U.S. patent application number 11/204196 was filed with the patent office on 2006-02-16 for raman chemical imaging of breast tissue.
Invention is credited to Matthew P. Nelson, Patrick J. Treado.
Application Number | 20060036181 11/204196 |
Document ID | / |
Family ID | 26880786 |
Filed Date | 2006-02-16 |
United States Patent
Application |
20060036181 |
Kind Code |
A1 |
Treado; Patrick J. ; et
al. |
February 16, 2006 |
Raman chemical imaging of breast tissue
Abstract
Apparatus and methods for spatially resolved Raman chemical
imaging of breast tissue is disclosed. A region of breast tissue is
illuminated with monochromatic light. A spatially organized area of
endogenous molecules in the tissue is then detected in the region
by detecting a Raman shifted light signal. The Raman shifted light
signal is spatially resolved in at least one direction and is thus
useful for examining breast tissue, especially to detect malignant
tissue.
Inventors: |
Treado; Patrick J.;
(Pittsburgh, PA) ; Nelson; Matthew P.;
(Pittsburgh, PA) |
Correspondence
Address: |
M. LISA WILSON;DUANE MORRIS LLP
380 LEXINGTON AVENUE
NEW YORK
NY
10168-0002
US
|
Family ID: |
26880786 |
Appl. No.: |
11/204196 |
Filed: |
August 9, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10184580 |
Jun 28, 2002 |
6965793 |
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11204196 |
Aug 9, 2005 |
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10185590 |
Jun 27, 2002 |
6729265 |
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11204196 |
Aug 9, 2005 |
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60301708 |
Jun 28, 2001 |
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60301708 |
Jun 28, 2001 |
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Current U.S.
Class: |
600/476 ;
356/301; 356/303; 600/310; 600/318; 600/473; 600/477; 606/10;
606/2; 606/3 |
Current CPC
Class: |
A61B 5/0091 20130101;
G01N 21/65 20130101; A61B 5/4312 20130101 |
Class at
Publication: |
600/476 ;
600/473; 600/477; 600/310; 600/318; 606/002; 606/003; 606/010;
356/301; 356/303 |
International
Class: |
A61B 6/00 20060101
A61B006/00; A61B 18/18 20060101 A61B018/18; G01J 3/44 20060101
G01J003/44; G01J 3/40 20060101 G01J003/40; A61B 5/00 20060101
A61B005/00 |
Claims
1. A method for detecting the distribution of one or more molecular
species within a breast lesion which comprises a) illuminating a
region of breast tissue with monochromatic light; b) detecting
Raman shifted light from the one or more molecular species in the
region with a Raman chemical imaging spectrophotometer; and c)
spatially resolving the Raman shifted light signal in at least one
direction to produce a Raman chemical image indicative of the
molecular species within said lesion in said at least one
direction.
2. The method of claim 1, wherein said one or more molecular
species is lipid, carbohydrate and/or protein.
3. The method of claim 1, wherein said breast lesion is a benign
tumor.
4. The method of claim 1, wherein said breast lesion is a malignant
tumor.
5. The method of claim 1, wherein said breast lesion is a pocket of
infection or inflammation.
6. The method of claim 1, wherein a two-dimensional chemical image
of the region is produced.
7. The method of claim 6, wherein a series of two-dimensional
chemical images are taken as a function of depth of the tissue.
8. A method for determining the morphology of a lesion in breast
tissue which comprises a) illuminating a region of breast tissue
containing said lesion with monochromatic light; b) detecting Raman
shifted light from endogenous molecules in the region with a Raman
chemical imaging spectrophotometer; and c) spatially resolving the
Raman shifted light signal to produce a Raman chemical image
indicative of the morphology of said lesion in said tissue in at
least two dimensions.
9. The method of claim 8, wherein a series of two-dimensional
chemical images are taken as a function of depth of the tissue.
10. The method of claim 9, wherein said series of images are
subject to morphometric analysis to thereby characterize the size
and shape of said lesion.
11. The method of any one of claims 1-10 wherein said molecular
species or said molecules are selected from the group consisting of
indoles, sulforaphanes, carotenoids, proteoglycans and
flavonoids.
12. The method of claim 11, where said molecular species or said
molecules are carotenoids.
13. A method for detecting a malignant breast tissue which
comprises a) illuminating a region of breast tissue with
monochromatic light; b) detecting Raman shifted light from
endogenous molecules in the region with a Raman chemical imaging
spectrophotometer; and c) spatially resolving the Raman shifted
light signal in at least one direction to produce a Raman chemical
image (RCI) in said at least one direction, d) ascertaining whether
said breast tissue has an RCI characteristic of malignant breast
tissue to thereby identify malignant breast tissue.
14. The method of claim 13 wherein said RCI discriminates between
benign and malignant tumors.
15. The method of claim 13, wherein a two-dimensional chemical
image of the region is produced.
16. The method of claim 15, wherein a series of two-dimensional
chemical images are taken as a function of depth of the tissue.
17. The method of any one of claims 13-16, wherein the molecules
are carotenoid molecules.
18. The method of any one of claims 1, 8 or 13, wherein the region
is prepared for illumination, in a step previous to step a), by
excision of the tissue and by placing a specimen prepared from the
tissue in position for illumination and imaging, wherein the tissue
is not treated with staining agents.
19. The method of any one of claims 1, 8 or 13, wherein the region
is prepared for illumination, in a step previous to step a), by
excision of the tissue and by placing a specimen prepared from the
tissue in position for illumination and imaging, wherein the tissue
is treated with staining agents.
20. The method of any one of claims 1, 8 or 13, wherein said region
is illuminated in vivo with monochromatic light introduced via an
endoscope or a fiberscope.
21. The method of claim 20, wherein a two-dimensional chemical
image of the region is produced.
22. The method of claim 20, where said molecular species or said
molecules are selected from the group consisting of indoles,
sulforaphanes, carotenoids, proteoglycans and flavonoids.
23. The method of claim 22, where said molecular species or said
molecules are carotenoids.
24. The method of claim 20, where a non-imaging endoscope or
fiberscope is moved through the tissue in vivo, and the Raman
shifted light signal is spatially resolved as the endoscope or
fiberscope is moved through the tissue.
25. The method of claim 20, where the Raman shifted light from the
region passes through an Evan's split element liquid crystal
tunable filter.
26. The method of claim 20, wherein the endoscope or fiberscope is
moved through the tissue to obtain a Raman chemical image from more
than one region of said tissue.
27. The method of claim 26, wherein a two-dimensional chemical
image is produced for each region of said tissue.
28. The method of claim 27, wherein said two-dimensional chemical
images represent a series of images taken as a function of depth of
the tissue.
29. The method of claim 28, wherein said method is for determining
the morphology of a lesion and said series of images are subject to
morphometric analysis to thereby characterize the size and shape of
said lesion.
30. A method of performing an optical biopsy which comprises a)
illuminating multiple regions of breast tissue in vivo with
monochromatic light introduced via an endoscope or a fiberscope; b)
detecting Raman shifted light from one or more molecular species in
the region with a Raman chemical imaging spectrophotometer; c)
spatially resolving the Raman shifted light signal in two
dimensions for each region to produce a Raman chemical image of
said tissue in two dimensions; and d) determining the location of
any lesion in said tissue.
31. The method of claim 30, wherein said multiple regions are in
series as a function of depth of the tissue.
32. A biopsy method which comprises a) obtaining the Raman chemical
image of breast tissue according to the method of claim 31; and b)
performing a biopsy of tissue in or around said lesion.
33. The method of claim 32, wherein said biopsy is a non-surgical
biopsy.
34. The method of claim 33, wherein said non-surgical biopsy is a
needle core biopsy or a fine needle biopsy.
35. The method of claim 32, wherein said biopsy is an excisional
biopsy.
36. The method of any one of claims 32-35 which further comprises
examining said biopsied tissue for signs of cancer.
37. The method of any one of claims 30-32, wherein said one or more
molecular species is an endogenous molecule indicative of a border
between normal tissue and a lesion.
38. The method of any one of claims 30-32, wherein said one or more
molecular species is hydroxyapatite.
39. The method of any one of claims 30-32, wherein said Raman
chemical image is of calcification in said tissue.
40. The method of any one of claims 1, 8 or 13, wherein the Raman
shifted light from the region passes through a FAST fiber array
spectral translator.
41. The method of any one of claims 1, 8 or 13, wherein the Raman
shifted light from the region passes through a filter selected from
the group consisting of a Fabry Perot tunable filter, an
acousto-optic tunable filter, a liquid crystal tunable filter, a
Lyot filter and an Evan's split element liquid crystal tunable
filter.
42. The method of any one of claims 1, 8 or 13, wherein the Raman
shifted light from the region passes through a
polarization-independent imaging interferometer.
43. The method of claim 42, wherein the Raman shifted light from
the region passes through an interferometer selected from the group
consisting of a Michelson interferometer, a Sagnac interferometer,
a Twynam-Green interferometer and a Mach-Zelmder interferometer.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
10/184,580, filed Jun. 28, 2002 and a continuation of U.S. Ser. No.
10/185,090, filed Jun. 28, 2002, both of which claim priority
pursuant to 35 U.S.C. 119(e) to U.S. Provisional Application No.
60/301,708, filed Jun. 28, 2001, each of which is incorporated
herein by reference in its entirety including incorporated
material.
FIELD OF THE INVENTION
[0002] The field of the invention is the field of tissue evaluation
and, more particularly, the field of tissue evaluation using
optical detection of light which has been Raman shifted in
frequency.
BACKGROUND OF THE INVENTION
[0003] Cancer is the second leading cause of death in the United
States and over 1.2 million people are diagnosed with this disease
annually. Cancer is significant, not only in lives lost, but also
in the $107 billion cost to the United States economy in 2000
according to the National Institutes of Health. It is widely
recognized among the cancer research community, that there is a
need to develop new tools to characterize normal, precancerous,
cancerous, and metastatic cells and tissues at a molecular level.
These tools are needed to help expand our understanding of the
biological basis of cancers. Molecular analysis of tissue changes
in cancer improve the quality and effectiveness of cancer detection
and diagnosis strategies. The knowledge gained through such
molecular analyses helps identify new targets for therapeutic and
preventative agents.
[0004] Diagnosis of cancer is the first critical step to cancer
treatment. Included in the diagnosis is the type and grade of
cancer and the stage of progression. This information drives
treatment selection. When cancer is suspected, a patient will have
the tumor removed or biopsied and sent for histopathology analyses.
Conventional handling involves the tissue undergoing fixation,
staining with dyes, mounting and then examination under a
microscope for analysis. Typically, the time taken to prepare the
specimen is of the order of one day. The pathologist will view the
sample and classify the tissue as malignant or benign based on the
shape, color and other cell and tissue characteristics. The result
of this manual analysis depends on the choice of stain, the quality
of the tissue processing and staining, and ultimately on the
quality of education, experience and expertise of the specific
pathologist.
[0005] Early definitive detection and classification of cancerous
growths is often crucial to successful treatment of this disease.
Currently, several biopsy techniques are used as diagnostic methods
after cancerous lesions are identified. In the case of breast
cancer, lesions are typically identified with mammography or self
breast exam. The most reliable method of diagnosis is examination
of macroscopic-sized lesions. Macroanalysis is performed in
conjunction with microscopic evaluation of paraffin-embedded
biopsied tissue which is thin-sectioned to reveal microscale
morphology.
[0006] The detection and diagnosis of cancer is typically
accomplished through the use of optical microscopy A tissue biopsy
is obtained from a patient and that tissue is sectioned and
stained. The prepared tissue is then analyzed by a trained
pathologist who can differentiate between normal, malignant and
benign tissue based on tissue morphology. Because of the tissue
preparation required, this process is relatively slow. Moreover,
the differentiation made by the pathologist is based on subtle
morphological differences between normal, malignant and benign
tissue based on tissue morphology. For this reason, there is a need
for an imaging device that can rapidly and quantitively diagnose
malignant and benign tissue.
[0007] Alternatives to traditional surgical biopsy include fine
needle aspiration cytology and needle biopsy. These non-surgical
techniques are becoming more prevalent as breast cancer diagnostic
techniques because they are less invasive than biopsy techniques
that harvest relatively large tissue masses. Fine needle aspiration
cytology has the advantage of being a rapid, minimally invasive,
non-surgical technique that retrieves isolated cells that are often
adequate for evaluation of disease state. However, in fine needle
biopsies intact breast tissue morphology is disrupted often leaving
only cellular structure for analysis which is often less revealing
of disease state. In contrast, needle biopsies use a much larger
gauge needle which retrieve intact tissue samples that are better
suited to morphology analysis. However, needle biopsies necessitate
an outpatient surgical procedure and the resulting needle core
sample must be embedded or frozen prior to analysis.
[0008] A variety of "optical biopsy" techniques have potential as
non-invasive, highly sensitive approaches that will augment, or
even be alternatives to current diagnostic methods for early
detection of breast cancer. "Optical biopsies" employ optical
spectroscopy to non-invasively probe suspect tissue regions in
situ, without extensive sample preparation. Information is provided
by the resultant spectroscopically unique signatures that may allow
differentiation of normal and abnormal tissues. Despite years of
research and development, two techniques that have not realized
their potential are: [0009] (1) fluorescence optical biopsies,
which fails due to the nonspecific nature of tissue auto
fluorescence; and [0010] (2) near-infrared optical diagnostics, in
particular non-invasive glucose sensing, which fails due to
interference from tissue major components, including predominantly
water.
[0011] In contrast to other techniques, Raman spectroscopy holds
promise as an optical biopsy technique that is anticipated to be
broadly applicable for characterization of a variety of cancerous
disease states, A number of researchers have shown that Raman
spectroscopy of masses of cells has utility in differentiating
normal vs. malignant tissue and differentiating normal vs. benign
tissue. In general, the Raman spectra of malignant and benign
tissues show an increase in protein content and a decrease in lipid
content versus normal breast tissue, demonstrating that cancer
disease states impact the chemistry of the tissue.
[0012] However, Raman spectroscopy has not been able to
differentiate benign vs. malignant tissues due to the spectral
similarities of these tissue types. In addition, Raman spectroscopy
of breast tissue samples requires large numbers of cell
populations. If only a small portion of the cells are cancerous, as
in the early stages of lesion development, then Raman spectroscopy
of a large number of such cells will be insensitive to the disease,
It would be advantageous to have a technique capable of the spatial
sensitivity needed for discrimination of cancerous from normal
cells in early stage breast cancer diagnosis.
[0013] Chemical imaging based on optical spectroscopy, in
particular Raman spectroscopy, provides the clinician with
important information. Chemical imaging simultaneously provides
image information on the size, shape and distribution (the image
morphology) of molecular chemical species present within the
sample. By utilizing molecular-specific imaging, based on chemical
imaging, the trained clinician can make a determination on the
disease-state of a tissue or cellular sample based on recognizable
changes in morphology without the need for sample staining or
modification.
[0014] Apparatus for Raman Chemical Imaging (RCI) has been
described by the inventors in U.S. Pat. No. 6,002,476, and in
co-pending U.S. Non-Provisional application Ser. No. 09/619,371
filed Jul. 19, 2000 which claims benefit of U.S. Provisional
Application 60/144,518 filed Jul. 19, 1999. The above identified US
patents, patent applications, and publications are hereby
incorporated by reference.
OBJECTS OF THE INVENTION
[0015] It is an object of the invention to produce apparatus and
methods using Raman shifted light for diagnosis of lesions in
tissue. It is an object of the invention to produce apparatus and
methods for diagnosis of tissue samples excised from a patient. It
is an object of the invention to produce apparatus and methods for
in vivo diagnosis of tissue. It is an object of the invention to
produce apparatus and methods for finding a lesion in vivo in
tissue. It is an object of the invention to produce apparatus and
methods for determining the borders of lesions in vivo and in
tissue samples excised from a patient. It is an object of the
invention to produce apparatus and methods for spatially resolving
Raman shifted light from tissue in vivo and in tissue samples. It
is an object of the invention to produce apparatus and methods for
imaging a lesion with light which has been Raman shifted.
SUMMARY OF THE INVENTION
[0016] Raman chemical imaging is used to differentiate between
normal tissue and benign and malignant lesions. In particular,
Raman chemical imaging is shown to be sensitive to calcified tissue
and to carotenoid molecules. Carotenoid molecules are concentrated
at the border of a lesion, and can be used to indicate the borders
of the lesion. Spatially resolved Raman signals indicate lesions
and borders of lesions. Other molecular species which may be
indicative of border regions are noted.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1A. Brightfield reflectance microscope image of a
calcified lesion from a five micron thick frozen section
biopsy.
[0018] FIG. 1B. Magnified region indicated from FIG. 1A.
[0019] FIG. 1C. Raman Chemical Image (RCI) of the spatial
distribution of calcification (calcium hydroxyapatite) and
background (microscope slide).
[0020] FIG. 2A. Brightfield reflectance microscope image of a
region of calcified lesion as indicated in FIG. 1A.
[0021] FIG. 2B. Polarized light image of the region of interest
indicated in FIG. 1A.
[0022] FIG. 2C. Raman Chemical Image (RCI) indicating calcified
tissue.
[0023] FIG. 2D. Raman spectral data from two regions of interest
indicated in FIG. 2C showing the different chemical composition of
these regions.
[0024] FIG. 3A. Brightfield reflectance microscope image of a 5
micron thin section of human breast tissue biopsy sample showing a
lesion and the adjacent tissue.
[0025] FIG. 3B. Magnified region of interest indicated in FIG.
3A.
[0026] FIG. 3C. Raman Chemical Image (RCI) of the endogenous
carotenoid (beta-carotene) that shows the border of the lesion.
[0027] FIG. 3D. Raman spectra obtained with a tunable filter for
two circled regions indicated in FIG. 3C.
[0028] FIG. 4. Amount of carotenoid as determined from its spectral
signal along the diagonal A-A' of the view shown in FIG. 1C.
[0029] FIG. 5. Preferred embodiment of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0030] Raman Spectroscopy
[0031] When light interacts with matter, a portion of the incident
photons are scattered in all directions. A small fraction of the
scattered radiation differs in frequency (wavelength) from the
illuminating light. If the incident light is monochromatic (single
wavelength) as it is when using a laser source or other
sufficiently monochromatic light source, the scattered light which
differs in frequency may be distinguished from the light scattered
which has the same frequency as the incident light. Furthermore,
frequencies of the scattered light are unique to the molecular or
crystal species present. This phenomenon is known as the Raman
effect.
[0032] In Raman spectroscopy, energy levels of molecules are probed
by monitoring the frequency shifts present in scattered light. A
typical experiment consists of a monochromatic source (usually a
laser) that is directed at a sample. Several phenomena then occur
including Raman scattering which is monitored using instrumentation
such as a spectrometer and a charge-coupled device (CCD). Similar
to an infrared spectrum, a Raman spectrum reveals the molecular
composition of materials, including the specific functional groups
present in organic and inorganic molecules and specific vibrations
in crystals. Raman spectrum analysis is useful because each
resonance exhibits a characteristic `fingerprint` spectrum, subject
to various selection rules. Peak shape, peak position and the
adherence to selection rules can also be used to determine
molecular conformation information (crystalline phase, degree of
order, strain, grain size, etc.). Unlike infrared spectroscopy, a
single Raman spectrometer can be applied to the molecular
characterization of organic and inorganic materials simultaneously.
Other advantages of Raman over traditional infrared spectroscopy
include the ability to analyze aqueous phase materials and the
ability to analyze materials with little or no sample preparation.
Deterrents to using Raman spectroscopy as opposed to infrared
spectroscopy include the relatively weak nature of the Raman
phenomenon and interferences due to fluorescence. In the past
several years, a number of key technologies have been introduced
into wide use that have enabled scientists to largely overcome the
problems inherent to Raman spectroscopy. These technologies include
high efficiency solid state lasers, efficient laser rejection
filters, and silicon charge coupled device (CCD) detectors.
[0033] In Raman spectroscopy instruments, a linear CCD array is
typically positioned at the exit focal plane of single stage, low f
number Raman monochromators for efficient collection of dispersive
Raman spectra. The monochromator disperses the Raman shifted light,
and the CCD array typically produces a signal which is proportional
to the intensity of the Raman signal vs. wavelength.
[0034] Raman Chemical Imaging (RCI)
[0035] In many respects, Raman chemical imaging is an extension of
Raman spectroscopy. Raman chemical imaging combines Raman
spectroscopy and digital imaging for the molecular-specific
analysis of materials. Much of the imaging performed since the
development of the first Raman microprobes has involved spatial
scanning of samples beneath Raman microprobes in order to construct
Raman "maps" of surfaces. Historically, Raman imaging systems have
been built using this so called flying spot ("point-scanning")
approach, where a laser beam is focused to a spot and is scanned
over the object field, or likewise a line scrolling approach, where
the laser spot is broadened in one direction by, for example, a
cylindrical lens, and the two dimensional image formed on a CCD
array has one spatial dimension and one wavelength dimension. Raman
chemical imaging techniques have only recently achieved a degree of
technological maturity that allows the collection of
high-resolution (spectral and spatial) data. Advancements in
imaging spectrometer technology and their incorporation into
microscopes that employ CCDs, holographic optics, lasers, and fiber
optics have allowed Raman chemical imaging to become a practical
technique for material analysis.
[0036] Imaging spectrometers include Fabry Perot angle rotated or
cavity tuned liquid crystal (LC) dielectric filters, acousto-optic
tunable filters, and other LC tunable filters (LCTF) such as Lyot
Filters and variants of Lyot filters such as Solc filters and the
most preferred filter, an Evan's split element liquid crystal
tunable filter, which is described in the March (1999) issue of
Analytical Chemistry on page 175A. Other preferred wavelength
filtering means comprise polarization-independent imaging
interferometers such as Michelson, Sagnac, Twynam-Green, and
Mach-Zehnder interferometers.
[0037] References describing the above identified techniques that
can be used to obtain chemical images include: [0038] Fiber Array
Filters (FAST)--M. P Nelson, M. L. Myrick, Appl. Spectroscopy 53,
751-759, (1999); [0039] Dielectric Interference filters--D
Batchelder, C Cheng, W Muller, B Smith, Makromol Chem Macromol.
Symp 46, 171, (1991); [0040] AOTF--P. J. Treado, I. W. Levin, E. N.
Lewis, Appl. Spectrosc. 46, 211-1216, (1992); [0041] Lyot--B. Lyot,
C. R. Acad. Sci. 197:1593 (1933); [0042] Fabry Perot--K. A.
Christainsen, N. L. Bradley, M. D. Morris, R. V. Morrison, Appl.
Spectrosc. 49, 120-1125 (1995); [0043] Solc filter--A. Yariv &
P. Yeh, Optical Waves in Crystals, (Wiley NY, 1984); [0044]
Michelson Interferometer--Sybil P. Parker, Optics Source Book,
(McGraw-Hill, NY, 1988, p. 143); [0045] Sagnac Interferometer--S.
Spielman, K. Fesler, C. B. Eom, T. H. Geballe, M. Fejer and A
Kapitulnik, Phys. Rev. Lett., 65, 123 (1990); [0046] Twyman-Green
Interferometer--M. Born and E. Wolf, Principles of Optics:
Electromagnetic Theory of Propogation of Light, 6th Ed, (Pergamon
Press, Oxford, 1980) pp. 302-305; [0047] Mach-Zehnder--James D.
Ingle, Jr., and Stanley R Crouch, Spectrochemical Analysis,
(Prentice Hall, Engelwood, NJ, 1988), p. 83.
[0048] Raman chemical imaging is a versatile technique that is well
suited to the analysis of complex heterogeneous materials. In a
typical Raman chemical imaging experiment, a specimen is
illuminated with monochromatic light, and the Raman scattered light
is filtered by an imaging spectrometer which passes only a single
wavelength range. The Raman scattered light may then be used to
form an image of the specimen. A spectrum is generated
corresponding to millions of spatial locations at the sample
surface by tuning an imaging spectrometer over a range of
wavelengths and collecting images intermittently. Changing the
selected passband (wavelength) of the imaging spectrometer to
another appropriate wavelength causes a different material to
become visible. A series of such images can then uniquely identify
constituent materials, and computer analysis of the image is used
to produce a composite image highlighting the information desired.
Although Raman chemical imaging is predominately a surface
technique, depth-related information can also be obtained by using
different excitation wavelengths or by capturing chemical images at
incremental planes of focus. Contrast is generated in the images
based on the relative amounts of Raman scatter or other optical
phenomena such as luminescence that is generated by the different
species located throughout the sample. Since a spectrum is
generated for each pixel location, chemometric analysis tools such
as correlation analysis, Principle Component Analysis (PCA) and
factor rotation, including Multivariate Curve Resolution (MCR) can
be applied to the image data to extract pertinent information
otherwise missed by ordinary univariate measures. A spatial
resolving power of approximately 250 nm has been demonstrated for
Raman chemical imaging using visible laser wavelengths. This is
almost two orders of magnitude better than infrared imaging which
is typically limited to 20 microns due to diffraction. In addition,
image definition (based on the total number of imaging pixels) can
be very high for Raman chemical imaging because of the use of high
pixel density detectors (often 1 million plus detector
elements).
[0049] Applications of Raman chemical imaging range from the
analysis of polymer blends, defect status analysis in semiconductor
materials, inclusions in human breast tissue and the
characterization of corrosion samples. RCI provides a potential
solution for obtaining both qualitative and quantitative image
information about molecular composition and morphology of breast
lesions allowing a more accurate medical diagnosis than traditional
imaging methods.
[0050] Breast Tissue Results
[0051] Raman spectra can potentially reveal a wealth of information
about molecular properties of tissues. RCI compounds this
information by allowing variations in these properties throughout
the tissue to be probed. FIG. 1 shows RCI data on a calcified
lesion. The tissue was excised from the patient, and frozen. A five
micron thick section was sliced from the tissue and prepared on a
microscope slide for imaging in a microscope. FIG. 1A shows a
brightfield reflectance image of a portion of the frozen sectioned
biopsy which is then magnified in FIG. 1B. The brightfield image
reveals light and dark regions resulting from differences in
refractive indices. These images, however, provide no insight into
the molecular makeup of the tissues at hand. A Raman chemical image
is shown in FIG. 1C and reveals the distribution of calcium
hydroxyapatite based on its Raman response. FIG. 2A and FIG. 2B
show dramatic differences in the optical microscopic image that
depend on the polarization of the light. However, the Raman
chemical image in FIG. 1C is unique in that it is derived from the
distinct spectral Raman shown in FIG. 2D. The Raman spectra in FIG.
1D shows the spectral "fingerprints" associated with the calcium
hydroxyapatite and the background, (the glass microscope slide)
respectively. Such Raman spectra are the basis that allow a Raman
Chemical image to be created. This ability to characterize
calcifications is a critical issue in the diagnosis of breast
carcinoma as calcification is a major element in mammographic
evaluation and early cancer detection, and is critical for the
diagnostic pathologist to identify. The Raman spectrum of calcium
salts and protein calcium complexes is an incompletely explored
area, in large part because of the previous unavailability of
instrumentation capable of simultaneous high resolution spatial
imaging and high wavelength resolution Raman spectrochemical
analyses.
[0052] Difficulties exist when trying to use non imaging Raman
spectroscopy alone to differentiate benign vs. malignant tissues
due to the spectral similarities of these tissue types and to the
spectrum of breast conditions that may mimic cancer. In addition,
non imaging Raman spectroscopy of breast tissue samples large
numbers of cell populations. If only a small portion of the cells
arc cancerous, as in the early stages of lesion development, then
non-imaging Raman spectroscopy will be insensitive to the disease.
It is very advantageous to have a technique capable of the spatial
imaging sensitivity needed for discrimination of cancerous from
normal cells in early stage breast cancer diagnosis.
[0053] We have developed an imaging optical biopsy approach based
on Raman chemical imaging. In comparison with non-imaging Raman
spectroscopy, our approach has the advantage that we efficiently
collect spatial resolved Raman spectra so that morphometric
analysis (characterization by size and shape) can be performed in
conjunction with Raman spectral analysis. The additional morphology
information is anticipated to add a critical component to the
analysis of disease states, in part because it builds upon
traditional cancer histopathology methods and could therefore be
readily adopted by pathologists. FIG. 3A shows a brightfield image
of a 5 .mu.m thin section human breast tissue biopsy sample viewed
under the microscope. An enlarged section of the lesion is
indicated and magnified in FIG. 3B to show the border or interface
between a tumor and normal tissue, where both cancerous and normal
cells are visible. The Raman chemical image of a carotenoid
molecule, .beta.-carotene, shown in FIG. 3C reveals the location of
the tumor and carotenoid molecules. Note that the carotenoid
molecules are associated with the border between the lesion and the
normal tissue. The LCTF-generated Raman spectra in FIG. 3D shows
the spectral "fingerprints" associated with the tumor and the
typical normal tissue, respectively. The ability to see this
boundary with an inherent chemical within human tissue is a unique
finding with potential biological and clinical significance
relating to the objective screening and characterization of tumor
margins
[0054] FIG. 4 shows the results of a scan of the carotenoid signal
along the diagonal A-A', i.e., along a line perpendicular to the
tumor normal tissue boundary of FIG. 3C.
[0055] It is very important to know where the tumor margins are,
and to know if the tumor has infiltrated beyond a well defined
boundary and into normal tissue. Detection of molecules indicative
of the boundary is of great importance. The nutritional literature
supports the idea that carotenoids are protective from cancer. It
is surmised but not proven by the inventors that such protective
molecules accumulate in the border region between a lesion and
normal tissue, and act to prevent the lesion from growing. Other
molecules suggested by the nutritional literature in relation to
breast cancer are indoles, sulforaphanes, and flavonoids.
Proteoglycans molecules have been noted to be associated with
prostate cancer. With the Raman chemical imaging, the position of
these molecules, and molecules which will be identified in the
future, may be clearly imaged and used to show the extent and the
stage of growth of the cancer or other lesion.
[0056] The cancerous cells shown in the lesion in FIGS. 3B and 3C
are also differentiated from adjacent cells in the Raman image
based on molecular compositional variations (lipid vs. protein
content primarily) and can also be used to create a Raman image of
the diseased tissue. As a result, the images are molecule-specific
and more specific than images derived from stains. Because the
Raman scattering of the tissues is intrinsic to the tissues, stains
are not required and the technique is suitable for in vivo use. The
Raman images are collected in only several seconds using laser
power density that does not modify the tissue samples.
[0057] An in vivo embodiment of the invention for examining a
breast 50 or other non-arterial soft tissue for a lesion 51 is
shown in FIG. 5. An endoscope or other instrument 52 is used to
introduce light carried by an optical fiber 53 from a monochromatic
light source 54. A dichroic mirror 55 and lens 56 are shown
schematically for introducing the light into the fiber 53. Raman
light from the breast is carried from the breast tissue back
through the lens 56 and mirror 55, through a filter 57 to a
detector 58. The signal from the detector 58 is analyzed by a
computer system 59 and displayed on a monitor 60.
[0058] Filter 57 is most preferably a Evan's split element liquid
crystal tunable filter, which is controlled by computer 59.
[0059] The endoscope 52 is preferably an imaging endoscope or
fiberscope, where light is conducted from the breast tissue to the
detector 58 in a coherent manner through a large plurality of
optical fibers. A series of two dimensional images is preferably
taken as a function of depth into the tissue and of the Raman
shifted wavelength.
[0060] Results of a preferable embodiment of the invention is shown
by an insert in FIG. 5, where the signal shown is a signal of a
molecule indicative of a border region between the breast 50 or
other non arterial soft tissue and the lesion 51. The spatially
resolved signal of calcified tissue or of, for example, carotenoid
molecules, is shown in the insert as a function of depth into the
breast as the needle carrying the optical fiber is moved into the
breast. The signal is shown displayed on the display device 60. In
this embodiment, a much finer needle is used than the needle
carrying an imaging endoscope. In the fine needle embodiment, the
location of the lesion may be more accurately determined, so that
fine needle aspiration cytology and/or needle core biopsy may be
performed. In the fine needle embodiment, the filter 57 may be a
normal spectrometer or a liquid crystal tunable filter, preferably
of the Evan's split element type.
[0061] Raman chemical imaging also has demonstrated utility for the
quantitative assessment of lesions in breast tissues. However,
there is a need to make systematic strides in the development of a
RCI optical biopsy. RCI of animal breast tissue models have been
analyzed, as well as studies of human cancer lesions. Other lesions
besides benign and malignant tumors, such as pockets of infection
and inflammation will show up in the Raman chemical images. Several
data treatment methods have been utilized to analyze the Raman
image data which include band ratioing, band shift analysis, and
classical linear least squares analysis. Comparisons have been made
between the various processing approaches that address the utility
of RCI for breast tissue component discrimination.
[0062] Applications
[0063] There is a great need for an instrument that can provide:
real time detection with accuracy, decreased patient discomfort and
recovery, minimal cosmetic defect of the breast, minimal distortion
of the breast tissue that might make interpretation of future
mammograms difficult and most importantly provide the patient with
rapid feedback on her condition.
[0064] The user base for an instrument suitable for objective
assessment of breast lesions will consist of medical research
laboratories, University and non-affiliated hospitals, and private
clinics.
[0065] On another level, the customer or end-user is the patient
that requires the procedure be completed to determine the disease
state of her breast tissues. At this level the numbers are as
follows: more than 1,000,000 biopsies were conducted in 1997; the
growth rate for biopsies is almost 20% annually as clinicians
struggle with how to determine the disease state of tissue early
enough to prevent radical measures; the typical "customer" is a
woman over the age of 40 that should be having annual breast exams
by a clinician; and the number of potential customers is
approximately 57 million (women between ages of 40 and 85).
[0066] The benefits to the target users of RCI systems will be
substantial. Configured in an endoscopic version of the technology,
RCI can be employed for "real-time" breast tissue evaluation tool
that is compatible with and complementary to existing, mature
clinical approaches (namely, needle core biopsies). When performed
in combination the effectiveness of breast cancer diagnosis will
likely be enhanced. Benefits will include, but are not limited to,
the following: [0067] Real-time evaluation of suspicious lesions
sites identified through self-breast exam and/or mammography that
are made accessible via needle core biopsy. [0068] Immediate
feedback to the clinician as to the severity of the clinical
situation. Results can be communicated to the patient by the
physician shortly after completion of Raman biopsy. [0069]
Potential information on prognostic indicators of disease such as
growth rate through quantitative evaluation of cellular nucleic
acid composition and proliferation associated peptides. [0070]
Minimal patient discomfort. [0071] Minimal to no cosmetic defect of
the breast. [0072] Reduced exposure to ionizing radiation (x-rays).
[0073] Specific applications of a RCI system for evaluating breast
lesions will include the following: [0074] Discrimination of
malignant vs. benign tumors [0075] Spatial distribution of
carotenoids in tissues [0076] Spatial distribution of calcified
tissue [0077] Spatial distribution of proteins, lipids and
carbohydrates in tissues
[0078] Advantages Over Currently Available Technology
[0079] Traditional approaches to identification of breast lesions
include self-breast exam and x-ray mammography. These techniques
are effective as initial screening techniques, especially when
performed in combination. Unfortunately, mammography is associated
with a high false positive rate, resulting in 3-7 patients being
biopsied for every patient cancer diagnosed. Although many
mammographic abnormalities are definitely benign, and others are
obviously malignant, there are many lesions in which the diagnosis
cannot be made with certainty based on the mammographic appearance
alone. To verify the disease-state of a detected lesion, tissue
must be sampled for pathologic examination. This may be done with
fine needle aspirates, core biopsies, or excisional biopsies. These
samples are then prepared, stained, and inspected by a trained
pathologist. This process can take several days to complete before
the patient is informed of the outcome. Raman chemical imaging
technology has the potential to assist diagnosis of the disease
state of breast lesions in real-time.
[0080] Currently, several biopsy techniques are used as diagnostic
methods after breast lumps are identified, typically with
mammography, ultrasound, or breast examination. The most reliable
method of diagnosis is examination of macroscopic-sized lesions.
Macroanalysis is performed in conjunction with microscopic
evaluation of paraffin-embedded biopsied tissue which is
thin-sectioned to reveal microscale morphology.
[0081] Alternatives to traditional surgical biopsy include fine
needle aspiration cytology and needle core biopsy. These
non-surgical techniques are becoming more prevalent as breast
cancer diagnostic techniques because they are less invasive than
conventional biopsy techniques that involve surgical incision. Fine
needle aspiration cytology has the advantage of being a rapid,
minimally invasive, non-surgical technique that retrieves cytologic
material that is often adequate for evaluation of disease state.
However, in fine needle biopsies breast tissue histologic features
are minimal, leaving only cytologic features for analysis of
disease state. In contrast, needle biopsies use a much larger gauge
needle which retrieve tissue samples that are better suited to
morphology analysis. However, needle biopsies necessitate an
outpatient surgical procedure and the resulting needle core sample
must be fixed, embedded and processed prior to analysis.
[0082] State-of-the-Art Raman Chemical Imaging Techniques
[0083] Several Raman chemical imaging technologies have evolved
that compete with widefield tunable filter-based RCI. These
techniques include point scanning RCI, line imaging RCI, RCI using
interference filters, Fourier-transform interferometry,
Hadamard-transform scanning and FAST technology.
[0084] Point scanning involves taking a complete spectrum for a
single X,Y position of a sample followed by raster-scanning the
sample for the remaining X,Y positions. This method offers
advantages of high spectral resolution and full spectral
resolution, but lacks high image definition capabilities and is
extremely time consuming. Line imaging involves collecting data
from vertical sections of the sample characterized by a single
value of X and all values of Y, followed by subsequent scanning in
the X direction. This method has the nearly the same advantages and
disadvantages as the point scanning approach, but can be done more
rapidly. Field curvature artifacts are a consequence of line
imaging which degrade image quality. The use of single or multiple
interference filters can be used to produce a wavelength specific
image(s). This method is rapid, cheap and produces high definition
images, but lacks spectral resolution and is susceptible to image
artifacts. Fourier-transform interferometers use a mechanically
driven interferometer with a CCD-based detection system.
Interferograms are imaged with the CCD for subsequent spectral
interpretation for each step of the interferometer. This method
boasts good spatial resolution but suffers from poor spectral
resolution (.about.100 cm.sup.-1). Hadamard transform chemical
imaging techniques couple Hadamard mask spatial multiplexing with
CCD-based detection to obtain two spatial and one spectral
dimension of data. This method offers S/N advantages for low-light
level applications such as Raman spectroscopy in addition to
sub-nanometer spectral resolution. However, the technique suffers
from fair spatial resolution and poor temporal resolution since the
latter involves scanning through numerous coding masks. Fiber array
spectral translators (FAST) use a two dimensional arrangement of
Raman collection fibers which are drawn into a one dimensional
distal array at the opposite end. The one dimensional fiber stack
is coupled to an imaging spectrograph. Software then extracts the
spectral/spatial information which is embedded in a single CCD
image frame. FAST is capable of acquiring thousands of full
spectral range, position-specific Raman spectra and wave
number-specific Raman chemical images in seconds. However, the
image definition of FAST is limited by the number of pixels in
anyone direction of the CCD chip used in the detection system
(typically no better than 45.times.45 (.about.2048) imaging
elements).
[0085] The ideal chemical imaging system for characterization would
provide fast acquisition times (seconds), high spatial resolution
(sub-micron) and good spectral resolution (<0.2 nm). To date,
systems equipped with liquid crystal tunable filters are the only
RCI system that meets these requirements.
[0086] Other Spectroscopy-Based Imaging Methods
[0087] Spectroscopic technologies that compete with Raman such as
fluorescence and infrared (IR) spectroscopy are not of great
concern based on the resolution needed to see molecules on the
order of 250 microns. Although fluorescence has showed some
promise, it suffers from low specificity without the use of
invasive dyes or stains that require FDA approval. IR spectroscopy
cannot compete due to the difficulty with water absorption in the
IR. Tissues do not image well because of their aqueous nature.
Systems equipped with LCTFs surpass any dispersive grating or
acousto-optic tunable filter (AOTF) technology on the market. The
spectral bandpass capability of the LCTF is 8 cm.sup.- allowing for
the most effective means to obtain image detail.
[0088] Traditional Biomedical Imaging Methods
[0089] Traditionally, biomedical imaging has been divided into
capturing images of live tissue (in vivo) at relatively low
resolution (from 10 to 1000 microns) and capturing images of
excised tissue at high resolution. In vivo imaging is usually
performed using non-optical modalities such as magnetic resonance
imaging, ultrasound, or x-ray tomography, which assess the general
shape and appearance of tissue in its native state; however, this
approach does not provide the cellular resolution necessary to
analyze cell types and tissue morphology. To image tissue at high
resolution using conventional optical or electron microscopes, one
had to slice the tissue into thin sections, otherwise the tissue
above and below the layer of interest will produce out-of-focus
reflections that seriously degrade image contrast. Confocal
techniques address this to some extent. Excising, fixing and
staining thin tissue sections is however static, is time-consuming
and by the very nature of the process involves tissues which have
been rendered non viable.
[0090] A RCI system will produce quantitative digital images of the
lesion tissue that will be recognizable to the clinician who makes
disease-state determinations, in large part, based on the visual
appearance of images. The appearance of suspect tissue, when viewed
by the naked eye if lesions are large enough, or via x-ray
mammography, or via magnetic resonance imaging (MRI) provides
important clues to the state of the tissue. After years of
training, clinicians can base diagnosis on these subtle visual
clues. Despite the best efforts of highly skilled professionals,
early stage disease-state determination is a difficult problem. By
aiding the pathologist with an image that maps the distribution of
certain molecular species, the large number of subjective
determinations of disease state in breast tissue biopsies can be
greatly reduced.
[0091] Although we have described certain present preferred
embodiments of our method for objective evaluation of breast tissue
using Raman imaging spectroscopy, it should be distinctly
understood that our invention is not limited thereto, but may
include equivalent methods. It is further to be distinctly
understood that the present invention is not limited to the
evaluation of breast tissue and applies to the evaluation of all
tissue. Obviously, many modifications and variations of the present
invention are possible in light of the above teachings. It is
therefore to be understood that, within the scope of the appended
claims, the invention may be practiced otherwise than as
specifically described. Publications, patents, and patent
applications noted herein are hereby incorporated by reference.
* * * * *