U.S. patent application number 12/334565 was filed with the patent office on 2009-07-23 for method and apparatus for examining tissue for predefined target cells, particularly cancerous cells, and a probe useful in such method and apparatus.
This patent application is currently assigned to Dune Medical Devices Ltd.. Invention is credited to Dan HASHIMSHONY.
Application Number | 20090187109 12/334565 |
Document ID | / |
Family ID | 27404543 |
Filed Date | 2009-07-23 |
United States Patent
Application |
20090187109 |
Kind Code |
A1 |
HASHIMSHONY; Dan |
July 23, 2009 |
METHOD AND APPARATUS FOR EXAMINING TISSUE FOR PREDEFINED TARGET
CELLS, PARTICULARLY CANCEROUS CELLS, AND A PROBE USEFUL IN SUCH
METHOD AND APPARATUS
Abstract
A method, apparatus and probe for examining tissue for the
presence of target cells, particularly cancerous cells, by
subjecting the tissue to be examined to a contrast agent containing
small particles of a physical element conjugated with a biological
carrier selectively bindable to the target cells. Energy pulses are
applied to the examined tissue. The changes in impedance and/or
optical characteristics of the examined tissue produced by the
applied energy pulses are detected and utilized for determining the
presence of the target cells in the examined tissue. In a described
preferred embodiment, the applied energy pulses include laser
pulses, and the physical element conjugated with a biological
carrier is a light-sensitive semiconductor having an impedance
which substantially decreases in the presence of light. The same
probe used for detecting the targeted cells may also be used for
destroying the cells so targeted.
Inventors: |
HASHIMSHONY; Dan; (Givat
Ada, IL) |
Correspondence
Address: |
MARTIN D. MOYNIHAN d/b/a PRTSI, INC.
P.O. BOX 16446
ARLINGTON
VA
22215
US
|
Assignee: |
Dune Medical Devices Ltd.
Caesaria
IL
|
Family ID: |
27404543 |
Appl. No.: |
12/334565 |
Filed: |
December 15, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10298196 |
Nov 18, 2002 |
7505811 |
|
|
12334565 |
|
|
|
|
60331548 |
Nov 19, 2001 |
|
|
|
60343583 |
Jan 2, 2002 |
|
|
|
Current U.S.
Class: |
600/476 ;
600/547 |
Current CPC
Class: |
A61B 5/0538 20130101;
A61B 5/416 20130101; A61K 49/00 20130101; A61B 5/065 20130101; A61B
5/0084 20130101; A61B 5/0075 20130101; A61B 5/411 20130101; A61B
5/0071 20130101; Y10S 607/901 20130101; A61B 5/0091 20130101 |
Class at
Publication: |
600/476 ;
600/547 |
International
Class: |
A61B 6/00 20060101
A61B006/00; A61B 5/053 20060101 A61B005/053 |
Claims
1. Apparatus for examining a tissue region for the presence of
predefined target cells therein, comprising a voltage pulse source;
an optical pulse source; a probe having an operative end for
applying optical pulses and voltage pulses from said sources to the
examined tissue region, and for detecting the reflections of said
voltage pulses; and a data processor system including an electrical
measuring sub-system coupled to said probe for detecting the
reflections of said voltage pulses, said reflections indicative of
changes in electrical properties of said examined tissue region
produced by at least one of said optical and voltage pulses, and
for determining therefrom the extent of the presence of said target
cells in the examined tissue region; the voltage pulses and optical
pulses being applied concurrently.
2. The apparatus according to claim 1, wherein said probe is also
operative for detecting optical reflections of said optical pulses;
and wherein said data processor system also includes an optical
analyzer sub-system coupled to said probe for detecting the
reflections of said optical pulses.
3. The apparatus according to claim 1, wherein said probe includes
a pair of spaced conductors at said operative end for applying said
voltage pulses to said examined tissue, and an optical fiber
centrally of said conductors for applying said optical pulses to
said examined tissue.
4. The apparatus according to claim 3, wherein said pair of spaced
conductors are coaxial conductors, and said optical fiber is a core
extending centrally of said coaxial conductors.
5. The apparatus according to claim 4, wherein one of said coaxial
conductors is an inner conductor in the form of a metal layer over
said optical fiber, and the other of said coaxial conductors is an
outer conductor enclosing said inner conductor and separated
therefrom by a dielectric material.
6. The apparatus according to claim 5, wherein said outer conductor
is in the form of a flexible metal braid, and the end thereof at
the operative end of the probe includes a rigid cap to facilitate
manipulating the probe.
7. The apparatus according to claim 3, wherein the end of at least
one conductor at said operative end of the probe extends slightly
past the respective end of said optical fiber to define an open
cavity at said operative end of the probe.
8. The apparatus according to claim 5, wherein said optical fiber
extends through an opening in said outer conductor for connection
to said optical pulse source.
9. The apparatus according to claim 3, wherein said pair of spaced
conductors are capacitor plates, and said optical fiber is located
between said capacitor plates at said operative end of the
probe.
10. The apparatus according to claim 3, wherein said pair of spaced
conductors at the operative end of the probe are configured to
clamp between them the tissue to be examined.
11. The apparatus according to claim 1, wherein said optical pulse
source includes a laser supplying laser pulses, and wherein said
probe includes an optical fiber for conducting said laser pulses to
the operative end of the probe for application to the examined
tissue.
12. The apparatus according to claim 3, wherein said optical fiber
in the probe also receives optical reflections of said optical
pulses; said probe thus being operative for detecting optical
reflections of said optical pulses; and wherein said data processor
system also includes an optical analyzer sub-system coupled to said
probe for detecting the reflections of said optical pulses.
13. The apparatus according to claim 2, wherein said optical
analyzer sub-system includes spectrum analyzer means and a
splitting box; wherein said splitting box includes a beam splitter
for directing a first part of the optical pulses from said optical
pulse source to said optical fiber in the probe, and a second part
to said spectrum analyzer means; and wherein said beam splitter
also directs to said spectrum analyzer means said optical
reflections received from the examined tissue region.
14. The apparatus according to claim 13, wherein said spectrum
analyzer means includes two spectrometers; and wherein said
splitting box includes a polarizer which polarizes into two forms
said second part of the optical pulse source and the optical
reflections from the examined tissue region, and directs each
polarization form to one of the two spectrometers.
15. The apparatus according to claim 1, wherein said optical pulse
source includes a light chopper for supplying said optical pulses
to the probe at a controlled repetition rate.
16. The apparatus according to claim 15, further comprising an
impedance measuring sub-system including a lock-in-amplifier which
is controlled in synchronism with said light chopper to increase a
signal-to-noise ratio output of the amplifier.
17. The apparatus according to claim 1, wherein said data processor
system includes an audio output device which is actuated according
to the determination of said data processor system as to the extent
cancerous cells are present in the examined tissue.
18. The apparatus according to claim 1, wherein said probe is
flexible so as to be capable of being introduced into a subject's
body via a catheter.
19. The apparatus according to claim 1, wherein said probe is
incorporated inside a biopsy needle.
20. The apparatus according to claim 19, wherein said biopsy needle
has a side cavity, and said operative end of the probe is aligned
with said side cavity.
21. The apparatus according to claim 19, wherein said biopsy needle
has a sharp front edge, and said operative end of the probe is
aligned with said sharp front edge.
22. The apparatus according to claim 1, wherein said optical pulse
source is a laser source capable of applying laser pulses of a
relatively low intensity for detecting the presence of target cells
in the examined tissue, and thereafter laser pulses of a high
intensity for destroying detected target cells.
23. The apparatus according to claim 22, wherein said laser source
is capable of applying femtosecond laser pulses of an intensity of
100 nj to 1 mj for destroying the detected target cells.
24. The apparatus according to claim 1, wherein said probe further
includes an array of optical sensors at a known location with
respect to said operative end of the probe for sensing the location
and orientation of said operative end of the probe.
25. A probe for use in examining a tissue region for the presence
of predefined target cells therein, comprising: an operative end
having at least one pair of spaced conductors for applying voltage
pulses to the examined tissue region, and that detects the
reflections of said voltage pulses, and that applies optical pulses
via an optical fiber to the examined tissue region; the voltage
pulses and the optical pulses being applied concurrently.
26. The probe according to claim 25, wherein said pair of spaced
conductors are coaxial conductors, and said optical fiber is a core
extending centrally of said coaxial conductors.
27. The probe according to claim 26, wherein one of said coaxial
conductors serving as an inner conductor is in the form of a metal
layer over said optical fiber, and the other of said coaxial
conductors serving as an outer conductor encloses said metal layer
and is separated therefrom by a dielectric material.
28. The probe according to claim 27, wherein said outer conductor
is in the form of a flexible metal braid, and the end thereof at
the operative end of the probe includes a rigid cap to facilitate
manipulating the probe.
29. The probe according to claim 25, wherein the end of at least
one conductor at said operative end of the probe extends slightly
past the respective end of said optical fiber to define an open
cavity at said operative end of the probe.
30. The probe according to claim 26, wherein said optical fiber
extends through an opening in outer one of said conductors for
connection to an optical pulse source.
31. The probe according to claim 25, wherein said pair of spaced
conductors are capacitor plates, and said optical fiber is located
between said capacitor plates at said operative end of the
probe.
32. The probe according to claim 25, wherein said pair of spaced
conductors at the operative end of the probe are configured to
clamp between them the tissue to be examined.
33. The probe according to claim 25, wherein the end of said probe
opposite to said operative end is manually graspable for
manipulation with respect to the tissue to be examined.
34. The probe according to claim 25, wherein said probe is flexible
so as to be capable of being introduced into a subject's body via a
catheter.
35. The probe according to claim 25, wherein said probe is
incorporated inside a biopsy needle.
36. The probe according to claim 35, wherein said biopsy needle has
a side cavity, and said operative end of the probe is aligned with
said side cavity.
37. The probe according to claim 35, wherein said biopsy needle has
a sharp front edge, and said operative end of the probe is aligned
with said sharp front edge.
38. The probe according to claim 25, wherein said probe further
includes an array of optical sensors at a known location with
respect to said operative end of the probe for sensing the location
and orientation of said operative end of the probe.
39. Apparatus for examining a tissue region, comprising a voltage
pulse source; an optical pulse source; a probe having an operative
end for applying optical pulses and voltage pulses from said
sources to the examined tissue region, and for detecting the
reflections of said voltage pulses; and a data processor system
including an electrical measuring sub-system coupled to said probe
for detecting the reflections of said voltage pulses, said
reflections indicative of changes in electrical properties of said
examined tissue region produced by at least one of said optical and
voltage pulses, the voltage pulses and optical pulses being applied
concurrently.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of pending U.S. patent
application Ser. No. 10/298,196 filed on Nov. 18, 2002, which
claims the benefit of U.S. Provisional Patent Application Nos.
60/331,548 filed on Nov. 19, 2001, and 60/343,583 filed on Jan. 2,
2002. The contents of the above applications are incorporated
herein by reference. The present application is also related to
U.S. patent application Ser. No. 10/035,428 filed on Jan. 4, 2002,
now U.S. Pat. No. 6,813,515, the contents of which are also
incorporated herein by reference.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present application relates to a method and apparatus
for examining tissue for the presence of predefined target cells
therein, and also to a probe for use in such method and apparatus.
The invention is particularly useful for detecting cancerous cells
in a real-time manner during a surgical operation for removing,
e.g., a breast tumor. The invention is therefore described below
with respect to such an application, but it will be appreciated
that the invention is useful in many other applications.
[0003] During a surgical operation for the removal of a tumor, it
would be highly desirable to provide the surgeon with a real-time
indication of the nature of the tissue at the surgical site, i.e.,
whether it is normal tissue or cancerous tissue. In the absence of
such a real-time indication, the surgeon may remove more tissue
than really necessary in order to provide better assurance that the
entire tumor is removed.
[0004] Existing medical instruments, such as computed tomography
(CT) scanners, magnetic resonance imagining (MRI) devices,
electrical bioimpedance scanning devices (T-scan), ultrasound, and
other similar instruments, are commonly used in pre-operative
guided biopsy procedures to obtain samples of tissues in order to
delineate the extent of the cancerous tissue. However, the accuracy
of such instruments and procedures for the delineation of cancerous
tissue depends to a high degree on the accuracy by which the sample
was taken, and the expertise of the surgeon in translating such
information to the actual conditions at the tumor site.
[0005] My above-cited U.S. patent application Ser. No. 10/035,428,
the contents of which are incorporated herein by reference, briefly
reviews various electrical techniques described in the prior art
for examining tissue in order to indicate its nature according to
the dielectric properties of the examined tissue. That patent
application is directed to an improved method of making such an
examination, by applying an electrical pulse (or a sequence of
pulses) to the tissue to be examined via a probe, which generates
an electrical field in the examined tissue and produces a reflected
pulse therefrom. The reflected electrical pulse is detected, and
its electrical characteristics are compared with those of the
applied electrical pulse to provide an indication of the dielectric
properties of the examined tissue, and thereby, the extent of the
presence of cancerous cells therein.
[0006] However, because of the critical importance of this
information to the surgeon during a surgical operation, efforts are
continually being made to provide methods, apparatus and probes,
which are capable of more accurately determining the extent of the
presence of cancerous cells in a real-time manner.
OBJECTS AND BRIEF SUMMARY OF THE PRESENT INVENTION
[0007] An object of the present invention is to provide a method
for examining tissue which is basically an electrical optical
measuring technique, but which is capable of more accurately
determining the extent of the presence of cancerous cells, as well
as other predefined target cells, in an examined tissue. Another
object of the invention is to provide a method for destroying the
target cells immediately after detection. Another object of the
invention is to provide apparatus for use in the novel method, and
a further object is to provide a probe particularly useful in such
apparatus.
[0008] According to one aspect of the present invention, there is
provided a method of examining tissue for the presence of
predefined target cells therein, comprising: subjecting the tissue
to be examined to a contrast agent containing small particles of a
physical element conjugated with a biological carrier, e.g., an
antibody, selectively bindable to the target cells; applying energy
pulses to the examined tissue; detecting changes in electrical
properties of the examined tissue produced by the applied energy
pulses; and utilizing the detected changes in electrical properties
for determining the presence of the target cells in the examined
tissue.
[0009] As indicated earlier, the method is particularly useful for
detecting cancer target cells in a realtime manner during a
surgical operation, and therefore the invention is described below
particularly with respect to this application.
[0010] As will be described more particularly below, the novel
method exploits the known technique for localization of tumors by
the injection of physical elements conjugated with a biological
carrier such as an antibody selectively bindable to the cancerous
cells. Such techniques have been used with radioisotopes in order
to target tumor tissue or cancerous cells when examined with
computer tomography. Techniques using nano/micro crystal particles
have also been used as novel intravascular probes for both
diagnostic (e.g., imaging) purposes and therapeutic (e.g., drug
delivery) purposes. For example, reference is made to the
publications Akerman, M. E., et al., Nanocrystal Targetting in
vivo, PNAS, Oct. 1, 2002, 12617-12621; and Beard, M. C., et al.,
Size-Dependent Photoconductivity in CdSe Nanoparticles as Measured
by Time-Resolved Terahertz Spectroscopy, American Chemical Society,
Aug. 14, 2002, the contents of which publications are incorporated
herein by reference.
[0011] The present invention utilizes such a technique (e.g., with
non-radioactive conjugated antibodies) in an electric optical
measuring procedure for detecting cancerous cells. Preferably, the
applied energy pulses include pulses of optical (e.g., laser)
energy, and the physical element conjugated with the antibody is
one which changes in impedance when illuminated by the optical
energy. As described more particularly below, especially good
results are obtainable when the physical element is a
light-sensitive semiconductor having an impedance which
substantially decreases in the presence of light. In the preferred
embodiments described below, changes in optical properties of the
examined tissue produced by the applied optical pulses are also
detected and utilized in determining the extent of the presence of
the target (e.g., cancer) cells in the examined tissue.
[0012] While particularly good results are obtainable when the
physical element conjugated with the antibody is a light-sensitive
semiconductor, the invention may also be implemented in
applications wherein the physical element is a metal having good
light reflecting characteristics, and wherein changes in an optical
characteristic (e.g., frequency, amplitude and/or phase) of the
reflected light are detected and utilized for determining the
extent of the presence of the target cells in the examined tissue.
The physical element may also be a fluorescent material which emits
radiation of a predetermined frequency when illuminated by light,
or a light absorption material which absorbs radiation of a
particular frequency, in which cases changes in frequency of the
reflected light would be detected and utilized for determining the
extent of the presence of the target cells in the examined
tissue.
[0013] According to further features in the described preferred
embodiments, voltage pulses may be applied to a probe area of the
examined tissue to detect the presence of the target cells in the
probe area, and optical pulses may be applied to a central region
of the probe area of the examined tissue to detect the presence of
the target, e.g., cancerous, cells in the central region. Such an
arrangement greatly aids the surgeon in determining, during a
surgical operation, the size of a tumor to be removed, and its
exact delineation from normal healthy tissue to be retained.
[0014] According to a further feature of the invention, as
described below, the target cells, e.g., cancerous cells, once
detected, may be subjected to optical energy of sufficient
intensity to destroy them. For example, where the target cells are
cancerous cells and the optical energy is laser energy, the same
probe as used for detecting the cancerous cells may also be used
for destroying such cancerous cells by applying femtosecond pulses
at an intensity of 100 nj-1 mj at the targeted cells. Longer pulses
can be also used but with more heat dissipation to the surrounding
area.
[0015] According to still further features in the preferred
embodiments described below, the optical pulses may be applied by
means of a flexible probe introduced into a subject's body via a
catheter, or incorporated in a biopsy needle.
[0016] According to another aspect of the present invention, there
is provided a method of examining tissue for the present of
cancerous cells therein, comprising: applying laser pulses to the
examined tissue; detecting the reflections of the laser pulses from
the examined tissue; comparing an optical characteristic of the
laser pulses applied to the examined tissue with that of the laser
reflections from the examined tissue; and utilizing the comparison
of optical characteristics for determining the presence of
cancerous cells in the examined tissue.
[0017] According to a still further aspect of the present
invention, there is provided a method of examining tissue for the
presence of cancerous cells therein, comprising: applying optical
energy pulses to the examined tissue in at least two polarization
forms; detecting changes in optical properties of the examined
tissue in each of the polarization forms; and utilizing the
detected changes for determining the presence of cancerous cells in
the examined tissue.
[0018] According to a still further aspect of the present
invention, there is provided a method of examining tissue for the
presence of cancerous cells therein, comprising: applying voltage
pulse and laser pulses to the examined tissue; detecting
reflections of the voltage pulses from the examined tissue;
comparing an electrical characteristic of the voltage pulse
reflections from the examined tissue with that of the voltage
pulses applied to the examined tissues with the laser pulses; and
utilizing the comparison of electrical characteristics for
determining the presence of cancerous cells in the examined
tissue.
[0019] According to yet another aspect of the present invention,
there is provided apparatus for examining tissue for the presence
of predefined target (e.g., cancer) cells therein, comprising: a
voltage pulse source; an optical pulse source; a probe having an
operative end for applying optical pulses and voltage pulses from
the sources to the examined tissue, and for detecting the
reflections of the voltage pulses produced by the examined tissue;
and a data processor system including an electrical measuring
sub-system coupled to the probe for detecting changes in the
electrical properties of the examined tissue produced by the
optical and voltage pulses, and for determining therefrom the
presence of the target cells in the examined tissue.
[0020] According to further features in the described preferred
embodiments, the probe also detects optical reflections of the
optical pulses from the examined tissue. In such case, the data
processor system also includes an optical analyzer sub-system
utilizing the detected optical reflections from the examined tissue
for detecting changes in optical characteristics of the examined
tissue produced by the applied pulses in determining the extent of
the presence of the target cells in the examined tissue.
[0021] According to yet another aspect of the present invention,
there is provided a probe for use in examining tissue for the
presence of cancerous cells therein, comprising: an operative end
having at least one pair of spaced conductors for applying voltage
pulses to the examined tissue; and an optical fiber at the
operative end for applying optical pulses to the examined
tissue.
[0022] Further features and advantages of the invention will be
apparent from the description below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The invention is herein described, by way of example only,
with reference to the accompanying drawings, wherein:
[0024] FIG. 1 is a block diagram illustrating one form of apparatus
constructed in accordance with the present invention for examining
tissue in a real-time manner for the presence of target cells,
particularly cancerous cells, therein;
[0025] FIGS. 2a-2e are pictorial illustrations illustrating the
mechanism of action involved in the impedance measuring technique
of the present invention, when using a contrast agent conjugated
with an antibody selectively bindable to the cancerous cells;
[0026] FIG. 3 is a block diagram illustrating a second form of
apparatus constructed in accordance with the present invention;
[0027] FIG. 4 illustrates generally the construction of the
electrical properties probe in the apparatus of FIG. 3;
[0028] FIG. 5 illustrates a preferred construction of the operative
end of the probe of FIG. 3;
[0029] FIG. 6a illustrates the probe of FIG. 9 when examining
tissue constituted only of normal cells; and FIG. 6b illustrates
the probe of FIG. 5 when examining tissues including cancerous
cells;
[0030] FIG. 7 schematically illustrates the probe in the apparatus
of FIG. 3 and its connections to the impedance measuring and
optical analyzer sub-systems;
[0031] FIG. 8 illustrates the optical splitting box in the optical
analyzer sub-system of FIG. 3;
[0032] FIGS. 9a and 9b illustrate examples of polarization
measurements from the two spectrometers in the optical analyzer
sub-system of FIGS. 3 and 8;
[0033] FIG. 10 is a pictorial illustration illustrating the manner
of using the apparatus of FIG. 3 for detecting cancer cells in a
real-time manner during a surgical operation for removing a
tumor;
[0034] FIGS. 11 and 12 illustrate possible variations in the
construction of the electrical probe;
[0035] FIG. 13a schematically illustrates a possible variation in
the probe construction that enables the use of the probe inside a
biopsy/needle/tool;
[0036] FIG. 13b schematically illustrates a possible variation in
the probe construction that enables the use of the probe in front
of the biopsy/needle/tool;
[0037] FIG. 14 illustrates another probe constructed to include a
3-D orientation sensor to enable determination of the position and
orientation of the operative end of the probe;
[0038] FIG. 14a is a fragmentary view illustrating the orientation
sensor in the probe of FIG. 14; and
[0039] FIG. 14b is a plan view schematically illustrating the
orientation sensor in the probe of FIG. 14.
[0040] It is to be understood that the foregoing drawings, and the
description below, are provided primarily for purposes of
facilitating understanding the conceptual aspects of the invention
and various possible embodiments thereof, including what is
presently considered to be a preferred embodiment. In the interest
of clarity and brevity, no attempt is made to provide more details
than necessary to enable one skilled in the art, using routine
skill and design, to understand and practice the described
invention. It is to be further understood that the embodiments
described are for purposes of example only, and that the invention
is capable of being embodied in other forms and applications than
described herein.
TECHNICAL DISCUSSION OF MECHANISM OF ACTION
[0041] As indicated earlier, the invention of the present
application provides an electro optic method and apparatus for
examining tissue for the presence of predefined target cells,
particularly cancerous cells, by using the known conjugation
technique presently used in imaging and drug-delivery procedures.
According to the present invention, the tissue to be examined is
subjected to a contrast agent containing small particles of a
physical element conjugated with an antibody selectively bindable
to the target cells. Energy pulses are applied to the examined
tissue; and changes in impedance of the examined tissue produced by
the applied energy pulses are detected; and utilizing for
determining the extent of the presence of the target cells in the
examined tissue.
[0042] Before describing the particulars of the method and
apparatus of the present invention as illustrated in the
accompanying drawings, it would be helpful first to provide a
technical discussion of the conjugation technique generally and of
the mechanism of action, as illustrated in FIGS. 2a-2e, involved
when this technique is used in the impedance-measuring method and
apparatus of the present invention for determining the extent of
the presence of target (e.g., cancer cells) in an examined
tissue.
[0043] One of the most promising developments in the biotechnology
field is the creation of MAbs, which are exquisitely selective
proteins (antigens or antibodies) that bind to only a single
target. Depending on the clinical setting, the specific antigens to
which MAbs may bind include bacteria, hormones, tumor cell
antigens, growth factors, and a variety of other substances. The
extraordinary specificity of MAbs has tremendous clinical value. As
reagents, Mabs appear ideal since they are homogeneous in nature,
recognize specific antigenic determinants, can be mass produced,
are relatively stable to conjugation methods, and are biocompatible
in vivo.
[0044] MAbs are created using cell fusion techniques in a process
called hybridoma technology. Cell fusion is a form of genetic
engineering that merges two types of cells to form a single
cell.
[0045] First, a mouse is immunized with an antigen that
specifically stimulates production of a desired antibody targeted
at that antigen. White blood cells that produce antibodies, called
B-lymphocytes, are isolated from the mouse's spleen. These cells
are then fused to myeloma cells, which are tumor cells that
replicate continuously and rapidly. Using cell fusion technology,
these two cell types are merged into a single cell, called a
hybridoma. The hybridoma contains the DNA of both of the original
cells and thus possesses their desirable qualities.
[0046] Each hybridoma is capable of producing large numbers of
identical antibody molecules, and also called MAbs because they are
produced by the identical offspring of a single, cloned,
antibody-producing cell. Like the lymphocytes from the immunized
animal, they produce antibodies targeted at the injected
antigen.
[0047] Efforts in hybridoma technology have created four different
types of MAbs. Murine (mouse) MAbs have been the primary focus of
MAb creation to date. However, they produce variable results.
Because mouse-produced antibodies are not identical to human
antibodies, they are eventually recognized as foreign proteins by
the human body and cleared from circulation by human antimouse
antibodies (HAMA). These reactions are not a serious problem with
MAb-based diagnostic and imaging products, where only a single
application may be required. However, they are a major obstacle to
the therapeutic use of murine antibodies. Most patients produce a
HAMA reaction, which significantly reduces therapeutic efficacy and
increases toxicity.
[0048] Human MAbs do not produce a HAMA reaction, so they tend to
succeed therapeutically and are less likely to produce allergic
reactions. Unfortunately, it is extremely difficult to fuse human
B-lymphocytes with myeloma cells. Several biotechnology companies
are investigating novel ways to produce human MAbs, but this
process appears to be much more expensive than murine-based
systems.
[0049] Chimeric MAbs use recombinant engineering technology and
involve the assembly of diverse gene segments not normally found
together in nature. With this approach, recombinant genes are
constructed that code for the production of specific proteins
(MAbs), in which selected segments from the mouse antibody are
fused to complementary segments from the human antibody. While the
chimeric antibody produced retains its binding specificity, it more
closely resembles a natural human antibody. Therefore, it is less
likely to produce a HAMA reaction.
[0050] Humanized MAbs incorporate only the genes for the specific
binding sites from the mouse.
[0051] Because MAbs act as specific probes that can be directed at
the protein that induced their formation, they can be used
successfully in clinical applications. For instance, MAbs can
direct immune system activity by seeking out targeted antigens and
attracting immune cells (such as monocytes, macrophages and
lymphocytes) to the targeted cell. Additionally, MAbs can be
directed at target molecules needed for cellular growth or
differentiation. For example, many patients with breast cancer
carry a specific protein on the surface of their tumor cells. When
a MAb directed at this protein is used in combination with
traditional chemotherapy, patients experience a greater degree and
duration of therapeutic response. This results in a greater rate of
overall survival when compared with treatment with chemotherapy
alone.
[0052] Conjugated MAbs are monoclonal antibodies that are combined
with some physical element like radioisotopes, toxins, metal,
semiconductors or. They are also can be combined with other
antibodies or drugs for targeted delivery to specific cells.
[0053] Because MAbs can be conjugated with a radioisotope, they are
well suited for use in diagnosing and monitoring disease. This was
one of the earliest uses for biotechnology. The first products
using MAbs to diagnose disease were approved by the FDA in 1981,
and MAbs have been used in diagnostic imaging since 1992. For
example, in cancer diagnostics, MAbs targeted at specific antigens
found on cancer cells, such as carcinoembryonic antigen, are
conjugated with a radioisotope. These MAbs are then administered to
patients, where they target tumor tissue when evaluated with
computer tomography. This approach has been successful in
diagnosing and monitoring patients with colorectal, ovarian and
prostate cancers.
[0054] As indicated earlier, in the present invention such a
technique is used (e.g., with a non-radioactive conjugated
antibody) in an impedance measuring procedure for detecting cancer
cells.
[0055] FIGS. 2a-2e schematically illustrate the process of
producing the conjugated MAbs, namely a physical element conjugated
with an antibody selectively bindable to certain defined target
cells, such as cancer cells; and the manner in which such MAbs,
when included in a contrast agent applied to the tissue to be
examined, affect the impedance measurement of such tissue to
provide an indication of the extent of the presence of the target
(e.g., cancer) cells in such examined tissue.
[0056] FIG. 2a illustrates the conjugation process generally,
wherein small particles (of nanometer or micrometer size, and
therefore hereinafter termed "nano/micro-particles) of a physical
element 2, such as silicon nano/micro-particles, are conjugated
with a specific monoclonal antibody 3, selectively bindable to the
target cells in the tissue to be examined, to produce a conjugation
of the antibodies to the silicon nano/micro-particles, as shown at
4.
[0057] FIG. 2b illustrates a cancer cell 5 containing specific
proteins 6 on its cell membrane to which the monoclonal antibodies
3 are selectively bindable or attachable.
[0058] FIG. 2c illustrates the attachment process occurring when
the conjugated physical elements 4 of FIG. 2a are included in a
contrast agent injected into the blood stream of the tissue 5 to be
examined, so that the conjugated elements 4 attach to the specific
proteins 6 in the cell 5. It will thus be seen that, because of the
high bonding selectivity of the monoclonal antibodies 3 of the
conjugated silicon particles 4 with respect to the specific
proteins 6 within the cancer cell 5, tissue having such cancer
cells will have a concentration of such nano/micro-particles bonded
thereto in proportion to the number of cancer cells, whereas tissue
not having cancer cells will not have such nano/micro-particles
bonded thereto.
[0059] Accordingly, the extent of the presence of cancer cells in
the examined tissue will be indicated by the extent to which the
conjugated nano/micro-particles 4 have been bonded to the tissue.
The extent of the presence of cancer cells in the examined tissue
can thus be determined by measuring the impedance, or changes in
impedance, of the examined tissue.
[0060] FIG. 2d schematically illustrates making such an impedance
measurement of the examined tissue 5 in order to determine the
extent of the target cells therein. Preferably in accordance with
the present invention, the nanoparticles 2 conjugated with the
antibodies 3 are particles which change in impedance when
illuminated by optical energy, particularly laser energy. Thus,
when the nano/micro-particles 2 are of a semiconductor, such as
silicon, silicon absorbs light photons above 1.1 ev and produces a
substantial increase in conductivity by transferring electrons from
the valance band to the conduction band.
[0061] In order to exploit this substantial change in the impedance
of the examined tissue for purposes of determining the extent of
cancer cells therein, an electro-optical probe, generally
designated P in FIG. 2d, applies both optical pulses and voltage
pulses to the examined tissue 5. In a preferred described
embodiment, the voltage pulses are applied by a pair of coaxial
conductors 7, 8 at the operative end of the probe, and the optical
pulses are laser pulses applied via an optical fiber 9 extending
through the inner conductor 8.
[0062] FIG. 2d schematically illustrates the situation when the
examined tissue 5 is not subjected to the laser, in which case the
impedance of the examined tissue would be relatively high; whereas
FIG. 2e illustrates the situation when a light beam is applied via
optical fiber 9 to the examined tissue 5, in which case the
impedance of the tissue would be substantially decreased.
[0063] FIGS. 2d and 2e illustrate a further important feature of
the present invention, namely that the voltage pulses produced by
the electrode 7, 8 are applied to a relatively large probe area of
the examined tissue to detect the extent of the presence of cancer
cells in the probe area; whereas the optical pulses from optical
fiber 9 are applied to a central region of the probe area of the
examined tissue to detect the extent of the presence of cancer
cells in the central region. This feature enables the probe to
detect, with relatively high accuracy, any cancer cells within the
probe area, as shown by the following example:
EXAMPLE
[0064] Let us assume that the area of the electrical probe P is
160.mu..times.160.mu., that silicon conjugated crystals are joined
to cancerous cells at that probe area, and that there are about 16
cancerous cells on the surface of that probe area. On each cell
there are many thousands of 10 to 100 nm diameter silicon crystals;
(in this example we are using the electrical parameter of the bulk
material; the exact electrical parameters will be different for the
various sizes of the crystals from the nanometer size to the
micrometer size). The conductivity [=1/resistivity] of normal cells
is, for example, 0.02-0.05 S/m for fat tissue, 0.1 S/m for brain
tissue, and 1.3 S/m for saline. Normal tissues of the above
dimension will have a resistance of about 10.sup.8.OMEGA. for fat,
7.5.times.10.sup.5.OMEGA. for saline, and 10.sup.4.OMEGA. for brain
tissue. The resistance of the probing zone is given by:
R=.rho.L/[w.times.d].
where: .rho. is the resistivity, L is the sample length, w is the
sample width, and d is the thickness of the layer.
[0065] When light irradiates the sample, the resistivity of the
crystal is reduced from about 1 K.OMEGA.cm to about
10.sup.-6.OMEGA. cm, and the resultant resistance will be about
1.OMEGA. to 0.1 {tilde over (.OMEGA.)}. It is clear that the change
is dramatically large. This abrupt change will be detected by the
impedance measurements, as well as by light reflection, as
described below.
[0066] Assuming only one cancerous cell exists between those 16
cells, and the one cell is only partially covered with silicon
crystal, even with a covering ratio of 1/1000 the total change can
be easily detected using a lock-in method, as also described
below.
[0067] Following is a simplified order of magnitude calculation.
[0068] 1 cell: 40 .mu.m.times.40 .mu.m=1600
.mu.m.sup.2=1600.times.10.sup.6 nm.sup.2 [0069] 1 average silicon
crystal: 40 nm.times.40 nm=1600 nm.sup.2
[0070] Assuming only 10.sup.3 crystals on one cell, the covering
ratio is 1600 nm.sup.2.times.10.sup.3/1600.times.10.sup.6
nm.sup.2=10.sup.-3; and the probe area is 160 mm.times.160 mm for
about 16 cells. If only one cell is cancerous, the effective
covering ratio is 10.sup.-3/16. The change in conductivity of each
crystal is of the order of 10.sup.4. Therefore one cell among 16
normal cells will induce 10.sup.4.times.10.sup.-3/16.about.0.5
change in conductivity averaged over the 16 cells area. Even if the
noise is of the order of 1000 times greater than the signal, by
using a lock-in method, as described more particularly below, the
signal is well in the detectable region.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The Embodiment of FIG. 1
[0071] The apparatus illustrated in FIG. 1 includes an
electro-optical probe, generally designated 10, having an operative
end 10a for applying optical pulses and voltage pulses to tissue 5
to be examined. The illustrated apparatus further includes a
voltage pulse source 11 and a laser source 12 coupled to the probe
10 for applying voltage pulses and optical (laser) pulses,
respectively, to the examined tissue 5. Probe 10 also detects
reflections of the voltage pulses and of the optical pulses from
the examined tissue 5.
[0072] The illustrated apparatus further includes a central data
processor system 13; an impedance measuring sub-system 14 coupled
to the probe for detecting the impedance of the examined tissue and
the change of impedance by the optical pulses; and an optical
analyzer sub-system 15 for detecting changes in the optical
characteristics of the examined tissue produced by the applied
optical pulses. As described above with reference to FIGS. 2a-2e,
and as will be described more particularly below, the detected
changes in impedance and in the optical characteristics of the
examined tissue 5, as a result of having been subjected to a
contrast agent containing small particles of a physical element
conjugated with an antibody selectively bindable to cancerous
cells, provides an indication of the extent of the presence of
cancerous cells in the examined tissue.
[0073] The apparatus illustrated in FIG. 1 further includes a
memory unit 15 for storing pre-prepared data, as well as data
produced during the operation of the apparatus. The apparatus
further includes a user interface 16 for producing a display output
and/or an audio output during the operation of the apparatus.
[0074] The impedance measuring sub-system 14 is preferably that
described in my above-cited U.S. application Ser. No. 10/035,428.
In such a system, the electro-optical probe 10 applies the
electrical pulses to the tissue 5 being examined such that the
probe generates an electrical fringe field in the examined tissue
and produces reflected pulses therefrom with negligible radiation
penetrating into neighboring tissues near the examined tissue. The
reflected electrical pulses are detected, and their electrical
characteristics are compared with respect to the applied electrical
pulses, to measure the impedance of the examined tissue relative to
the impedance of its neighborhood and by comparison to find
similarities to the pre-recorded data.
[0075] Such electrical property measurements, involving the
complete area of the examined tissue contacted by the operative end
of the probe 10, are capable of distinguishing between fat, muscle,
bone, cancerous tissue and other human tissue, and therefore can
serve to guide the surgeon on the way to the tumor and to define
the tumor margin with a typical accuracy of about 90%. The optical
beam produced by the laser 12 is applied to the center region of
the probe area, and is capable of detecting, with much better
accuracy, cancerous cells conjugated to the physical particles at
the central region.
[0076] Preferably, the small particles are nano/micro-particles in
size and are of a light-sensitive semiconductor, such as Silicon
Germanium or CdTe crystals, having a conductivity that
substantially decreases in the presence of light. While the use of
a light-sensitive semiconductor is particularly advantageous, as
described more particularly below, other materials can be used for
the nano/micro-particles conjugated with the antibody. For example,
nano/micro-particles of a metal, such as gold, having good
reflecting characteristics can be used, whereupon an optical
characteristic (e.g., amplitude, frequency or phase) of the
reflected light could also be detected and utilized for determining
the extent cancerous cells are present in the examined tissue. The
nano/micro-particles may also be of a fluorescent material, which
emits radiation of a predetermined frequency, such as fluorescent
dye Hoechst 33258 which emits light at 470 nm when exited at 360
nm, or a semiconducting-fluorescent material like CdSe and Cds
could be of a light absorption material which absorbs radiation of
a particular frequency, such as diamond nanoparticles which absorbs
radiation of from 200 to 700 nm, or CdSe and Cds. The emission
wavelength of a nanocrystal depends on its size, and therefore by
controlling its size, it is possible to tune the emission
wavelength. The excitation spectrum of nanocrystals is very broad.
This has the advantage that nanocrystals can be excited at many
wavelengths shorter than the emission peak. It further means that a
mixture of nanocrystals with different emission peaks may be
excited efficiently by light of a single wavelength. Therefore, the
numbers set forth above can be tuned and adjusted.
[0077] In the above cases, changes in the spectrum of the reflected
light would be detected and utilized for determining the extent
cancer cells are present in the examined tissue.
[0078] Another possible application of the invention would be to
use nano/micro-particles of a dielectric material, such as Iodine
(which has a large dielectric constant (about 120), and to apply
only voltage pulses, in which case the optical analyzer sub-system
could be omitted or not used, and only the impedance measuring
sub-system would be used for measuring the impedance of the
examined tissue.
The Apparatus of FIGS. 3-8
[0079] The apparatus of FIGS. 3-8 also includes an electro-optic
probe 20 having an operative end 20a for applying optical pulses
and voltage pulses to tissue 5 to be examined, and for detecting
both voltage reflections and optical reflections of such pulses in
order to determine the extent cancerous cells are present within
tissue 5. As indicated earlier, such an examination is made after
the tissue has been subjected to a contrast agent containing small
particles (nano/micro-particles) of a physical element conjugated
with an antibody selectively bindable to the cancerous cells.
[0080] The voltage pulses are applied from a voltage pulse source
30, and the optical pulses are applied by a laser 40 via an optical
chopper 41 and an optical fiber 42. The illustrated apparatus
further includes a data processor system, generally designated 50,
having an impedance measuring sub-system 60. Data processor system
50 further includes an optical analyzer sub-system, generally
indicated by the broken-lines 701 having a light splitting box 80.
Further illustrated in FIG. 3 is a user interface 90, which
includes a visual display as well as an audio output.
[0081] The electro optic probe 20 is more particularly illustrated
in FIG. 4. It is in the form of an elongated member adapted to be
grasped by the surgeon during a surgical operation for the removal
of a tumor, as shown in FIG. 10, with its operative end 20a brought
into direct contact with the tissue at the surgical site.
[0082] Probe 20 is preferably of a construction similar to the
electrical probe described in my above-cited patent application
Ser. No. 10/035,428, to include outer and inner coaxial conductors
21, 22, insulated from each other by a body of dielectric material
23, for generating an electrical fringe field in the examined
tissue as described in that patent application. Probe 20
illustrated in FIG. 4, however, further includes an optical fiber
24 extending centrally through the probe to conduct optical pulses
from laser 40 to the examined tissue in the central region of the
tissue area covered by the operating end 20a of the probe. As
schematically shown in FIG. 5, the outer conductor 21 is preferably
tapered at the operative end of the probe to provide a tapered tip
21a. it also extends slightly past the respective end of the inner
conductor 22, as well as of the central optical fiber 24, to define
an open cavity closed by the tissue 5 being examined.
[0083] The inner conductor 22 is preferably a silver or other metal
coating over the outer surface of the central optical fiber 24,
whereas the outer conductor 21 is preferably in the form a flexible
metal braid. At the operative end of the probe, the outer conductor
21 is enclosed by a rigid metal cap 25. The complete outer surface
of the outer conductor 21, including its cap 25, is preferably
covered by an insulating jacket 26.
[0084] As described above, the two coaxial conductors 21, 22 apply
voltage pulses from the voltage source 30 to the examined tissue 5,
whereas the central optical fiber 24 applies optical pulses from
the laser 40 to the central region of the probe area. The voltage
pulses produce voltage reflections, and the optical pulses produce
optical reflections, both of which are detected by the probe
20.
[0085] FIGS. 6a and 6b schematically illustrate the results
produced by such an examination with respect to tissue that has
been previously subjected to a contrast agent as described above,
namely one containing small particles (nano/micro-particles) of a
physical element, particularly a light-sensitive semiconductor such
as silicon, conjugated with an antibody selectively bindable to
cancerous cells. Thus, when the examined tissue contains cancerous
cells, the physical element nano/micro-particles are bonded to the
cells, according to the extent the cancerous cells are present in
the examined tissue, as shown in FIG. 6a; whereas examined tissue
not containing cancerous cells are relatively free of such physical
element nano/micro-particles, as shown in FIG. 6b.
[0086] As shown in FIG. 7, and also in FIG. 3, the inner and outer
conductors 21, 22 of the probe are connected by a connector 27,
e.g., an SMA connector, to the voltage pulse source 30. The central
optical fiber 24 is passed through an opening in the inner
conductor 22 and outer conductor 21 to its external extension 24'
which includes a connector 27, e.g., an SMA connector, for
connection to the laser source 40.
[0087] As shown in FIG. 3, the coaxial conductors 21, 22 of the
probe 20 are connected, via connector 26 and a transmission line
28, to the voltage pulse source 30, and to the impedance measuring
sub-system 60. The voltage pulse source 30, and the impedance
measuring sub-system 60, are basically the same as described in the
above-cited patent application Ser. No. 10/035,428, except that the
impedance measuring sub-system includes a lock-in amplifier
locked-in with chopper 41 of the laser source 40, to increase the
signal-to-noise ratio of the impedance measurement by measuring the
mutual optical-electrical effect (mode 3), as will be described
more particularly below.
[0088] The laser source 40 produces a sequence of femtosecond to
nanosecond pulses of viz-nir (Visible-Near Infra-Red) light (the
duration of the laser pulse is controlled by the CPU). Laser source
40 further includes an optical chopper 41 for chopping the train of
nanosecond pulses at a desired frequency or modulation. The
chopping modulation could be a constant modulation, for example
1000 HZ modulation, or other kind of modulation for example
information-like modulation. The chopper itself is a standard
mechanical chopping device (or an electro optic device) and is
mounted inside the laser source box 40. As one example, chopper 41
could be Model 360C OEM Ultra Miniature Optical Chopper made by
Sciatic Instruments Ltd. As seen in FIG. 3, the train of laser
pulses exiting from chopper 41 are transmitted, via an optical
fiber 42 and optical splitting box 80 of the optical analyzer
sub-system 70, to the external extension 24' of optical fiber 24
within the probe 20.
[0089] As further shown in FIG. 31 the optical analyzer sub-system
70 includes two spectrometers 71, 72, each coupled by a separate
connector 73, 74 to the optical splitting box 80. The optical
splitting box 80 is more particularly described below with respect
to FIG. 8. As further shown in FIG. 3, optical fiber 42 includes
another connector 75 connecting the chopped laser pulses from laser
source 40 to the optical splitting box 80 for transmission
therethrough to the optical fiber 24 within probe 20, via connector
27 and external extension 24' of the optical fiber.
[0090] FIG. 8 more particularly illustrates the optical splitting
box 80, including its connector 75 to the laser source 40, its
connector 27 to optical fiber 24 within probe 20, and its connector
73 and 74 to the two spectrometers 71, 72.
[0091] As shown in FIG. 8, the optical splitting box 80 includes a
beam splitter 81 which splits the beam of laser pulses from laser
40 and chopper 41 into two beams: a main beam, carrying most of the
laser energy, is directed to optical fiber 24 in the probe, via
connector 27 and the external extension 24' of the optical fiber;
whereas a secondary beam is directed to a polarizing cube 82.
Polarizing cube 82 splits the secondary beam into two differently
polarized beams, one being directed via connector 73 to
spectrometer 71, and the other being directed by mirror 83 and
connector 74 to spectrometer 72. As will be described below, this
arrangement including polarizing cube 82 and the two spectrometers
71, 72 measures the spectrum of the incident optical (laser) pulses
at each polarization separately.
[0092] The light reflected back from the examined tissue is
detected by optical fiber 24 and is directed, by its extension 24'
and connector 27, to the backside of beam splitter 81. This
reflected light is in turn reflected by beam splitter 81, via
mirrors 84, 85 and 86, to the polarizing cube 82. Cub 82 again
splits the reflected light according to two polarizations, one
being passed via connector 73 to spectrometer 71, and the other
being passed via mirror 83 and connector 74 to the other
spectrometer 72.
[0093] The two spectrometers 71, 72 thus measure the frequency
spectrum of both the incident optical pulses and reflected optical
pulses at each polarization. As will be described more particularly
below, this information is also utilized, in addition to the
impedance-measurement information, in determining the extent the
cancerous cells are present in the examined tissue.
[0094] Each spectrometer 71, 72 is a fiber optic spectrometer
equipped with computer software that allows real-time detection of
optical properties of the light directed into the spectrometer,
namely a portion of each incident optical pulse, and all of each
reflected pulse. As one example, each spectrometer may be Ocean
Optic pc2000 fiber optic spectrometer or Wavestarv750 made by
Ophir. The main spectrometer reading is the power spectrum of the
light (amplitude at each optical frequency). Both spectrometers are
preferably the same. FIGS. 9a, 9b illustrate typical outputs of the
two spectrometers 71, 72 during a typical examination
procedure.
[0095] The optical fiber 24 may be a commercial Silica core silica
clad fiber, optimized for the NIR-VIZ (near infrared, visible light
range of the electromagnetic spectrum). The outer diameter of the
fiber is preferably 45 .mu.m.
[0096] Following is one manner of making the electro-optical-probe
20.
[0097] A length of the commercially available optical fiber 24 is
coated with a silver layer to define the inner conductor 22, is
then covered with a thick dielectric layer, preferably Teflon.TM.,
and is inserted into the flexible metal braid serving as the outer
conductor 21 of the coaxial cable so produced. A metal rigid cap 25
is then applied at one of the coaxial cable, constituting the
operative end of the probe to be produced, and a thin layer of a
polymer is then applied over the coaxial cable to serve as the
outer jacket 26.
[0098] In view of the flexible nature of the probe, the metal cap
facilitates grasping and moving the probe by the surgeon. As one
example, the metal cap may be a gold-coated aluminum cylinder of 8
mm in length.
[0099] The optical fiber 24 is split into two portions at the
opposite end of the probe. One portion coated with the inner
conductor 22 continues to the opposite end of the probe where it is
connected, together with the outer conductor 21, to connector 26
for connection, via transmission line 28 (FIG. 3), to the voltage
pulse source 30 as well as to the impedance measuring sub-system
60. The other part of optical fiber 24 is passed through an opening
in the outer conductor braid 21, and serves as the external
extension 24' of the probe and carries connector 27 for connection,
via the optical splitting box 80 described above, to the laser
source 40.
[0100] Transmission line 28, illustrated in FIG. 3, may be a
standard coaxial cable such as the RG-174, with the inner conductor
constituted of a silver coating applied to the outer surface of the
optical fiber 24.
[0101] The impedance-measuring method performed by the impedance
measuring sub-system 60 in FIG. 3 is preferably basically the same
as in my above-cited U.S. patent application Ser. No. 10/035,428,
the contents of which are incorporated herein by reference. In this
case, however, the impedance measurement is affected by the type of
nano/micro-particles selectively bonded to the cancerous cells of
the examined tissue, and the extent to which such
nano/micro-particles have been bonded. The latter factors thereby
provide an indication of the extent of the cancerous cells present
in the examined tissue.
[0102] For example, if the nano/micro-particles are merely
particles of dielectric material, then the extent to which they
have become bonded to the examined tissue will merely affect the
impedance of the examined tissue. However, if such
nano/micro-particles are light-sensitive such that their impedance
is of one value in the absence of light and another value in the
presence of light, then the presence of light pulses from the laser
40 will also affect the impedance of the examined tissue. Therefore
the light can modulate the impedance of the tissue. The ability to
modulate the measured impedance, only of cancerous tissue, is what
make this mode so powerful. In the preferred embodiment of the
invention, the nano/micro-particles are preferably of a
semi-conductor crystal, e.g., silicon, exhibiting a very
substantial increase in electrical conductivity when exposed to
light.
The Lock-In Method
[0103] As indicated earlier, laser source 40 includes a chopper 41,
and the impedance measuring sub-system 60 includes a lock-in
amplifier. These features enable a lock-in examination to be
performed to increase the sensitivity of the measurement
particularly with respect to background noise. Thus, the lock-in
measurement method can provide high-resolution measurements
producing relatively clean signal outputs having a high
signal-to-noise ratio over several orders of magnitude and
frequency.
[0104] A lock-in amplifier, commonly used with most AC indicating
instruments, provides a DC output proportional to the AC signal
under investigation. In practice, the DC output may be presented as
a reading on a digital panel meter or as a digital value
communicated over a computer interface, rather than a voltage at an
output connector, but the principle remains the same. A special
rectifier, called a phase-sensitive detector (PSD), which performs
this AC to DC conversion, forms the heart of the instrument. It is
special in that it rectifies only the signal of interest, while
suppressing the effect of noise or interfering components, which
may accompany that signal.
[0105] The traditional rectifier in a typical AC voltmeter makes no
distinction between signal and noise and produces errors due to
rectified noise components. The noise at the input to a lock-in
amplifier, however, is not rectified but appears at the output as
an AC fluctuation. This means that the desired signal response, now
a DC level, can be separated from the noise accompanying it in the
output by means of a simple low-pass filter. Hence in a lock-in
amplifier, the final output is not affected by the presence of
noise in the applied signal.
[0106] In order to function correctly, the detector must be
"programmed" to recognize the signal of interest. This is achieved
by supplying it with a reference voltage of the same frequency and
with a fixed phase relationship to that of the signal. This is most
commonly done by ensuring that they are derived from the same
source. The use of the reference frequency ensures that only
signals at the reference frequency will be measured. In our case
the reference frequency is given by the optical modulation
frequency so the impedance changes in the tissue are in the
reference frequency while all other "noises" are not in that
frequency. This inherent tracking ability allows extremely small
bandwidths to be defined for the purpose of signal-to-noise ratio
improvement since there is no frequency "drift", as is the case
with analog "tuned filter/rectifier" systems. Because of the
automatic tracking, lock-in amplifiers can give effective "Q"
values (a measure of filter selectivity) in excess of 100,000,
whereas a normal band pass filter becomes difficult to use with Q's
greater than 50.
[0107] The following mathematical description of a typical lock-in
operation will aid in understanding this measurement method.
[0108] Consider the case where a noise-free sinusoidal signal
voltage Vin is being detected, where Vin=A cos(.omega.t).omega. is
the angular frequency of the signal which is related to the
frequency, F, in Hertz by the equality: .about..omega.=2.pi.F.
[0109] The lock-in amplifier is supplied with a reference signal at
frequency F derived from the same source as the signal, and uses
this to generate an internal reference signal of: -Vref=B
cos(.omega.t+q). The reference frequency is the modulation
frequency of the optical chopper, where q is a user-adjustable
phase-shift introduced within the lock-in amplifier.
[0110] The detection process consists of multiplying these two
components together so that the PSD output voltage is given:
by : - V psd = A cos ( .omega. t ) B cos ( .omega. t + q ) = 1 / 2
AB cos .theta. + 1 / 2 Ab cos ( 2 .omega. t + q ) ##EQU00001##
[0111] If the magnitude, B, of the reference frequency is kept
constant, then the output from the phase-sensitive detector is a DC
signal which is:
[0112] Proportional to the magnitude of the input signal A
[0113] Proportional to the cosine of the angle, q, between it
and
The Reference Signal
[0114] Modulated at 2.omega.t, i.e., it contains components at
twice the Reference frequency.
[0115] The output from the PSD then passes to a low-pass filter
which removes the 2.omega.t component, leaving the output of the
lock-in amplifier as the required DC signal.
[0116] In a practical situation the signal will usually be
accompanied by noise, but it can be shown that as long as there is
no consistent phase (and therefore by implication frequency)
relationship between the noise and the signal, the output of the
multiplier due to the noise voltages will not be steady and can
therefore be removed by the output filter that integrates the
signal over a time interval. The out of phase components which
contains the noise will be summed towards zero, while the in phase
signal will be summed.
[0117] Thus, a condition in the use of the lock-in measurement
method is that the detected signal should be modulated at a
reference frequency while all other signals from the surrounding
environment should not be modulated. This condition is satisfied in
above described method and apparatus by providing modulation to the
physical element conjugated to the antibody. In all other respects,
the lock-in method used herein is the same as in traditional
lock-in measurements.
[0118] A preferred amplifier in the impedance measuring sub-system
60 is the commercial Lock-in Model SRS850.
EXAMINATION PROCEDURE
[0119] FIG. 10 illustrates a typical examination procedure for
examining a patient in order to determine the extent of the
presence of cancerous cells in examined tissue during a surgical
operation for removing a tumor.
[0120] Before the surgical operation, the contrast agent is
prepared and injected into the patient's bloodstream. As described
above, the contrast agent includes a tumor specific antibody
conjugated to nano/micro-particles of a physical element having a
characteristic which affects the impedance measurements of the
examined tissue, and preferably also of the optical measurements of
the examined tissue.
[0121] The present invention integrate synergistically two
modalities in the same device: (1) local electrical measurement,
and (2) optical detection. Both modalities can work independently
to detect the existence of cancerous tissue, or together, in
conjunction with a pre-injected contrast agent to detect, with high
accuracy the cancerous cells. The contrast agent consists of
antibodies conjugated to small particles, or nano/micro-particles,
of physical elements. The synergetic combination of the two
detection technologies allows the following three detection Modes
of operation, depending on the material used for the
nano/micro-particles and one cell destruction mode (Mode 4): [0122]
1. Electrical measurement only; [0123] 2. Light reflection
measurement only; [0124] 3. Light induced impedance change
measurement, preferably, also with light-reflection
measurement.
[0125] Thus, if the nano/micro-crystals are merely non-switchable
material like a dielectric or a conductor material, such as Iodine
or gold, the extent of their presence in the examined tissue would
affect the impedance of the examined tissue, in which case merely
an impedance measurement may be made of the examined tissue to
provide an indication of the extent cancerous cells are present
therein. In the case of iodine, the measurement will detect its
impedance because of the high dielectric constant of the iodine. In
the case of gold, the measurement will detect the very low
conductivity of the gold particles. Such an examination may be
called a Mode 1 examination.
[0126] On the other hand, if the nano/micro-particles are of a
light-reflecting material, such as nano/micro-particles of gold or
of a light fluorescent material, such as CdSe, Cds, or of a light
absorptive material such as diamond nano particles, an optical
examination and analysis of the light detected from the examined
tissue would also provide an indication of the extent cancerous
cells are present in the examined tissue. Such an examination may
be called a Mode 2 examination.
[0127] Preferably, however, the nano/micro-particles are light
sensitive, such as Silicon, Germanium, ZnSe, ZnS or GaAs crystals,
which substantially affect the impedance of the examined tissue
when irradiated with light (e.g., laser pulses). When such
materials are used as the nano/micro-particles, a Mode 3 type
examination may be performed. In this mode any sequence of light
pulses will induce a similar sequence of impedance-change pulses.
Since the light pulses are controllable, the changes in the
impedance pulses can be expected. This knowledge regarding the
impedance modulation is used to improve the signal-to-noise ratio
of the system.
[0128] While the first two modes can be measured, in principle,
sequentially by two different devices, the third mode is a unique
one and can be realized only with a device that combines the 1 and
2 modalities. The ability to control cells impedance using light,
switching on and off, make this mode an enabling tool for
ultra-high accuracy tumor cells detection.
[0129] It will be appreciated that the contrast agent could include
just one of the above-type nano/micro-particles conjugated with the
tumor-specific antibody, or could include a plurality of such types
of nano/micro-particles, to permit more than one of the above
mode-type examination to be performed on the tissue. Furthermore a
few different types of nanocrystals can be attached together to
form a microsphere.
[0130] Accordingly, during the course of a surgical operation for
the removal of a tumor, the surgeon may move the probe 20 over
different tissues in order to examine such tissue to determine the
extent of cancerous cells therein, and thereby to decide whether
such tissue should be considered as cancerous tissue to be removed,
or healthy tissue not to be removed. As the surgeon moves probe 20
to different tissues of the patient's body, the data processor
system housed within the user interface unit 90 shown in FIG. 10
examines the tissue and provides a visual display and/or audio
signals corresponding to the results of the examination of the
specific tissue. For example, the tissue type and dielectric
properties may displayed on the user interface 90 such that when a
cancerous cell is detected, the surgeon hears a sharp tone from the
user interface.
[0131] The broad determination of tissue type by electrical
properties measurement, and the accurate detection of cancerous
cells by physical element detection, gives the surgeon excellent
background knowledge for decision-making during the operation as to
the delimitation of the cancerous tissue. Using that knowledge, the
surgeon can manipulate the surgical cutting tool to maximizing the
removal of cancerous tissue and minimize the removal of healthy,
non-cancerous tissue.
[0132] In the embodiment of the invention described below, the
nano/micro-particles are of a light-sensitive semi-conductor, such
as silicon, which drastically changes in impedance when exposed to
light immediately after absorbing the light. In addition, both
optical (laser) pulses and voltage pulses are applied together to
the tissue being examined. This permits first a Mode 1 examination,
and then a Mode 2 and Mode 3 examination to be performed.
[0133] Thus a Mode 1 examination, involving merely measuring the
impedance of the tissue, can be performed by the application only
of the voltage pulses from voltage source 30 (FIG. 3), since such
an impedance measurement has an excellent ability of distinguishing
between fat, muscle, bone and other human tissues and to define
tumor margins in an accuracy scale of about 200 cells. Such an
impedance measuring mode may be that described in the above-cited
patent application Ser. No. 10/035,428. As described therein, the
voltage pulse source sends a pulse via transmission line 28 to the
probe 20 which applies the voltage pulses to the examined tissue,
detects the impedance of the examined tissue, and utilizes the
detected changes in impedance for determining the extent target
cells are present in the examined tissue, thereby distinguishing
the type of tissue examined.
[0134] As more particularly described in that patent application,
the probe generates an electrical fringe field in the examined
tissue and produces reflected pulses therefrom. The reflected
electrical pulses are detected and compared with respect to the
applied electrical pulses to provide an indication of the
dielectric properties of the examined tissue, and thereby of the
type of tissue then contacted by the probe. The voltage pulses are
measured by a high-speed digitizer, and are then transformed to the
frequency domain mathematically. Since the present invention
preferably includes, but does not require, the specific biological
tissue impedance measuring technique in that patent application,
further details of that biological tissue impedance measuring
system and method are not set forth herein.
[0135] When the probe 20 is determined to be within the area of the
tumor site, the apparatus is switched-over to a Mode 2 and 3
operation in order to more accurately determined the extent of the
target cells present in the examined tissue. During this Mode 3
operation, the laser source 40 is actuated to produce a train of
laser pulses which are applied to the examined tissue via the
optical fiber 24 within probe 20 concurrently with the application
of the voltage pulses applied by voltage pulse source 30 to the
examined tissue via the coaxial conductors 21, 22 of the probe 20.
In this mode, the laser source 40 produces a train of nanosecond
pulses of small viz-nir (visible-near infra-red) light via optical
chopper 41 which modulates the frequency of the optical pulses in
order to produce a more precise indication of the extent target
cells are present in the examined tissue.
[0136] Thus during the Mode 3 operation, the electrical impedance
measurement is set to detect impedance changes that are at the same
frequency as the optical pulse train modulation frequency of
chopper 41. For example: if the repetition rate of the electrical
pulses is 500 KHz and the repetition of the laser pulse is 2 KHz
with modulation of 500 Hz, the impedance measurement is set to
detect changes in the 500 Hz band. In this mode, the digitizing
process is as in Mode 1, e.g., as described in the above-cited
patent application Ser. No. 10/035,428, but the system no longer
compares pair of electrical voltage pulses (incident and
reflected). Instead it looks for the changes in reflected pulse
amplitude only at the modulation frequency as described above in
the description of the lock-in method.
[0137] This feature produces a new important advantage: If the
probe does not point on cancerous cells, no electrical impedance
changes will be detected; but if the probe points on cancerous
cells containing the attached nano/micro-particles of
light-sensitive material, a substantial change of impedance is
induced. Since the detection method looks for changes at the
modulation frequency, its sensitivity increases dramatically such
that even one cancerous cell will induce a total change in the
detected impedance.
[0138] Thus, as described above, the lock-in method utilizing the
lock-in amplifier in the impedance measuring sub-system 60 of FIG.
3 utilizes the signal generated by the impedance changes at the
examined tissue by a sinus function at the modulation frequency and
integrates this signal over a time interval. This known lock-in
measurement technique reduces the noise many orders of magnitude
since the noise is not modulated and therefore its integral is
summed towards zero.
[0139] The change of reflected amplitude at each measurement point
is saved in the memory (e.g., 15, FIG. 1) and is sent to the
analysis program.
[0140] The analysis program during the Mode 1 operation (i.e.,
wherein only voltage pulses are applied to the examined tissue), is
the same as described in the above-cited patent application Ser.
No. 10/035,428. As indicated earlier, the Mode 1 operation is
capable of detecting changes in impedance of the examined tissue
sufficiently for determining the type of tissue sufficient to
locate the tumor and to remove it. However, in order to determine
whether tissue adjacent to the removed tissue may also be cancerous
and should therefore be removed, a Mode 2 and Mode 3 examination is
performed to detect changes in the electrical characteristics and
in the optical characteristics of the examined tissue, and to
utilize such detected characteristics for more accurately
determining the presence of cancerous cells in the examined
tissue.
[0141] In Modes 2 and 3, the analysis program looks for different
features in order to detect cancerous cells.
[0142] Thus, the analysis checks the data array and looks for
local, same point, changes of impedance during the light
activation. If a change exists, it is due the existence of the
physical element nano/micro-particles, and therefore confirms
cancer cell presence in the tissue under the tip of the probe. The
surgeon thus receives a clear indication of cancerous cell, e.g.,
by an audio output from the user interface unit 90.
[0143] The analysis also compares the light reflection from each
tissue point with respect to that from the previous tissue point.
It is also looks for a distinct features of light reflection due
the existence of the physical elements attached to the cancerous
cells; absorption at certain frequency, emission at certain
frequency (florescence), a change of polarization as a function of
frequency, and total change at reflection (average) of the
reflection over the full spectrum. When the analysis finds a
parameter that is a unique for the physical element in the contrast
agent, this provides a clear indication of a cancerous cell.
[0144] The measurement of changes in impedance during the Mode 3
examination is performed by the impedance-measuring sub-system 60
of FIG. 3. As described above, sub-system 60 includes a digitizing
unit and a lock-in amplifier for producing a measurement which is
characterized by a high signal-to-noise ratio.
[0145] The measurement of changes in the optical characteristics of
the examined tissue, according to the Mode 2 examination, is
performed by the optical analyzer sub-system 70 in FIG. 3,
including the optical splitting box 80, its polarizing cube 82, and
the two spectrometers 71 and 72.
[0146] As briefly described above, and as more particularly
illustrated in FIG. 8, the modulated laser beam from the optical
chopper 41 is split by beam splitter 81 in the splitting box 80
into a main beam which carries most of the optical energy to the
tissue being examined, and a secondary beam to the two
spectrometers 71, 72, via the polarizing cube 82 which produces two
polarizations of the beams. This is done in order to measure the
pulse spectrum of the incident optical beam and of the reflected
optical beam at each polarization separately. Such information
enables an analysis to be made of the changes in the optical
characteristics of the examined tissue produced by the
nano/micro-particles therein, which changes in optical
characteristics may be utilized particularly with the changes in
the electrical properties characteristics measured by sub-system
60, to provide a relatively accurate determination of whether any
cancerous cells are present in the examined tissue.
[0147] FIGS. 9a and 9b show how the polarization function is
calculated from the readings of the two spectrometers 71, 72. Each
spectrometer digitizes the power amplitude of light at each
frequency. The light directed to each of the spectrometers is of a
different polarity. Accordingly, at certain frequencies the
relation between the amplitudes of the two spectrometers gives the
polarity. The relation calculation is repeated at all measured
frequencies, and the polarization function is measured. This is
done for the incident pulse. For the reflected pulse, the change in
polarities is again calculated and recorded.
[0148] Since the apparatus, when operated as described above, has
detected cancerous cells with a relatively high accuracy, the
apparatus may now be operated according to a fourth mode (Mode 4)
for destroying the detected cells by ablation without the need for
aiming it again since it is already in the place. This may be done
automatically when detecting cancerous cells, or on demand, by
activating the laser to transmit high-power short duration pulses
at the detected spot of the examined tissue. The ablation pulses
should be of femtosecond pulse duration with energy per pulse of
about 100 nj up to about 1 mj.
Modifications in the Probe Structure
[0149] FIG. 11 schematically illustrates a possible modification in
the construction of the electro-optic probe, therein generally
designated 120, for use in examining tissue 105. Probe 120 includes
two capacitor plates 121, 122, mounted on a dielectric mounting 123
and connected to a voltage source 130 for applying the voltage
pulses to the examined tissue. Probe 120 further includes an
optical fiber 124 received within an opening of the dielectric
mounting plate 123 between the two capacitor plates 121, 122, and
connected to a light source 140, such as a laser, for applying the
optical pulses to the examined tissue. Probe 120 may be used in
apparatus constructed and operating as described above.
[0150] FIG. 12 illustrates another variation wherein the probe,
therein generally designated 220, includes an outer conductor 221
and an inner conductor 222 in the form of a metal coating on an
optical fiber 223. The outer conductor 221 is closed by an end wall
221a at the operative end of the probe, which end wall is formed
with an opening 221b for receiving the tissue 205 to be
examined.
[0151] Probe 220 illustrated in FIG. 12 is otherwise the same as
described above, including an electrical pulse source 230 connected
to the two conductors 221, 222 for applying voltage pulses to the
tissue, and a laser source 240 for applying optical pulses via
optical fiber 223 to the tissue.
[0152] FIG. 13a illustrates the probe, therein generally designated
320, incorporated in a biopsy needle 300 having a needle cavity
302. Such a biopsy needle may be about 2 mm in diameter. As shown
in FIG. 13a, the probe 320 is located within the needle with the
operative end 320a of the probe facing the cavity 302 for examining
the biopsy specimen obtained by the needle.
[0153] FIG. 13b illustrates a biopsy needle 400 including a side
cavity 402 and a front sharp edge 404. As shown in FIG. 13b, the
probe 420 is located within the biopsy needle such that the
operative 420a of the probe is in front of the sharp front edge
404.
[0154] Probe 20 illustrated in FIG. 10 is constructed so as to be
manually graspable and manipulatable with respect to the tissue to
be examined. Such a probe, however, can be constructed so as to be
of a size and of sufficient flexibility to enable it to be
introduced into the body of a subject via a catheter for examining
selected tissue in accordance with the above-described Modes 1, 2
or 3, and/or for ablating selected tissue in order to destroy
detected cancerous cells as described above with respect to Mode
4.
[0155] In addition, the probe could equipped with a 3-D coordinate
orientation sensor for determining the location and orientation of
the operative end of the probe, and thereby of the tissue being
examined. FIG. 14 illustrates such a probe, therein generally
designated 500, including an orientation sensor in the form of a
rectangular array or matrix of optical sensor elements 520 at the
back end of the probe and at a known location with respect to the
operative end 500a of the probe. FIG. 14a more particularly
illustrates the back end of the probe 520 carrying the matrix of
optical sensor elements 530, and FIG. 14b schematically illustrates
the matrix of the optical sensor elements. It will be appreciated
that any 3-D orientation sensor construction and measuring system
could be used with this application of the invention.
[0156] It will also be appreciated that the sensor array 530 could
all be used for imagining the examined tissue 505 and for
displaying its image in a real-time manner during the course of the
surgical operation.
[0157] Another variation would be to provide the probe with a
thimbal-type mounting device for mounting it on the surgeon's
fingertip. Further, while the embodiments described above included
only one pair of electrodes for applying voltage pulses, the probe
could include a plurality of such pairs, e.g., in the form of
micro-electrodes, at the operative end of the probe.
[0158] While the invention has been described with respect to
several preferred embodiments, it will be appreciated that these
are set forth merely for purposes of example, and that many
variations may be made. For example, the apparatus could be used
for targeting other cells, such as bacteria, etc., which are
selectively bindable to an antibody conjugated with the
nano/micro-particles. In addition, the apparatus and method could
be used for examining cells off-line, e.g., in examining
pre-operation biopsies or post-operation samples of removed tissue.
Further, while the above selective-bonding technique is described
above with respect to using antibodies, it will be appreciated that
other biological carriers could be used for this purpose instead of
antibodies. Other electrical-characteristic measuring techniques
and/or light-measuring techniques may be used. Many other
variations and applications of the invention will be apparent.
* * * * *