factors
(i.e., for example, type of subject imaged).
[0065] Administration of contrast imaging agents of the present
invention can be carried out in various fashions, such as
intravascularly, intralymphatically, parenterally, subcutaneously,
intramuscularly, intraperitoneally, interstitially, hyperbarically,
orally, or intratumorly using a variety of dosage forms. One
preferred route of administration is intravascularly. For
intravascular use the contrast agent is generally injected
intravenously, but may be injected intraarterially as well. The
useful dosage to be administered and the mode of administration may
vary depending upon the age and weight of the subject, and on the
particular imaging application intended. The dosage can be
initiated at lower levels and increased until the desired contrast
enhancement is achieved.
[0066] The contrast agent can be administered in the form of an
aqueous suspension such as in water or a saline solution (e.g.,
phosphate buffered saline). The water can be sterile and the saline
solution can be a hypertonic saline solution (e.g., about 0.3 to
about 0.5% NaCl), although, if desired, the saline solution may be
isotonic. The solution also may be buffered, if desired, to provide
a pH range of pH 6.8 to pH 7.4. In addition, dextrose may be
included in the media.
[0067] Exemplary dosing can be based on the body weight of the
subject and on composition administered. Generally, however, the
dosage can vary with the imaging protocol and the desired imaging
characteristics, and can be determined by one skilled in the art.
The dosage can be adjusted by the individual researcher. It is
further contemplated that the dosage can vary, and can be
administered in one or more dose administrations daily, for one or
several days.
[0068] The contrast agent provided herein, while not limited to a
particular use, can be administered intravenously to a laboratory
animal. The contrast agent can administered intravenously to a
mouse, rat or rabbit. The contrast agent can also be administered
to a human patient.
[0069] If a small animal subject is used it can be positioned on a
heated platform with access to anesthetic equipment. Thus, the
methods can be used with platforms and apparatus used in imaging
small animals including "rail guide" type platforms with
maneuverable probe holder apparatuses. For example, the described
systems can be used with multi-rail imaging systems, and with small
animal mount assemblies as described in U.S. patent application
Ser. No. 10/683,168, now U.S. Pat. No. 7,133,713, entitled
"Integrated Multi-Rail Imaging System," U.S. patent application
Ser. No. 10/053,748, U.S. publication No. 20050215878, entitled
"Integrated Multi-Rail Imaging System," U.S. patent application
Ser. No. 10/683,870, now U.S. Pat. No. 6,851,392, issued Feb. 8,
2005, entitled "Small Animal Mount Assembly," and U.S. patent
application Ser. No. 11/053,653, entitled "Small Animal Mount
Assembly," which are incorporated herein by reference.
[0070] Small animals can be anesthetized during imaging and vital
physiological parameters such as heart rate and temperature can be
monitored. Thus, the system can include means for acquiring ECG and
temperature signals for processing and display. The system can also
display physiological waveforms such as an ECG, respiration blood
pressure waveform. If a small animal is used, contrast agent can be
optionally injected either through the tail vein, through the
jugular in a cannulation procedure or directly into the heart.
[0071] Also provided is the use of a system for producing an
ultrasound image using line-based image reconstruction with the
contrast agents and the methods provided herein. One example of
such a system may have the following components as described in
U.S. patent application Ser. No. 10/736,232, U.S. patent
application publication 20040236219, now U.S. Pat. No. 7,052,460,
which is set forth in part below and is incorporated herein by
reference. The system for producing an ultrasound image using line
based image reconstruction can provide an ultrasound image having
an effective frame rate in excess of 200 frames per second. The
system incorporates an ECG based technique that enables
significantly higher time resolution than what was previously
available, thus allowing the accurate depiction of a rapidly moving
structure, such as a heart, in a small animal, such as a mouse,
rat, rabbit, or other small animal, using ultrasound (and
ultrasound biomicroscopy).
[0072] The intravenous injection can be administered as a single
bolus dose, or by repeated injection or continuous infusion.
Effective dosages and schedules for administering a given contrast
agent can be determined empirically, and making such determinations
is within the ordinary skill in the art. The dosage range for the
administration of the contrast agents are those large enough to
produce the desired ultrasound imaging effect. Such an effect
typically includes an increased return from the contrast agent.
Such an increased return or intensity of signal from a contrast
agent can be indicated by increased brightness on an ultrasound
image, which can be represented by coloration of an ultrasound
image.
[0073] A microbubble contrast agent can be disrupted or destroyed
by a pulse of ultrasound. The pulse of ultrasound can be produced
by the same or a different transducer as the transducer producing
the imaging frequency ultrasound. Therefore, the methods
contemplate using a plurality of ultrasound probes and frequencies.
The microbubbles can be disrupted or popped by the ultrasound
energy at a frequency above or below 20 MHz. As used throughout,
"disrupted" or "destroyed" means that a microbubble is fragmented,
ruptured, or cracked such that gas escapes from the micro bubble.
The micro bubble contrast agent can also be disrupted or destroyed
via other means such as chemical interactions with other
administered drugs or by altering the oxygenation level of the
blood. In some cases this may result in gas diffusion from the
bubble. Contrast agent expulsion from the subject can also occur
and includes being filtered by the liver and or kidneys or via the
contrast agent breaking down or binding with other molecules and
organelles already present in the subject including cells, oxygen,
and carbon dioxide. Herein the destruction of micro bubbles and
other contrast agents will includes both physical destruction
methods, such as ultrasound pulses, and chemical destruction
methods and expulsion from the subject via other methods and
combinations thereof.
[0074] The ultrasound or other imaging modality used can be
transmitted immediately after administration of contrast agent or
at any time interval subsequent to contrast agent administration.
Imaging can also begin prior to administration, continue throughout
the administration process, and continue subsequent to the
completion of administration. The imaging can also take place at
any discrete time prior to, during or after administration of the
contrast agent.
[0075] Any portion of a subject can be imaged. For example, the
organ can be selected from the group consisting of a heart, a
brain, a kidney, and a muscle. One non-limiting example of an organ
that can be imaged is a heart. A non-limiting example of a muscle
type that can be imaged is a skeletal muscle. For example, muscles
of the limbs can be imaged. As would be clear to one skilled in the
art, however, other muscle types can also be imaged, including
smooth muscle, and cardiac muscle, such as when the heart is
imaged. Other organs that can be imaged include, but are not
limited to a lung, a brain, a liver and blood and blood vessels.
The organs imaged or portions thereof can be that of a mouse, rat,
or other small animal. The systems and methods can also be used to
image physiological or pathological processes such as angiogenesis
or inflammation.
[0076] Other subjects and portions of subjects can also be imaged
including individual cells or collections of cells either in vivo
or ex vivo as might be grown or collected into a Petri dish. In
this case the imaging modality can optionally be optical imaging
either with or without a magnifying lens and the contrast agent
might be a fluorescent dye which could be used to identify
particular parts of the cells, or individual cells expressing a
particular genetic marker.
[0077] Contrast agents can be targeted or non-targeted or lodging.
Several strategies can be used to direct ultrasound contrast agent
to a desired target including lodging of bubbles in tissues of the
subject based on size.
[0078] In regard to microbubble contrast agents, one exemplary
targeting strategy takes advantage of the inherent chemical
properties of the microbubble shell components. For example,
albumin or lipid microbubbles can attach to the surface of target
cells via cell receptors.
[0079] Contrast agents can also be targeted by conjugation of
specific ligands or antibodies that bind to desired markers. A
further strategy takes advantage of the physical size of the
contrast agent. For example, in regard to microbubbles, bubbles of
a certain size can lodge in the microvasculature of a subject,
wherein they can be imaged. Lodging can be further augmented by
alteration of the microbubble shell charge in order to further
enhance the percentage of microbubbles lodging within the
microcirculation.
[0080] A contrast agent can advantageously be employed as delivery
agents for bioactive moieties such as therapeutic drugs (i.e.,
agents having a beneficial effect on a specific disease in a living
human or non-human animal). Thus, for example, therapeutic
compounds can be located in a microbubble, may be linked to part of
an encapsulating wall or matrix, e.g., through covalent or ionic
bonds, if desired through a spacer arm, or may be physically mixed
into such encapsulating or matrix material. To deliver an agent a.
microbubble can be disrupted as described herein. For example, when
microbubbles are disrupted or destroyed, drugs or genes that are
housed within them or bound to their shells can be released to the
blood stream are then delivered to tissue by convective forces
through the permeabilized microvessels. Moreover, if the agent is
linked or otherwise attached to the microbubble, the agent can be
delivered without disrupting the microbubble. For example, a lodged
microbubble can deliver a therapeutic agent linked to its shell
without being disrupted.
[0081] A targeted contrast agents used in the methods described can
be targeted to a variety of cells, cell types, antigens, cellular
membrane proteins, organs, markers, tumor markers, angiogenesis
markers, blood vessels, thrombus, fibrin, and infective agents. For
example, targeted microbubbles can be produced that localize to
targets expressed in a subject. Desired targets are generally based
on, but not limited to, the molecular signature of various
pathologies, organs and/or cells. For example, adhesion molecules
such as integrin .alpha..sub.v.beta..sub.3, intercellular adhesion
molecule-1 (I-CAM-1), fibrinogen receptor GPIIb/IIIa and VEGF
receptors are expressed in regions of angiogenesis, inflammation or
thrombus. These molecular signatures can be used to localize high
frequency ultrasound contrast agents through the use of targeting
molecules, including but not limited to, complementary receptor
ligands, targeting ligands, proteins, and fragments thereof. Target
cell types include, but are not limited to, endothelial cells,
neoplastic cells and blood cells. The methods described herein
optionally use microbubbles targeted to VEGFR2, I-CAM-1,
.alpha..sub.v.beta..sub.3 integrin, .alpha..sub.v integrin,
fibrinogen receptor GPIIb/IIIa, P-selectin, mucosal vascular
adressin cell adhesion molecule-1. Moreover, using methods known in
the art, complementary receptor ligands, such as monoclonal
antibodies, can be readily produced to target other markers in a
subject. For example, antibodies can be produced to bind to tumor
marker proteins, organ or cell type specific markers, or infective
agent markers. Thus, the targeted contrast agents can be targeted,
using antibodies, proteins, fragments thereof, or other ligands, as
described herein, to sites of neoplasia, angiogenesis, thrombus,
inflammation, infection, as well as to diseased or normal organs or
tissues including but not limited to blood, heart, brain, blood
vessel, kidney, muscle, lung and liver. Optionally, the targeted
markers are proteins and may be extracellular or transmembrane
proteins. The targeted markers, including tumor markers, can be the
extracellular domain of a protein. The antibodies or fragments
thereof designed to target these marker proteins can bind to any
portion of the protein. Optionally, the antibodies can bind to the
extracellular portion of a protein, for example, a cellular
transmembrane protein. Antibodies, proteins, or fragments thereof
can be made that specifically or selectively target a desired
target molecule using methods known in the art.
[0082] Such selective or specific binding can be readily determined
using the methods and devices described herein. For example,
selective or specific binding can be determined in vivo or in vitro
by administering a targeted contrast agent and detecting an
increase ultrasound scattering from the contrast agent bound to a
desired target. Thus a targeted contrast agent can be compared to a
control contrast agent having all the components of the targeted
contrast agent except a targeting ligand. By detecting increased
resonance or scattering from the targeted contrast agent versus a
control contrast agent, the specificity or selectivity of binding
can be determined. If an antibody or similar targeting mechanism is
used, selective or specific binding to a target can be determined
based on standard antigen/epitope/antibody complementary binding
relationships. Further, other controls can be used. For example,
the specific or selective targeting of the microbubbles can be
determined by exposing targeted microbubbles to a control tissue,
which includes all the components of the test tissue except for the
desired target ligand or epitope. To compare a control sample to a
test sample, levels of non-linear resonance can be detected by
enhanced ultrasound imaging.
[0083] Illustrative targeting mechanisms that can be targeted to
particular targets and indicated areas of use for targetable
diagnostic and/or therapeutic agents include, but are not limited
to, antibodies to: CD34, ICAM-1, ICAM-2, ICAM-3, E-selectin,
P-selectin, PECAM, CD18 Integrins, VLA-1, VLA-2, VLA-3, VLA-4,
VLA-5, VLA-6, GIyCAM, MAdCAM-1, fibrin, and myosin. These and other
targeting molecule molecules are identified and discussed in U.S.
Pat. No. 6,264,917, which is incorporated by reference herein
generally and specifically for purposes of identifying useful
targeting molecule molecules.
[0084] Specific or selective targeted contrast agents can be
produced by methods known in the art, for example, using the
methods described.
[0085] For example, targeted microbubble contrast agents can be
prepared as perfluorocarbon or other gas-filled microbubbles with a
monoclonal antibody on the shell as a ligand for binding to target
ligand in a subject as described in Villanueva et al.,
"Microbubbles Targeted to Intracellular Adhesion Molecule-1 Bind to
Activated Coronary Artery Endothelial Cells," Circulation (1998)
98: 1-5. For example, perfluorobutane can be dispersed by
sonication in an aqueous medium containing phosphatidylcholine, a
surfactant, and a phospholipid derivative containing a carboxyl
group. The perfluorobutane is encapsulated during sonication by a
lipid shell. The carboxylic groups are exposed to an aqueous
environment and used for covalent attachment of antibodies to the
microbubbles by the following steps. First, unbound lipid dispersed
in the aqueous phase is separated from the gas-filled microbubbles
by floatation. Second, carboxylic groups on the microbubble shell
are activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodimide,
and antibody is then covalently attached via its primary amino
groups with the formation of amide bonds.
[0086] Targeted microbubbles can also be prepared with a
biotinylated shell as described in Weller et al., "Modulating
Targeted Adhesion of an Ultrasound Contrast Agent to Dysfunctional
Endothelium," Ann. Biomed, Engineering, (2002) 30: 1012-1019. For
example, lipid-based perfluorocarbon-filled microbubbles can be
prepared with monoclonal antibody on the shell using avidin-biotin
bridging chemistry using the following protocol. Perfluorobutane is
dispersed by sonication in aqueous saline containing phosphatidyl
choline, polyethylene glycol (PEG) stearate, and a biotinylated
derivative of phosphatidylethanolamine as described in the art. The
sonication results in the formation of perfluorobutane microbubbles
coated with a lipid monolayer shell and carrying the biotin label.
Antibody conjugation to the shell is achieved via avidin-biotin
bridging chemistry. Samples of biotinylated microbubbles are washed
in phosphate-buffered saline (PBS) by centrifugation to remove the
lipid not incorporated in the microbubble shell. Next, the
microbubbles are incubated in a solution (0.1-10 .mu.g/mL of
streptavidin of in PBS. Excess streptavidin is removed by washing
with PBS. The microbubbles are then incubated in a solution of
biotinylated monoclonal antibody in PBS and washed again. The
resultant microbubble have antibody conjugated to the lipid shell
via biotin-streptavidin-biotin linkage. In another example, for
targeted microbubbles, biotinylated microbubbles can be prepared by
sonication of an aqueous dispersion of decafluorobutane gas,
distearoylphodphatidylcholine, polyethyleneglycol-(PEG-) state, and
distearoyl-phosphatidylethanolamine-PEG-biotin. Microbubbles can
then be combined with streptavidin, washed, and combined with
biotinylated echistatin.
[0087] Targeted microbubbles can also be prepared with an
avidinated shell, as is known in the art. In a preferred
embodiment, a polymer microbubble can be prepared with an
avidinated or streptavidinated shell. For example, a polymer
contrast agent comprising a functionalized polyalkylcyanoacrylate
can be used as described in patent application PCT/EP01/02802.
Streptavidin can be bonded to the contrast agent via the functional
groups of the functionalized polyalkylcyanoacrylate. In a preferred
embodiment, avidinated microbubbles can be used in the methods
disclosed herein. When using avidinated microbubbles, a
biotinylated antibody or fragment thereof or another biotinylated
targeting molecule or fragments thereof can be administered to a
subject. For example, a biotinylated targeting ligand such as an
antibody, protein or other bioconjugate can be used. Thus, a
biotinylated antibody, targeting ligand or molecule, or fragment
thereof can bind to a desired target within a subject. Once bound
to the desired target, the contrast agent with an avidinated shell
can bind to the biotinylated antibody, targeting molecule, or
fragment thereof. When bound in this way, high frequency ultrasound
energy can be transmitted to the bound contrast agent, which can
produce non-linear scattering of the transmitted ultrasound energy.
An avidinated contrast agent can also be bound to a biotinylated
antibody, targeting ligand or molecule, or fragment thereof prior
to administration to the subject.
[0088] When using a targeted microbubble contrast agent with a
biotinylated shell or an avidinated shell a targeting ligand or
molecule can be administered to the subject. For example, a
biotinylated targeting ligand such as an antibody, protein or other
bioconjugate, can be administered to a subject and allowed to
accumulate at a target site. A fragment of the targeting ligand or
molecule can also be used.
[0089] When a targeted contrast agent with a biotinylated shell is
used, an avidin linker molecule, which attaches to the biotinylated
targeting ligand can be administered to the subject. Then, a
targeted contrast agent with a biotinylated shell is administered
to the subject. The targeted contrast agent binds to the avidin
linker molecule, which is bound to the biotinylated targeting
ligand, which is itself bound to the desired target. In this way a
three step method can be used to target contrast agents to a
desired target. The intermediate targeting ligand can bind to all
of the desired targets detailed above as would be clear to one
skilled in the art.
[0090] Targeted contrast agents or non-targeted contrast agents or
microbubbles can also comprise a variety of markers, detectable
moieties, or labels. Thus, a microbubble contrast agent equipped
with or without a targeting ligand or antibody incorporated into
the shell of the microbubble can also include another detectable
moiety or label. As used herein, the term "detectable moiety" is
intended to mean any suitable label, including, but not limited to,
enzymes, fluorophores, biotin, chromophores, radioisotopes, colored
particles, electrochemical, chemical-modifying or chemiluminescent
moieties. Common fluorescent moieties include: fluorescein, cyanine
dyes, coumarins, phycoerythrin, phycobiliproteins, dansyl chloride,
Texas Red, and lanthanide complexes. Of course, the derivatives of
these compounds which are known to those skilled in the art also
are included as common fluorescent moieties.
[0091] The detection of the detectable moiety can be direct
provided that the detectable moiety is itself detectable, such as,
for example, in the case of fluorophores. Alternatively, the
detection of the detectable moiety can be indirect. In the latter
case, a second moiety reactable with the detectable moiety, itself
being directly detectable can be employed. The detectable moiety
may be inherent to the molecular probe. For example, the constant
region of an antibody can serve as an indirect detectable moiety to
which a second antibody having a direct detectable moiety can
specifically bind.
[0092] Other image modalities, as set forth herein, can be used to
create reference and data sets. Before and after events or other
changes captured by imaging can distinguish between the reference
set and the data set. The infusion of contrast agents can be such a
"before and after event" whereby the reference set can be acquired
using ultrasound imaging prior to the infusion of the contrast
agent and the data set can be acquired after the infusion of the
contrast agent. Optionally, changes captured by imaging which can
determine the timing of a reference set acquisition or a data set
acquisition can be any change which can be captured by an imaging
modality, such as by non-limiting example a change in thermal
characteristics, a change in radar reflectivity, a change in blood
flow, a change in magnetic resonance, a change in fluid flow, a
change in fluorescence, a change in intensity of visible and
non-visible spectra, and a change in proton density, proton spin
state, tissue density, tissue elasticity, and attenuation of tissue
to X-Rays.
[0093] The identified set of reference images and the identified
set of data images can be compared. One or more reference images
can be compared to a plurality of images of the data image set.
[0094] The comparison step of the methods provided for herein can
comprise summing the absolute difference of pixel intensity levels
and paring the reference set image frame with the data set image
frame which yields the smallest absolute difference value. Other
methods to compare images can also be used.
[0095] The image processing step can comprise the application of a
median filter to the image. In one exemplary aspect, the median
filter can comprise a 3 by 3 median filter. The image processing
step can comprise the application of a blurring filter to the
image. In one aspect, the blur filter can comprise a 5 by 5 box
filter. In another aspect, the blur filter can comprise a Gaussian
filter. The image processing step can comprise the application of
image decimation to the image. The image decimation can comprise
the use of four adjacent pixels to create one representative pixel.
The image decimation can comprise the use of the maximum intensity
value of four adjacent pixels as the intensity value of one
representative pixel.
[0096] Further provided herein is a method for blending the
contrast overlay, calculated from a post-event image with respect
to a pre-event image, with its corresponding post-event source
image.
[0097] Also provided herein is a method of creating a persisted
overlay frame comprising the steps described above for creating a
contrast overlay wherein the persisted overlay frame is created
from a moving average of two or more adjacent overlay frames.
Persistence can be applied either as a post-processing step, after
all images have been acquired, or as an real time acquisition
step.
[0098] Still further provided herein is a method of creating
ultrasound images comprising the steps of acquiring a set of
ultrasound reference images of a subject; interposing into the
subject contrast agent; acquiring a set of ultrasound data images
of the subject; comparing a data set image to each of several
reference set images wherein each data set image is associated with
the reference set image or images which most closely resembles the
data set image; performing image processing on each image of the
associated collection; performing image comparison between
reference and data set images; performing image subtraction of the
two processed images or collection of images wherein a contrast
overlay image is created. In the case where one data set image is
to be associated with more than one reference set images, the
resulting contrast overlays can be combined into a single overlay
using averaging, as one non limiting example.
[0099] Also provided herein is a method to use an optionally
acquired ECG signal to improve the association between data set
images and reference set images. For imaging a subject or portions
thereof where the subject motion is predominantly cardiac the ECG
signal can be used to improve the matching of reference set to data
set images. For example, if each reference set image is tagged with
a temporal quantifier describing which portion of the heart cycle
the image was acquired in, and each data set image that is
similarly tagged, the temporal information can be used to restrict
the number of reference set images each data set image is compared
with. For example, a data set image would only be compared with
reference set images which were acquired during the same portion of
the heart cycle as the data set image. In this way, the comparison
images are known a priori to represent the anatomy in similar
positions and state. Reducing the set of comparison images to those
that are known to be most similar reduces false positives and also
increases processing speed and efficiency. Similar temporal tagging
can also be done using other physiological parameters, or
biodynamic cycles, such as the respiration signal, blood pressure,
temperature, or blood oxygen level.
[0100] One non limiting example of the method is shown in FIG. 1.
FIG. 1 is a flow diagram in block form showing an exemplary
embodiment of the invention. The method 100 is a process which can
be performed in a computer or other electronic processing device as
described more fully herein. Images can be acquired using methods
generally known to one of ordinary skill in the art. These images
are commonly stored in frames of a digital format which are
arranged in two dimensions with each individual two dimensional
point of the frame called a pixel. Multiple frames can be combined
in a sequential in time loop to form a movie or as is commonly
called in imaging systems, a cine clip.
[0101] These images can be acquired using a wide range of
modalities including but not limited to diagnostic ultrasound,
x-ray fluoroscopy, MRI, optical imaging, and any other modality
capable of acquiring a cine loop of images.
[0102] In exemplary method 100, a reference set 101 is identified
for use in the method. This reference set 101 comprises of
individual images 1 . . . n with an example of one image shown as
102. The reference set 101 can be a subset of a longer data set
103. The reference set 101 can be an image set of a "before" event;
that is, an image set taken before some change takes place to or
within the subject being imaged. The reference set can be a sub-set
of a larger set, for example, in the case where during a continuous
acquisition contrast agent is administered at some point during the
acquisition. In this case the reference set might consist of the
images at the beginning of the set before contrast is administered
up to the point where contrast is administered. Another example is
to select as the reference set images which occur immediately after
or before a destruction sequence. Another further example is to
select as the reference set at the end of a long set of separate
acquisitions when the level of contrast agent has reached a stable
state in the subject or region of imaging. The reference set
selected can be applied to data images that occur after the images
of the reference set were acquired, or before.
[0103] A corresponding data set 103 is identified for use in the
method. The data set 103 can be acquired using the same imaging
modality and methods that were used for acquisition of the
reference set 101. Data set 103 can comprise "after" images; that
is, images of the same subject of the reference set acquired after
some event, such as the interposition of a contrast agent in the
subject or the destruction event designed to remove the contrast
agent from the subject. The event can include creating an area of
increased contrast in the subject. An area of increased contrast
can be created by increasing the intensity of the image in an area
of the subject or by decreasing the intensity of the image in an
area of the subject. The after event can also be the injection of
contrast agents in other modalities. For example, in MRI imaging,
after the injection of gadolinium, manganese, iron, gadopentetate
dimeglumine, gadoteridol, gadoterate meglumine, mangafodipir
trisodium, gadodiamide, perfluorocarbons or other MRI specific
contrast agent which would be known to one skilled in the art. For
optical imaging an exemplary contrast agent can be a fluorescent
dye. The event could also be, in MRI imaging, after a temperature
change, change to the applied pulse sequence, gradient coil fields,
or pulse frequency. The event is not limited to conditions which
cause an increase in image intensity but as well, a decrease in
image intensity. For CT and X-Ray based imaging modalities, the
event can occur after the injection of barium sulfate, gadolinium,
iodine, or other contrast agent as would be known to one skilled in
the art.
[0104] The data set 103 comprises individual image frames 1 . . . m
with an example of one image shown as 104. While the number of
reference set images can equal the number of data set frames (n=m),
they do not have to be equal. The number of reference set images
can also be larger or less than the number of data set images.
[0105] The reference set 101 and the data set 103 can comprise
three-dimensional (3-D) images. The methods described herein are
also applicable to imaging in 3D. For example, in an ultrasound
modality, using a separate 3D stepper motor (like the VisualSonics
Inc 3D Acquisition Motor), or other moving transducer, the imaging
transducer can be stepped across the length of an organ of the
subject (call this the Y axis). At each point along the Y axis a
number of images can be acquired as a reference set. Upon
completion of acquisition, there are a number of reference sets
acquired at different positions along the length of the organ. The
data set images can be acquired in the same way. For example, in
the case of the use of contrast agents, after injection of the
contrast agent, data sets at each Y axis position can be acquired.
Creation of the contrast overlay can be done as described herein
and can be done independently for each acquired position. With the
use of 3-D images, the Y axis position can be out of phase with the
others in terms of respiration and cardiac cycles. In that event,
respiration gating can be used to start the acquisition at the
beginning of a respiration cycle. In another aspect, 3-D images
acquired using two dimensional phased array transducers can also
comprise the reference set and data set.
[0106] Once the reference set 101 and data set 103 are identified,
a comparison step takes place at block 105. This comparison process
105 creates a difference error measurement for each image in the
reference set 101 as compared to each image in the data set 103.
The difference error is a measure of the similarity of one image to
another. The difference error can be computed using a sum of the
absolute difference of intensity levels for corresponding pixels on
the reference set image 102 and data set image 104 being compared.
Every image of the data set can be compared with every image of the
reference set. Using ECG and respiratory signals, the number of
comparisons can be reduced to include only those images which occur
during the same phase of the ECG and respiration cycles. Other
difference error calculations can be simple sum of differences, or
correlation techniques (sum of pixel to pixel multiplications).
Image comparisons can include image shifts (in any direction) or
minor non-linear transforms which morph features while keeping the
overall image structures consistent.
[0107] In block 106 the difference error is used to associate
images in the reference set 101 with images in the data set 103.
Optionally, a data set image is paired with a reference set image
wherein the two images have the smallest difference error as
compared to other possible data set image/reference set image
combinations. A data set image can also be paired with a reference
set image based on similarity of the two images. For example,
substantially similar reference set and data set images can be
paired. Thus, an individual data set image 102 is associated with
an individual reference set image 104 which optionally gives the
smallest difference error (Equation 1).
Sum of absolute difference squared Net Error = i AllPixels (
ReferenceImage [ i ] - DataImage [ i ] ) 2 Equation 1
##EQU00001##
[0108] Alternatively, the sum of absolute differences can be used
to determine image similarity (Equation 2).
Sum of absolute differences Net Error = i AllPixels Abs (
ReferenceImage [ i ] - DataImage [ i ] ) Equation 2
##EQU00002##
[0109] Alternatively, other comparison techniques can be used such
as frequency domain methods, convolution methods such as the cross
correlation, or pattern matching methods.
[0110] Association of multiple reference set images to a data set
image can also be done. Multiple image associations can be done
with a frame rate of, for example 15 fps, however any frame rate
can be used, or with a frame rate whereby there exists enough image
redundancy to perform analysis across multiple images. For example,
for each data set image there can be more than one reference set
image which is a similar match. In this case the two reference
images can be combined to reduce noise and spurious uncorrelated
changes. The combination can be a simple average, or a maximum
filter (take the maximum pixel value from each of the images to
create a third) or median filter. The same process can be applied
to the data set images. Typically for each data set image the next
acquired image (which can be on the order of 10-60 ms delayed from
the previous) is similar. These images can also be averaged or a
max filter applied to generate a third target image.
[0111] The associated images next undergo individual image
processing. The associated reference image 107 is image processed
108 and the associated data image 109 is image processed 110. While
the images are called "associated" the image processing at this
point in the method is done on each individual image. Image
processing 108 and 110 can be the same processing techniques. Image
processing 108 and 109 can include decimation which is the process
of reducing the frame size. Decimation can provide for increased
processing efficiency, more efficient memory usage, and removal of
local image intensity differences on the pixel resolution scale.
Decimation techniques can include taking the average of an
intensity for a pixel neighborhood, or the maximum pixel intensity
for a pixel neighborhood, as the representative pixel intensity for
the decimated frame.
[0112] Exemplary image processing 108 and 110 techniques include
the application of one or more of noise reduction filters, contrast
enhancement filters, blurring filters, low pass filters, high pass
filters, and non-linear filters such as median filters or maximum
filters. Noise reduction filters can be of a type such as median
filters, averaging or mean filters, mode filters, low pass filters,
or Monte Carlo filters. Blurring filters can be of a type such as
box filters, or Gaussian filters. Contrast enhancement filters can
be of a type such as histogram equalization filters. Some imaging
modalities suffer from image and hardware noise. Ultrasound images
can be inherently noisy due to speckle which is manifested from the
tissue structure itself and not by any flaws in hardware or
processing. Filters such as those described above can remove this
type of noise and other high frequency noise information. Removal
of such noise can improve subsequent image subtraction techniques
and can be used to emphasize changes due to contrast enhancement
only.
[0113] Additional image processing techniques can include linear
image transformations to bring dominant image structures closer
inline. Exemplary examples of linear transformations include
resizing, rotations, and shifts. Non-linear image transformations
can also be used and include image morphing.
[0114] After each associated image 107 and 109 are processed, an
image subtraction is performed 111 whereby the processed reference
image is subtracted from the processed data image. The image
subtraction 111 can be a subtraction of pixel intensities. For the
case of ultrasound imaging, the pixel intensities can represent log
compressed envelope data. Non-logged, or linear envelope data can
also be processed as well as raw RF data. The subtracted image can
optionally have its pixel intensity values of less than zero
replaced by an intensity value of zero at block 112. Optionally,
pixel intensity values that are greater than zero can be replaced
by a value of zero. The choice can depend on whether it is expected
that the signal intensity should increase or decrease and the
result is improved reduction of false positives. For example, when
infusing micro bubble contrast agent the expected result is that
blood vessels and tissue increase in brightness. However, at the
same time tissue located beneath the blood vessels might undergo
shadowing due to attenuation of the ultrasound beam through the now
contrast enhanced vessels. This reduction of tissue intensity can
be suppressed by zeroing out intensity values of less than zero
from the subtracted image. The resulting image produced from the
image subtraction 111 is called a contrast overlay or enhanced
medical image 113.
[0115] The contrast overlay or enhanced medical image 113 can be
displayed on top of the source (data) set image from which it was
derived as a semi transparent, or opaque overlay. In one aspect,
the overlay can be a color overlay that can be overlaid on top of a
gray scale image. In another aspect, the B-Mode image can be
depicted in color and the contrast overlay can be depicted in a
distinguishable color. The display can be done in a blending
fashion so that the contrast overlay highlights via intensity
(brightness) the regions of change.
[0116] In a further aspect, the contrast overlay 113 can be
processed by applying a predetermined threshold. Here, the
intensity of each pixel of the contrast overlay can be compared to
the predetermined threshold, and only those pixels within the
contrast overlay that exceed the threshold can be displayed. It is
contemplated that the threshold value can be a fixed predetermined
value, or it can be under user control.
[0117] Image persistence can also be shown via the steps
illustrated in FIG. 2. Two or more contrast overlays can be
combined by taking a moving average 207 to create a persisted
contrast overlay 208. For example, four contrast overlays, 201,
202, 203, and 204 can be combined as shown in FIG. 2, removing
spurious image differences and leaving regions which are similar
among the four.
[0118] Image persistence can also be performed using a peak hold
approach where for each pixel a frame to frame maximum is
determined over a moving window of several frames.
[0119] Alternatively, frame to frame persistence can be performed
using a method comprising the following steps: in the subtracted
image (the contrast overlay), evaluate the sign of the difference
in pixel level frame to frame; if the sign is positive, meaning the
brightness level of that particular pixel has increased compared to
the previous frame, set the persistence to zero, i.e., the pixel
level in the display frame is equal to the pixel level of the most
recent frame; if the sign is negative, meaning that the brightness
level of the pixel has decreased, set the persistence to non-zero,
so that the pixel level in the display frame decreases
exponentially.
[0120] Image persistence can also be performed using a maximum
intensity projection. In this method, if the intensity of a pixel
of a previous frame is larger than the intensity of a pixel in the
current frame, the current frames pixel is replaced with the
previous pixel value. This has the effect of causing moving
reflectors, micro bubbles in blood for example, to cause its path
to be traced. An analogous process is keeping the shutter open on a
camera while moving a flashlight beam across its field of view. The
path of the light beam will be exposed onto the film. The advantage
of this type of persistence is to form the ability to map out
vessel paths as the micro bubbles progress though the vascular
system.
[0121] The methods described herein can be implemented in a
computer architecture of various types generally known in the art.
The several images and information calculated there from can be
stored in various forms of data storage generally known in the art
including magnetic media or electronic memory.
[0122] Aspects of the exemplary systems shown in the Figures and
described herein, can be implemented in various forms including
hardware, software, and a combination thereof. The hardware
implementation can include any or a combination of the following
technologies, which are all well known in the art: discrete
electronic components, a discrete logic circuit(s) having logic
gates for implementing logic functions upon data signals, an
application specific integrated circuit having appropriate logic
gates, a programmable gate array(s) (PGA), a field programmable
gate array (FPGA), etc. The software comprises an ordered listing
of executable instructions for implementing logical functions, and
can be embodied in any computer-readable medium for use by or in
connection with an instruction execution system, apparatus, or
device, such as a computer-based system, processor-containing
system, or other system that can fetch the instructions from the
instruction execution system, apparatus, or device and execute the
instructions.
[0123] Aspects of the exemplary systems can be implemented in
computerized systems. Aspects of the exemplary systems can be
operational with numerous other general purpose or special purpose
computing system environments or configurations. Examples of well
known computing systems, environments, and/or configurations that
may be suitable for use with the system and method include, but are
not limited to, personal computers, server computers, laptop
devices, and multiprocessor systems. Additional examples include
set top boxes, programmable consumer electronics, network PCs,
minicomputers, mainframe computers, distributed computing
environments that include any of the above systems or devices, and
the like.
[0124] Aspects of the exemplary systems can be described in the
general context of computer instructions, such as program modules,
being executed by a computer. Generally, program modules include
routines, programs, objects, components, data structures, etc. that
perform particular tasks or implement particular abstract data
types. The system and method may also be practiced in distributed
computing environments where tasks are performed by remote
processing devices that are linked through a communications
network. In a distributed computing environment, program modules
may be located in both local and remote computer storage media
including memory storage devices.
[0125] Aspects of the exemplary systems disclosed herein can be
implemented via a general-purpose computing device in the form of a
computer 301 shown in FIG. 3. The components of the computer 301
can include, but are not limited to, one or more processors or
processing units 303, a system memory 312, and a system bus 313
that couples various system components including the processor 303
to the system memory 312.
[0126] The system bus 313 represents one or more of several
possible types of bus structures, including a memory bus or memory
controller, a peripheral bus, an accelerated graphics port, and a
processor or local bus using any of a variety of bus architectures.
By way of example, such architectures can include an Industry
Standard Architecture (USA) bus, a Micro Channel Architecture (MCA)
bus, an Enhanced ISA (EISA) bus, a Video Electronics Standards
Association (VESA) local bus, and a Peripheral Component
Interconnects (PCI) bus also known as a Mezzanine bus. This bus,
and all buses specified in this description can also be implemented
over a wired or wireless network connection. The bus 313, and all
buses specified in this description can also be implemented over a
wired or wireless network connection and each of the subsystems,
including the processor 303, a mass storage device 304, an
operating system 305, application software 306, data 307, a network
adapter 308, system memory 312, an Input/Output Interface 310, a
display adapter 309, a display device 311, and a human machine
interface 302, can be contained within one or more remote computing
devices 314 a,b,c at physically separate locations, connected
through buses of this form, in effect implementing a fully
distributed system.
[0127] The computer 301 typically includes a variety of computer
readable media. Such media can be any available media that is
accessible by the computer 301 and includes both volatile and
non-volatile media, removable and non-removable media. The system
memory 312 includes computer readable media in the form of volatile
memory, such as random access memory (RAM), and/or non-volatile
memory, such as read only memory (ROM). The system memory 312
typically contains data such as data 307 and/or program modules
such as operating system 305 and application software 306 that are
immediately accessible to and/or are presently operated on by the
processing unit 303.
[0128] The computer 301 may also include other
removable/non-removable, volatile/non-volatile computer storage
media. By way of example, FIG. 5 illustrates a mass storage device
304 which can provide non-volatile storage of computer code,
computer readable instructions, data structures, program modules,
and other data for the computer 301. For example, a mass storage
device 304 can be a hard disk, a removable magnetic disk, a
removable optical disk, magnetic cassettes or other magnetic
storage devices, flash memory cards, CD-ROM, digital versatile
disks (DVD) or other optical storage, random access memories (RAM),
read only memories (ROM), electrically erasable programmable
read-only memory (EEPROM), and the like.
[0129] Any number of program modules can be stored on the mass
storage device 304, including by way of example, an operating
system 305 and application software 306. Each of the operating
system 305 and application software 306 (or some combination
thereof) may include elements of the programming and the
application software 306. Data 307 can also be stored on the mass
storage device 304. Data 304 can be stored in any of one or more
databases known in the art. Examples of such databases include,
DB2.RTM., Microsoft.RTM. Access, Microsoft.RTM. SQL Server,
Oracle.RTM., mySQL, PostgreSQL, and the like. The databases can be
centralized or distributed across multiple systems.
[0130] A user can enter commands and information into the computer
301 via an input device (not shown). Examples of such input devices
include, but are not limited to, a keyboard, pointing device (e.g.,
a "mouse"), a microphone, a joystick, a serial port, a scanner, and
the like. These and other input devices can be connected to the
processing unit 303 via a human machine interface 302 that is
coupled to the system bus 313, but may be connected by other
interface and bus structures, such as a parallel port, game port,
or a universal serial bus (USB).
[0131] A display device 311 can also be connected to the system bus
313 via an interface, such as a display adapter 309. For example, a
display device can be a monitor or an LCD (Liquid Crystal Display).
In addition to the display device 311, other output peripheral
devices can include components such as speakers (not shown) and a
printer (not shown) which can be connected to the computer 301 via
Input/Output Interface 310.
[0132] The computer 301 can operate in a networked environment
using logical connections to one or more remote computing devices
314a,b,c. By way of example, a remote computing device can be a
personal computer, portable computer, a server, a router, a network
computer, a peer device or other common network node, and so on.
Logical connections between the computer 301 and a remote computing
device 314a,b,c can be made via a local area network (LAN) and a
general wide area network (WAN). Such network connections can be
through a network adapter 308. A network adapter 308 can be
implemented in both wired and wireless environments. Such
networking environments are commonplace in offices, enterprise-wide
computer networks, intranets, and the Internet 315. The remote
computer 314a,b,c may be a server, a router, a peer device or other
common network node, and typically includes all or many of the
elements already described for the computer 301. In a networked
environment, program modules and data may be stored on the remote
computer 314a,b,c. The logical connections include a local area
network ("LAN") and a wide area network ("WAN"). Other connection
methods may be used, and networks may include such things as the
"world wide web" or internet.
[0133] For purposes of illustration, application programs and other
executable program components such as the operating system 305 are
illustrated herein as discrete blocks, although it is recognized
that such programs and components reside at various times in
different storage components of the computing device 301, and are
executed by the data processor(s) of the computer. An
implementation of application software 306 may be stored on or
transmitted across some form of computer readable media. Computer
readable media can be any available media that can be accessed by a
computer. By way of example, and not limitation, computer readable
media may comprise "computer storage media" and "communications
media." "Computer storage media" include volatile and non-volatile,
removable and non-removable media implemented in any method or
technology for storage of information such as computer readable
instructions, data structures, program modules, or other data.
Computer storage media includes, but is not limited to, RAM, ROM,
EEPROM, flash memory or other memory technology, CD-ROM, digital
versatile disks (DVD) or other optical storage, magnetic cassettes,
magnetic tape, magnetic disk storage or other magnetic storage
devices, or any other medium which can be used to store the desired
information and which can be accessed by a computer. An
implementation of the disclosed method may be stored on or
transmitted across some form of computer readable media.
[0134] The processing of the disclosed method can be performed by
software components. The disclosed method may be described in the
general context of computer-executable instructions, such as
program modules, being executed by one or more computers or other
devices. Generally, program modules include computer code,
routines, programs, objects, components, data structures, etc. that
perform particular tasks or implement particular abstract data
types. The disclosed method may also be practiced in grid-based and
distributed computing environments where tasks are performed by
remote processing devices that are linked through a communications
network. In a distributed computing environment, program modules
may be located in both local and remote computer storage media
including memory storage devices.
[0135] FIG. 4 is a block diagram illustrating an exemplary
ultrasound imaging system 400 for creating an image overlay. The
ultrasound imaging system is exemplary only. As described
throughout, other imaging modalities can also be used. The imaging
system 400 operates on a subject 402. An ultrasound probe 412 is
placed in proximity to the subject 402 to obtain ultrasound image
information. The ultrasound probe 412 can comprise a mechanically
moved transducer, or an array that can be used for collection of
ultrasound data 410. For example, the transducer can both transmit
ultrasound waves to the subject 402 and receive ultrasound waves or
backscatter from the subject 402 and can receive a return from
contrast agent located in the subject. An ultrasound system 431 can
cause the transducer 412 to emit ultrasound by sending a
transmitter control signal, USTX signal.
[0136] The transducer within the probe 412 can be an array, single
element transducer or some other suitable transducer. The
transducer can transmit ultrasound at a low frequency, such as
frequencies less than or equal to 20 megahertz (MHz). For example,
the transducer can transmit ultrasound at or below about 20 MHz, 15
MHz, 10 MHz, 5 MHz, or some other suitable frequency. Further,
transducer operating frequencies significantly lower than those
mentioned are also contemplated. The transducer can also transmit
ultrasound at a high frequency, such as frequencies greater than or
equal to 20 megahertz (MHz). For example, the transducer can
transmit ultrasound at or above about 20 MHz, 25 MHz, 30 MHz, 35
MHz, 40 MHz, 45 MHz, 50 MHz or some other suitable frequency.
Further, transducer operating frequencies significantly higher than
those mentioned are also contemplated.
[0137] The ultrasound system 431 includes a control subsystem 427,
an image construction subsystem 429, a transmit subsystem 418, a
receive subsystem 420, and a user input device in the form of a
human machine interface 436. A processor 434 is coupled to the
control subsystem 427 and the display 416 is coupled to the
processor 434.
[0138] A memory 421 is coupled to the processor 434. The memory 421
can be any type of computer memory, and is typically referred to as
random access memory "RAM," in which the software 423 of the
invention executes. Software 423 controls the acquisition,
processing and display of the ultrasound data allowing the
ultrasound system 431 to display an image. The software also allows
for the processing and comparison of images, as described in the
disclosed methods.
[0139] The method and system for creating an image overlay can be
implemented using a combination of hardware and software. The
hardware implementation of the system can include any or a
combination of the following technologies, which are all well known
in the art: discrete electronic components, a discrete logic
circuit(s) having logic gates for implementing logic functions upon
data signals, an application specific integrated circuit having
appropriate logic gates, a programmable gate array(s) (PGA), a
field programmable gate array (FPGA), etc.
[0140] The software for the system 400 comprises an ordered listing
of executable instructions for implementing logical functions, and
can be embodied in any computer-readable medium for use by or in
connection with an instruction execution system, apparatus, or
device, such as a computer-based system, processor-containing
system, or other system that can fetch the instructions from the
instruction execution system, apparatus, or device and execute the
instructions.
[0141] In the context of this document, a "computer-readable
medium" can be any means that can contain, store, communicate,
propagate, or transport the program for use by or in connection
with the instruction execution system, apparatus, or device. The
computer readable medium can be, for example but not limited to, an
electronic, magnetic, optical, electromagnetic, infrared, or
semiconductor system, apparatus, device, or propagation medium.
More specific examples (a non-exhaustive list) of the
computer-readable medium would include the following: an electrical
connection (electronic) having one or more wires, a portable
computer diskette (magnetic), a random access memory (RAM), a
read-only memory (ROM), an erasable programmable read-only memory
(EPROM or Flash memory) (magnetic), an optical fiber (optical), and
a portable compact disc read-only memory (CDROM) (optical). Note
that the computer-readable medium could even be paper or another
suitable medium upon which the program is printed, as the program
can be electronically captured, via for instance optical scanning
of the paper or other medium, then compiled, interpreted or
otherwise processed in a suitable manner if necessary, and then
stored in a computer memory.
[0142] The ultrasound system 431 includes software 423 stored in
the memory 421. This software can include system software, as well
as, software to process and compare ultrasound backscatter and to
formulate images, as described herein, to perform the described
methods. The software 423 can also include image or overly
comparison software and frame selection software.
[0143] Memory 421 also includes the ultrasound data 410 obtained by
the ultrasound system 431. A computer readable storage medium 438
is coupled to the processor 434 for providing instructions to the
processor 434 to instruct and/or configure the processor 434 to
perform algorithms related to the operation of ultrasound system
431. The computer readable medium can include hardware and/or
software such as, by the way of example only, magnetic disk,
magnetic tape, optically readable medium such as CD ROMs, and
semiconductor memory such as PCMCIA cards. In each case, the medium
may take the form of a portable item such as a small disk, floppy
disk, cassette, or may take the form of a relatively large or
immobile item such as a hard disk drive, solid state memory card,
or RAM provided in the support system. It should be noted that the
above listed example mediums can be used either alone or in
combination.
[0144] The ultrasound system 431 includes a control subsystem 427
to direct operation of various components of the ultrasound system
431. The control subsystem 427 and related components may be
provided as software for instructing a general purpose processor or
as specialized electronics in a hardware implementation. The
ultrasound system 431 includes an image construction subsystem 429
for converting the electrical signals generated by the received
ultrasound echoes (or backscatter) to data that can be manipulated
by the processor 434 and that can be rendered into an image or
graphical depiction on the display 416. The control subsystem 427
is connected to a transmit subsystem 428 to provide ultrasound
transmit signal, USTX signal, to the ultrasound probe 412. The
ultrasound probe 412 in turn provides an ultrasound receive signal
to a receive subsystem 420. The receive subsystem 420 also provides
signals representative of the received signals to the image
construction subsystem 429. The received subsystem 420 is also
connected to the control subsystem 427. The image construction
subsystem 429 is directed by the control subsystem 427 to operate
on the received data to render an image for display using the image
data 410.
[0145] The receive subsystem 420 is connected to the control
subsystem 427 and an image construction subsystem 429. The image
construction subsystem 429 is directed by the control subsystem
427. The ultrasound system 431 transmits and receives ultrasound
data with the ultrasound probe 412, provides an interface to a user
to control the operational parameters of the imaging system 400,
and processes data appropriate to formulate still and moving images
that represent anatomy and/or physiology. Images are presented to
the user through the display 416.
[0146] The human machine interface 436 of the ultrasound system 431
takes input from the user and translates such input to control the
operation of the ultrasound probe 412. The human machine interface
436 also presents processed images and data to the user through a
display. Software 423 in cooperation with the image construction
subsystem 429 operate on the electrical signals developed by the
receive subsystem 420 to develop an ultrasound image and/or
representations and/or comparisons of ultrasound backscatter data
received from areas of interest of the subject.
EXPERIMENTAL
[0147] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, articles, devices
and/or methods claimed herein are made and evaluated, and are
intended to be purely exemplary of the invention and are not
intended to limit the scope of what the inventors regard as their
invention. Efforts have been made to ensure accuracy with respect
to numbers (e.g., amounts, temperature, etc.), but some errors and
deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, temperature is in .degree. C. or is at
ambient temperature, and pressure is at or near atmospheric.
Example 1
[0148] Embodiments of the invention comprise a method for the
imaging of contrast agents for high-resolution, high-frequency
ultrasound imaging of animal subjects including rodent models.
[0149] As described above, contrast agents can flow in the blood
stream of the animal and can be deposited at any site that the
blood flows. Contrast agents can also be prepared in such a way
that they can attach themselves to specific markers in the animal.
This "targeted" contrast agent technique uses ligands and
antibodies to latch onto specific targets. Contrast agents can also
be used for image enhancement to allow a user to easily distinguish
small vessels in the organ. In one aspect, perfusion imaging is a
way of injecting contrast agent into the animal, allowing the agent
to circulate and then the agent can be destroyed using a
destruction event. The time taken for the re-perfusion is
meaningful to the researcher in quantifying perfusion. This image
processing method is not specific to the imaging of targeted or
untargeted contrast agents.
[0150] Embodiments exploit the brightness change or increase in
intensity of an image, to determine how much, and where the
contrast agent flows and where it is deposited. The methods
described herein can be used with or without ECG or respiration
gating.
[0151] Contrast agents can be gas filed bubbles with diameters
ranging from about 1 to about 4 microns. The bubble size
distribution can range from sub-micron size to up to about 10
micron in size. Exemplary contrast agents can be acquired from
ImaRx, Targeson, Bracco, or VisualSonics Inc. The ImaRx bubble has
an average size of 0.9 microns, the Targeson bubble has a range of
2 to 4 microns in size.
[0152] The animal is typically anesthetized and contrast agent can
be injected through the tail vein, through the jugular in a
cannulation procedure or directly into the heart. The typical image
procedure is as follows:
Ultrasound Setup
[0153] The ultrasound machine is set up according to the following
parameters: position the ultrasound scanhead to image the organ of
interest. (In this example, the hind limb and the kidney); set the
frame rate to approximately 15 frames per second or optionally,
increased frame rates may be used if the total power deposited does
not affect the contrast agent itself; and set the power to 50% or
such level as not to significantly disturb the contrast agent.
[0154] Sequence of Events
TABLE-US-00001 Step Action Observation 1 Start capturing a cine
clip. The organ of interest is displayed live on the ultrasound
monitor. 2 Inject a bolus of contrast The blood vessels turn bright
due to the agent after about 10 seconds enhancement. The ultrasound
image intensity of imaging. increases wherever contrast agent is
present. 3 Continue image capture for The penetration of the agent
is observed into the approximately 1 minute and organ of interest.
then stop imaging. 4 Save the cine clip. The ultrasound machines
saves approximately 800 frames of data. 5 Wait for approximately 4
The image is frozen on the screen as the agent minutes. dissipated
through the body and is trapped in the lungs or metabolized in the
body. 6 Start image capture again The agent is not as concentrated
as seen in step 2. Agent is seen in the organ either by lodging in
the small capillaries or by binding to the appropriate target for
the targeted case. 7 Initiate a destruction The destruction
sequence distorts the entire sequence approximately 20 image.
seconds after starting 8 Continue imaging The image has returned to
a state as seen before the bolus injection. 9 Stop imaging and save
the cine clip. 10 Retrieve the first cine clip saved in step 4 11
Mark the first 40 to 60 frames No contrast agent is observed in the
blood as the reference cine clip vessels. 12 Process the entire
cine clip by using the image processing algorithm described below.
13 Play back the processed cine All contrast agent is displayed as
an overlay on clip. the regular B-Mode image. The overlay can be
colorized. 14 Retrieve and process the second Before the
destruction phase a much lower cine clip saved in step 9 amount of
contrast is visible. After the destruction sequence only very small
traces of agent are visible.
[0155] The method of the exemplary invention uses two sets of
images: a reference loop of image frames 101, and a post-injection
data loop of image frames 103. The data loop 103 typically refers
to a series of sequential frames organized as a cine clip. The
position of the animal can be as static as possible to minimize
false readings. The processing method also has inherent stability
for small changes in image positioning due to things such as
respiration and cardiac motion. The reference sets and data sets
are then compared with each other to find the associated frames
which are more similar 105 and 106. Each image of the associated
frames, 107 and 108, is then processed with filters to remove
certain local image features 108 and 109. They are then subtracted
111 and a difference map generated which forms the contrast overlay
113. The contrast overlay can be a colorized version of the result
that is then overlaid onto the original B-Mode image. The flow
diagram for this process is shown in FIG. 100 and described
generally herein.
Reference Loop
[0156] The reference loop 101 is a set of n images acquired when
contrast agent has not permeated the tissue of interest: either
before contrast agent has been injected, after the bubbles have
passed through the animal, or after a destruction sequence (bubbles
are destroyed in real-time by applying a high power pulse through
the transducer. The reference loop 101 can be long enough to
encompass 2-3 respiration cycles, and 2-3 cardiac cycles. At a
frame rate of 15 frames per second, 30-60 frames are
sufficient.
[0157] The destruction sequence can comprise a sequence of
ultrasound pulses which cause the contrast agent to be destroyed.
For micro bubble imaging this involves transmitting ultrasound
pulses, either from the imaging transducer itself, or an external
second transducer aligned along the plane of imaging or designed to
insonate the entire animal. The transmit pulses can cause a high
mechanical pressure (sometimes referred to as mechanical index--MI)
to the micro bubbles causing them to burst. This can be
accomplished by maximizing the transmit power and number of pulses
transmitted. The bandwidth of the pulses can be kept high as to
incite any frequency dependent resonances across the largest range
of bubble sizes.
[0158] The reference loop 101 can be a sub-loop of a larger set,
captured at the beginning, middle, or end of the loop. The
reference loop can comprise a set of images of any size as required
by the operator. Its size can be prescribed by the largest amount
of images which can be seen to not contain contrast enhancement.
Determining which frames based on the presence of contrast is known
to a person of ordinary skill in the art. The reference can be a
retrospective look at the mouse, after the contrast agent has
flushed from the mouse or after a destruction sequence.
[0159] The reference loop 101 is a snap shot of the state of the
mouse over a small period of time. During this time the animal goes
through a number of motion cycles and general image adjustments.
These image differences are all captured in the reference loop.
Data Loop
[0160] The data loop 103 comprises images outside of the reference
loop 101. It can occur before the reference loop 101, after, or
include images from the reference loop 101. Typically the reference
loop 101 is acquired during a period where there is no contrast
agent in the animal. The data loop 103, however, can be acquired as
contrast agent is flowing into the tissues. This causes the tissues
and vessels to increase in reflectivity and consequently brightness
on the image. The data loop 103 can be longer than the reference
loop 101 and can account for several minutes' worth of data.
Multiple data loops 103 can also exist at different time points
after contrast injection or after a destruction sequence.
Reference Loop--Data Loop Association
[0161] After identification of the reference loop 101 and the data
loop 103 a next step is to associate images from the data loop to
their partner image in the reference loop. It is expected that
images in the data loop 103 undergo small motion changes. In order
to do a final comparison between post and pre contrast agent
injections, similar features are compared. Each data loop frame 104
is compared with each reference loop frame 102, using an
absolute-sum-of-differences technique 105. Alternatively, other
comparison techniques previously described herein can be used. The
two images demonstrating the smallest total difference (Equation 3)
can be associated. These are the images that are most similar of
the two sets.
Sum of absolute difference Net Error = i AllPixels ( ReferenceImage
[ i ] - DataImage [ i ] ) 2 Equation 3 ##EQU00003##
Image Subtraction
[0162] Once the associated frames have been determined, the images
can be processed 108 and 110 to generate difference maps. A number
of image processing algorithms are applied in steps 108 and 109 as
shown in FIG. 5.
[0163] Images 107 or 109 are first decimated 502 by a factor of 2
in each direction (horizontal and vertical) to increase processing
efficiency, memory usage, and to remove local image intensity
differences on the pixel-resolution scale. Changes at this scale
are evident regardless of the modality or accuracy of the
experiment. Image decimation selects the maximum intensity of 4
adjacent pixels as the representative pixel intensity.
[0164] A median filter 503 of size 3.times.3 is then used to
further reduce image noise and small resolution changes. An
addition box blur 504 filter of size 5.times.5 smoothes out image
features. A Gaussian filter may optionally replace the box filter
used here as well. The processed images are then subtracted in
block 111. Subtracting these processed images gives a difference
map showing the intensity changes.
[0165] The reference images 101 represent the non-contrast enhanced
images and thus are less bright than the data loop images 103. This
information allows for the exclusion of regions of the difference
map where the data image is less bright than the reference image
112. The final result is an overlay 113 showing the regions of the
image that show increased brightness over the reference loops.
[0166] Image persistence can be done as described herein. Also,
quantification of data can be done by selecting a
region-of-interest from the final processed data set. This can be
over the target organ or vessel. From each image the mean intensity
of the image difference overlay is determined. This information is
plotted as a function of time to give an intensity profile. The
shape of this profile gives quantitative information regarding the
dynamics, and quantity of contrast markers in the target
tissue.
[0167] Additionally, the wash out rate of the contrast agent can be
observed over a period of time. Once the reference frames have been
acquired, and the contrast agent has been injected, a specified
number of image frames can be collected at specified time
intervals; e.g., 1 second of image data is collected every 10
seconds. This allows for observation of the wash out over a long
period of time without an excessive amount of data collected.
[0168] In a further aspect, the contrast overlay 113 can be
processed by applying a predetermined threshold. Here, the
intensity of each pixel of the contrast overlay can be compared to
the predetermined threshold, and only those pixels within the
contrast overlay that exceed the threshold can be displayed. It is
contemplated that the threshold value can be a fixed predetermined
value, or it can be under user control.
[0169] The contrast overlay 113 is displayed on top of the B-Mode
image using a blending algorithm. This method is designed to allow
the contrast overlay 113 to be displayed as a semi transparent
color map on top of the grayscale B-Mode image. The level of
transparency is user controlled.
[0170] FIG. 6 shows images created using the exemplary method
described herein. FIG. 6A shows a pre-contrast agent injection
reference loop ultrasound image. FIG. 6B shows a post contrast
agent injection data loop ultrasound image. FIG. 6C shows a
contrast overlay. FIG. 6D shows the data loop image of FIG. 6B with
the contrast overlay of FIG. 6C blended in. As discussed above,
colorization can be used in the methods of the present invention,
particularly where colorization can help to visually represent the
areas of contrast agent presence.
[0171] An exemplary blending algorithm in pseudo-code for an
exemplary 8-bit image is:
TABLE-US-00002 iColour = intensity of Contrast overlay pixel (0 -
255) iGray = intensity of B-Scan pixel (0 - 255) iBlendValue =
level of transparency (0-255) iAtemp = iColour - iBlendValue;
if(iAtemp<0) iAtemp=0; iBtemp = 256 - iAtemp; iTemp = iGray *
iBtemp; NewPixelRed = (OverlayPalette[iColour].Red*iAtemp + iTemp)
/ 256; NewPixelBlue = (OverlayPalette [iColour].Green*iAtemp +
iTemp) / 256; NewPixelGreen = (OverlayPalette [iColour].Blue*iAtemp
+ iTemp) / 256;
Example 2
Materials and Methods
[0172] Inflammation in the mouse hindleg was induced by a one or
three hour treatment with TNF-alpha injected subcutaneously into
the hindpaw. Inflammation in the kidney was induced by
ischemia-reperfusion injury. The left kidney was exposed and the
renal artery was clamped for 32 minutes, followed by 2 hours of
re-perfusion as described in Singbartl K, Green S A, Ley K. (2000)
"Blocking P-selectin protects from ischemia/reperfusion induced
acute renal failure" FASEB J. 14: 48-54. The wound was closed in
layers and, covered with a saline-soaked gauze.
[0173] Targestar.sup.B microbubbles from Targeson (Charlottesville,
Va.) were targeted to P-selectin by conjugating an anti-P-selectin
monoclonal antibody to the surface of the MB per manufacturer's
instructions. The MB were diluted to a concentration of 10.sup.7 or
10.sup.8 MB in 100 .mu.L of phosphate-buffered saline, and injected
as a bolus through a cannula placed in the left jugular vein.
[0174] Ultrasound imaging was performed using a VisualSonics.TM.
(Toronto, CA) model VEVO.RTM. 770 at 30 or 40 MHz. Cine loops of
800 frames were recorded for all studies. Frame rate was held
constant at 17 Hz. The Vevo.RTM. system included software for
comparing, matching and subtracting data set images and reference
set images. For example, Vevo.RTM. Contrast Mode software was used.
Wash-in of MB through the imaged tissue immediately after injection
was recorded, and imaging was suspended for 4 minutes to allow
circulating MB to accumulate at the target site. The targeted
tissue was then imaged for about 100 frames, and a pulsing sequence
to destroy microbubbles in the ultrasound field was applied. The
tissue was imaged for several hundred frames after destruction to
assess the contrast due to circulating (not adherent)
microbubbles.
[0175] The first 100 frames following the destruction sequence were
selected as a reference set, and represented the contrast signal
due to freely-circulating MB. A region of interest (ROI)
encompassing the kidney or saphenous vein and medial large muscle
was selected. The spatially-averaged pixel intensities of the
reference images within the ROI were subtracted from the 100 frames
before destruction (data set images) to derive the contrast signal
due to adherent microbubbles. Wash-in following MB injection was
assessed by setting 100 frames prior to injection as a reference
set, and subtracting this from succeeding frames.
Results
[0176] FIG. 7 shows results of a bolus injection of microbubbles in
healthy kidney at 40 MHz. FIG. 7A shows kidney before injection and
1C shows the kidney after injection. Background-subtracted contrast
enhancement after injection (green scale) is shown in FIG. 7D. FIG.
7B shows pixel intensity averaged over a ROI encompassing the
kidney for bolus injections of 10.sup.5, 10.sup.6, 10.sup.7, and
10.sup.8 MB.
[0177] FIG. 8 shows ultrasound images before MB destruction in a
kidney. The top row shows post-ischemic in FIG. 8A and healthy
contra lateral kidney in FIG. 8B before a destructive pulse. The
bottom row shows background-subtracted images of post-ischemic in
FIG. 8C and healthy contra lateral kidney FIG. 8D before a
destructive pulse.
[0178] FIG. 9 shows background-subtracted mean pixel intensity of
ROI around kidney showing contrast enhancement in a post-ischemic
kidney in FIG. 9A and in a healthy contralateral kidney in FIG.
9B.
[0179] FIG. 10 shows pixel intensity within ROI averaged over 100
frames before a destructive pulse and after MB destruction. The
error bars show standard deviation over 100 frames.
[0180] FIG. 11 shows ultrasound images before MB destruction in
hindleg. The top row shows 3-hour TNF-alpha treated in FIG. 11A and
healthy untreated hindleg in FIG. 11B before a destructive pulse.
The bottom row shows background-subtracted images of 3-hour
TNF-alpha treated in FIG. 11C and healthy untreated hindleg in FIG.
11D before a destructive pulse.
[0181] FIG. 12 shows pixel intensity within an ROI averaged over
100 frames before a destructive pulse and after MB destruction in
inflamed and non-inflamed (untreated) hindleg. The error bars show
standard deviation over 100 frames.
Example 3
Image Enhancement by Bolus Injection of Vascularized Tissue
[0182] A Vevo 770.RTM. (VisualSonics Inc. Toronto, CA)
high-resolution imaging system was used to acquire data set images
and reference set images. The Vevo.RTM. system included software
for comparing, matching and subtracting data set images and
reference set images. For example, Vevo.RTM. Contrast Mode software
was used.
[0183] The Default Cine Loop Size for Contrast Mode was set at 800,
which defined the size of the Contrast Mode cine loop. A high
frequency ultrasound imaging probe was connected to the to the
Vevo.RTM. 770 imaging system. In this example, an RMV.TM.-706 probe
(VisualSonics Inc., Toronto, CA) was used.
[0184] The subject was positioned for scanning, and while scanning,
the Field of View was adjusted to be 9.times.9 mm. Images were
acquired at a frame rate between 10 and 20 Hz and the transmit
power was set to 50%.
[0185] Contrast agent was prepared according to the instructions
provided in VisualSonics, Inc. (Toronto, CA) Preparation
Protocol--Preparation for Bolus Injection using the Vevo
MicroMarker.TM. Contrast Agent Kits.
[0186] The MicroMarker.TM. Microbubbles, made by Bracco Research SA
(Amsterdam, Netherlands) and available from VisualSonics (Toronto,
CA), were used for improved vascular enhancement and perfusion
imaging. MicroMarker.TM. Microbubbles are lyophilized microbubbles
with a lipid based shell containing polyethylene Glycol,
Phospholipids and fatty acid. The bubbles are stored in a glass
vial containing a gas head consisting of nitrogen and
perfluorobutane gas. The Microbubbles will become gas filled
contrast enhancing agents when reconstituted with saline, agitated
and allowed to incubate for 10 mins.
[0187] The Vevo MicroMarker.TM. Contrast Agent Kits were used to
prepare the microbubble agent in order to observe image enhancement
due to the introduction of the contrast agent at the fundamental
imaging frequency. Upon bolus injection, opacification of larger
vessels were demonstrated with gradual infiltration of contrast
into the anatomical area of interest.
[0188] The MicroMarker.TM. solution was prepared for Mouse Imaging
using a 1 ml syringe pre-filled with 0.7 ml of sterile saline. A
21G 5/8'' needle was attached and the sterile saline was injected
into the MicroMarker.TM. vial. The syringe was removed and the
needle was left to vent before removal of the needle. The vial was
gently agitated hand for one minute and then the vial was allowed
to rest for 10 minutes. A 27G 1/2'' needle was attached to a 1 ml
syringe pre-filled with sterile saline to be used as a flush
syringe.
[0189] An empty 1 ml syringe and a second 21G 5/8'' needle was used
to draw up approximately 120 .mu.L of prepared MicroMarker.TM. from
the vial to compensate for the dead space in the needle hub. The
vial was gently agitated in a top-to-bottom manner before
collecting a sample. The air that is drawn up as well was removed
and the volume was adjusted to 50 .mu.l. This was the bolus amount
used for injection.
[0190] The reconstituted MicroMarker.TM. vial contained
2.times.10.sup.9 microbubbles/ml. A 50 .mu.L bolus was delivered
using a 271/2 G needle to give a final working solution of
1.0.times.10.sup.8 microbubbles for imaging most tumors and less
vascularized areas like the hind limb.
[0191] The Microbubbles can be further diluted with saline in order
to perform a dose response curve or to determine the optimal number
of microbubbles. For example, 1.0.times.10.sup.7 microbubbles/50
.mu.l can be used for well vascularized tissues such as the kidney
and liver. This can be achieved by performing a 1:10 dilution of
the original stock concentration. For each injection a 1:10
dilution of the stock solution includes 15 .mu.L of contrast agent
to be diluted to 150 .mu.L with saline (i.e. add 15 .mu.L of
contrast agent to 135 .mu.L of saline). The dilution can be
performed immediately before the injection is to be delivered in
order to avoid destabilization of the microbubbles. Any additional
contrast agent removed from the vial can be discarded.
[0192] Bolus injection of Microbubbles can follow the following
protocols depending on the area of interest. For tumors:
1.times.10.sup.8 bubbles/50 .mu.L bolus; for the Retina (eye):
1.times.10.sup.8 bubbles/50 .mu.l bolus; for cardiovascular:
1.times.10.sup.7 bubbles/50 .mu.l bolus; for the liver:
1.times.10.sup.7 bubbles/50 .mu.l bolus; for the hind limb
(muscle): 1.times.10.sup.8 bubbles/50 .mu.l bolus.
[0193] A small animal subject was prepared for imaging. Contrast
agent was injected via tail vein, jugular vein or into a
retro-orbital sinus. An appropriate imaging plane with the target
to be imaged was centered inside the focal zone and an 800 frame
baseline or reference set cine loop was acquired.
[0194] Another acquisition of an 800 frame data set image cine loop
was started and a bolus of contrast agent was slowly and gently
injected into the subject. The injection took about 5 seconds. Care
was taken to inject the bolus slowly to avoid destruction of the
microbubbles in the administered contrast agent. Acquisition
stopped after 800 frames were acquired.
[0195] The Vevo 770, including the contrast mode feature and
software, was used to process the acquired Contrast Mode data by
comparing the acquired data set image cine loop with the selected
images of the reference frames from the reference set cine loop. A
"contrast overlay" or enhanced ultrasound image was created to
identify the differences in image intensity between the reference
and the data loops. This overlay represented the change in B-mode
imaging caused by injection of the MicroMarker.TM. contrast
agent.
[0196] To generate a contrast overlay the bolus-injection or data
image set cine was loaded into the processing system of the
Vevo.RTM. imaging system. A reference cine loop was created by
selecting the first 25-100 frames from the loop during which no
contrast agent has entered in the imaging plane. Persist options to
apply the contrast overlay were selected based on the desired
imaging protocol. For example, no filters were applied to visualize
the movement of microbubbles with in the tissue. A smoothing filter
was used to suppress moving microbubbles, which was useful in
observing adherent bubbles by applying a 7 frame average display.
MIP (Maximum Intensity Persistence) was used to show the track of
the microbubbles through the vascular structure by using a
subtracted display that averages frames together.
[0197] The acquired Contrast Mode data was analyzed. The processed
bolus-injection cine loop to be analyzed was loaded into the Vevo
processing system. A contrast region measurement was created as
shown in FIG. 13. To create the contrast region measurement,
measurement calipers were used to trace the edge of the tissue. A
contrast region intensity curve vs time graph was also create as
shown in FIG. 14.
Example 4
[0198] A Vevo 770.RTM. (VisualSonics Inc. Toronto, CA)
high-resolution imaging system was used to acquire data set images
and reference set images. The Vevo.RTM. system included software
for comparing, matching and subtracting data set images and
reference set images. For example, Vevo.RTM. Contrast Mode software
was used.
[0199] The Default Cine Loop Size for Contrast Mode was set at 800,
which defined the size of the Contrast Mode cine loop. A high
frequency ultrasound imaging probe was connected to the Vevo.RTM.
770 imaging system. In this example, an RMV.TM.-706 probe
(VisualSonics Inc., Toronto, CA) was used. The transmit power was
set to 100% and the maximum available frame rate was selected.
[0200] The contrast agent was prepared according to the
instructions provided in the appropriate VisualSonics Inc.
(Toronto, CA) Preparation Protocol.
[0201] The MicroMarker.TM. Ultrasound Contrast agents are made by
Bracco Research SA (Amsterdam, Netherlands) for improved vascular
enhancement imaging and are available from VisualSonics Inc.
(Toronto, CA). DEPO.TM. MicroMarker.TM. agents are lyophilized
microbubbles with a lipid based shell containing polyethylene
glycol, phospholipids and fatty acids. The agents are stored in a
glass vial containing a gas head-space consisting of nitrogen and
perfluorobutane. The microbubbles are gas filled agents when
reconstituted with saline. MicroMarker.TM. DEPO.TM. Kits can be
stored at room temperature and have a shelf life of 6 months. The
DEPO.TM. contrast agents that have been opened and reconstituted
are stable within the vials for 4-6 hrs.
[0202] The DEPO.TM. agents were used to observe image enhancement
of the myocardium due to the introduction of the contrast agent at
the fundamental imaging frequency that lodges in the capillaries.
Upon bolus injection, opacification of larger vessels was
demonstrated with gradual infiltration of contrast into the
structure/tissue.
[0203] DEPO.TM. MicroMarker.TM. Contrast Agent was prepared for
Mouse Imaging. 1.4 ml of sterile saline was injected into the
DEPO.TM. contrast agent vial using a prefilled syringe containing
0.7 ml of sterile saline and a 21G 5/8'' needle. The syringe was
removed first to allow for ventilation, and then the needle was
removed. The glass vial was gently agitated by hand for approx. 10
seconds and then left to rest for 10 minutes. A 27G 1/2'' needle
was attached to a 1 ml syringe pre-filled with sterile saline to be
used as a flush syringe.
[0204] The vial was gently agitated in a top to bottom manner to
allow for even mixing. Using an empty 1 ml syringe and a second 21G
5/8'' needle, 90 .mu.L, of prepared DEPO.TM. contrast agent was
drawn up from the vial. A 50 .mu.L bolus from this amount was
injected. The 21G 5/8'' needle was replaced with the second 27G
1/2'' needle and air was removed air for the injection.
[0205] A 50 .mu.L bolus provided a final count of
1.2.times.10.sup.7 microbubbles. The microbubbles can be further
diluted with saline in order to perform a dose response curve or to
determine the optimal number of microbubbles for a specific model.
For example, a 1:2 dilution yields 6.times.10.sup.6 bubbles/50
.mu.L, which provides good results. Dilutions with adequate and
gentle mixing can be performed in an eppendorf tube immediately
prior to an injection into the animal, to minimize microbubble
destabilization.
[0206] A small animal subject was prepared for imaging. Contrast
agent was injected via tail vein, jugular vein or into a
retro-orbital sinus.
[0207] An appropriate imaging plane with the target to be imaged
was centered inside the focal zone and an 800 frame cine loop
acquisition was begun. A bolus was slowly and gently injected into
the subject. The injection took about 5 seconds. Injecting the
bolus too quickly can destroy the contrast agent. Acquisition stops
after 800 frames have been acquired. A second cine loop is acquired
approximately 11 minutes after the final injection. This cine loop
is compared during processing against the loop baseline saved
initially to visualize areas enhanced by DEPO.TM. contrast
agent.
[0208] The acquired cine loops including reference set images and
data set images were compared and a "contrast overlay" was created
to identify the differences in image intensity between these two
loops. To generate a contrast overlay the baseline cine loop in
which the injection occurred was identified as a reference cine
loop. The cine loop acquired approximately 11 minutes after the
final injection was used as the data cine loop. 11 minutes after
DEPO.TM. injection, the ventricle was clear of circulating bubbles,
and the myocardium became clearly defined, with the microbubbles
deposited in areas where there is blood flow.
[0209] The Vevo MicroMarker Depo.TM. Contrast Agent Kits enable for
the assessment of relative spatial perfusion of the myocardium in
the mouse heart using a single intravenous contrast agent injection
and the Vevo.RTM. 770 micro-ultrasound system. Using the
MicroMarker.TM. Depo.TM. kit, mouse models of myocardial viability
and left ventricular remodeling were analyzed.
Micromarker.TM. Contrast Agent Characteristics
[0210] Vevo.RTM. MicroMarker.TM. is a contrast agent optimized for
small animal micro-ultrasound imaging. It has been specifically
developed for use on small animals using high frequency
micro-ultrasound (>20 MHz). After reconstitution, the contrast
agent contains gas-filled micro-bubbles that are administered
intravenously to the subject. Ultrasound-based micro-bubbles are
confined to the vascular compartment and provide strong reflections
of the sound waves thereby providing detailed imaging of the blood
circulation using the Vevo.RTM. 770. Due to the size distribution
of the micro-bubbles in the MicroMarker.TM. DEPO.TM. contrast
agent, a population of sufficiently large micro-bubbles evade
pulmonary vascular filtering and are entrapped in the microvessels
of the myocardium. The retention (or deposition) of the
micro-bubbles in the myocardium is primarily due to lodging of a
fraction of "large" (>5 .mu.m) micro-bubbles.
[0211] The MicroMarker.TM. DEPO.TM. formulation used included a gas
mixture of nitrogen and perfluorobutane and an excipient or
polyethylene glycol, phospholipids, fatty acid and surfactant. The
solvent used was sodium chloride 0.9% w/v in water.
[0212] The excipient constituents were contained in a lyophilized
powder. After the micro-bubbles were reconstituted in saline and
the vial was agitated, the micro-bubbles were then administered
through the animal's venous system. These micro-bubbles contained a
gas mixture and were stabilized by a phospholipid monolayer. The
median diameter in volume of the administered micro-bubbles was
approximately 5 .mu.m.
[0213] Myocardial assessment using the MicroMarker.TM. DEPO.TM.
contrast agent was performed in real-time with the full analysis
workflow being completed in less than 20 minutes. The protocol used
included DEPO.TM. contrast agent preparation, animal preparation
(anesthetize animal, remove hair from area of imaging when
required, gain vascular access for introduction of agent), imaging
and acquiring baseline cineloop of myocardium (typically short axis
view), injection of 50 .mu.L bolus of DEPO.TM. contrast agent,
imaging and acquiring cineloop of bolus injection, and acquiring a
cineloop of myocardium opacification after waiting 10 minutes where
the DEPO.TM. contrast agent has lodged in the microvessels.
[0214] FIG. 15 shows a baseline image prior to DEPO.TM. bolus
injection. Infarct regions in the anterolateral and posterolateral
walls are illustrated as is the area of viable myocardium. FIG. 16
shows an image of initial bolus injection as the left ventricular
cavity is filled with DEPO.TM. contrast agent. FIG. 17 shows an
image of the left ventricle 10 minutes following injection of
DEPO.TM.. The DEPO.TM. contrast agent has perfused into the viable
myocardial microcirculation and has cleared the left ventricular
cavity. FIG. 18 shows a processed image showing the myocardial
infarction using the DEPO.TM. data; the overlay indicates regions
of well perfused myocardium and areas without the overlay indicate
areas of myocardial infarction and tissue damage. FIG. 19 shows a
polar coordinate graph of processed images demonstrating the
location of the region of infarction. Strong image intensity
(y-axis) indicates viable myocardium and conversely low signal
intensity illustrates damaged myocardial tissue.
[0215] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains.
[0216] Unless otherwise expressly stated, it is in no way intended
that any method set forth herein be construed as requiring that its
steps be performed in a specific order. Accordingly, where a method
claim does not actually recite an order to be followed by its steps
or it is not otherwise specifically stated in the claims or
descriptions that the steps are to be limited to a specific order,
it is no way intended that an order be inferred, in any respect.
This holds for any possible non-express basis for interpretation,
including: matters of logic with respect to arrangement of steps or
operational flow; plain meaning derived from grammatical
organization or punctuation; and the number or type of embodiments
described in the specification.
[0217] It will be apparent to those skilled in the art that various
modifications and variations can be made in the present invention
without departing from the scope or spirit of the invention. Other
embodiments of the invention will be apparent to those skilled in
the art from consideration of the specification and practice of the
invention disclosed herein. It is intended that the specification
and examples be considered as exemplary only, with a true scope and
spirit of the invention being indicated by the following
claims.
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