U.S. patent application number 11/301222 was filed with the patent office on 2006-04-27 for multi-modal optical tissue diagnostic system.
This patent application is currently assigned to SpectRx, Inc.. Invention is credited to Anant Agrawal, Shabbir B. Bambot, Mark L. Faupel, Tim Harrell.
Application Number | 20060089556 11/301222 |
Document ID | / |
Family ID | 26796580 |
Filed Date | 2006-04-27 |
United States Patent
Application |
20060089556 |
Kind Code |
A1 |
Bambot; Shabbir B. ; et
al. |
April 27, 2006 |
Multi-modal optical tissue diagnostic system
Abstract
An apparatus and method according to the invention combine more
than one optical modality (spectroscopic method), including but not
limited to fluorescence, absorption, reflectance, polarization
anisotropy, and phase modulation, to decouple morphological and
biochemical changes associated with tissue changes due to disease,
and thus to provide an accurate diagnosis of the tissue
condition.
Inventors: |
Bambot; Shabbir B.;
(Suwanee, GA) ; Faupel; Mark L.; (Alpharetta,
GA) ; Harrell; Tim; (Norcross, GA) ; Agrawal;
Anant; (Atlanta, GA) |
Correspondence
Address: |
Michael B. Lasky;Altera Law Group
Suite 100
6500 City West Parkway
Minneapolis
MN
55344-7704
US
|
Assignee: |
SpectRx, Inc.
|
Family ID: |
26796580 |
Appl. No.: |
11/301222 |
Filed: |
December 12, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10603597 |
Jun 26, 2003 |
6975899 |
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11301222 |
Dec 12, 2005 |
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09786781 |
Mar 9, 2001 |
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PCT/US99/20646 |
Sep 10, 1999 |
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10603597 |
Jun 26, 2003 |
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60143579 |
Jul 13, 1999 |
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60099875 |
Sep 11, 1998 |
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Current U.S.
Class: |
600/476 ;
600/407; 600/473; 600/477; 606/15; 606/16; 607/89 |
Current CPC
Class: |
A61B 5/0071 20130101;
A61B 5/0084 20130101; A61B 1/043 20130101; A61B 5/0075 20130101;
A61B 5/7264 20130101 |
Class at
Publication: |
600/476 ;
600/473; 600/477; 600/407; 606/015; 606/016; 607/089 |
International
Class: |
A61B 6/00 20060101
A61B006/00; A61B 18/18 20060101 A61B018/18; A61N 5/06 20060101
A61N005/06; A61B 5/05 20060101 A61B005/05 |
Claims
1-37. (canceled)
38. A method for diagnosing a condition of a target tissue,
comprising the steps of: a.) irradiating a target tissue with
excitation electromagnetic radiation; b.) sensing a returned
electromagnetic radiation returned from the target tissue; c.)
determining characteristics of the returned electromagnetic
radiation using at least two spectroscopic methods; d.) combining
the characteristics determined by the at least two spectroscopic
methods, thereby decoupling biochemical changes from morphological
changes in the target tissue; and e.) determining a condition of
the target tissue based on the combined determined
characteristics.
39. The method of claim 38, wherein the at least two spectroscopic
methods are selected from the group consisting of absorption
measurements, scattering measurements, reflection measurements,
polarization anisotropic measurements, steady state fluorescence
measurements, and time resolved fluorescence measurements.
40. The method of claim 39, wherein the time resolved fluorescence
measurements comprise at least one of phase modulation techniques,
polarization anisotropic techniques and techniques that directly
monitor the decay profile of fluorescent emissions.
41. The method of claim 38, wherein step b.) comprises
simultaneously sensing electromagnetic radiation emitted from the
target tissue in response to the excitation electromagnetic
radiation and excitation electromagnetic radiation that is
scattered from the target tissue.
42. The method of claim 41, wherein step c.) comprises making
intensity based measurements on both said electromagnetic radiation
emitted from the target tissue in response to the excitation
electromagnetic radiation and said excitation electromagnetic
radiation that is scattered from the target tissue.
43. The method of claim 38, wherein step b.) comprises sensing
electromagnetic radiation emitted from the target tissue in
response to the excitation electromagnetic radiation and then
subsequently sensing excitation electromagnetic radiation that is
scattered from the target tissue.
44. The method of claim 38, wherein step b.) comprises sensing
electromagnetic radiation returned from a plurality of
interrogation points distributed over the target tissue.
45. The method according to claim 44, further comprising a step of
dividing the target tissue into a first set of field areas, wherein
step c.) comprises determining characteristics of the returned
electromagnetic radiation in each of said first set of field areas
using at least two spectroscopic methods, step d.) comprises
combining the characteristics determined by the at least two
spectroscopic methods for each of said first set of field areas and
step e.) comprises determining a condition of the target tissue by
comparing the combined determined characteristics of each of said
first set of field areas.
46. The method of claim 45, further comprising, after determining a
condition of the target tissue by comparing the combined determined
characteristics of each of said first set of field areas,
re-dividing the target tissue into a second set of field areas,
different from said first set of field areas and the determining
characteristics of the returned electromagnetic radiation in each
of said second set of field areas using at least two spectroscopic
methods, combining the characteristics determined by the at least
two spectroscopic methods for each of said second set of field
areas and determining a condition of the target tissue by comparing
the combined determined characteristics of each of said second set
of field areas.
47. The method of claim 44, wherein the method is performed using
an apparatus comprising an irradiation source, a detector and a
processor, wherein the step of sensing electromagnetic radiation
returned from a plurality of interrogation points comprises the
steps of: sensing electromagnetic radiation returned from the
target tissue from a first subset of the plurality of interrogation
points; moving at least one of the apparatus and the tissue;
sensing electromagnetic radiation returned from the target tissue
from a second subset of the plurality of interrogation points;
again moving at least one of the apparatus and the tissue; and
continuing this process until sensing has been peformed at all of
the plurality of interrogation points.
48. A system for determining a condition of a target tissue in a
human or animal, comprising: a electromagnetic radiation source for
providing excitation electromagnetic radiation; a device that
couples the excitation electromagnetic radiation to a target
tissue; a device that senses electromagnetic radiation returned
from the target tissue; a processor configured to determine
characteristics of the returned electromagnetic radiation using at
least two spectroscopic methods, wherein the processor combines the
characteristics determined by each of the at least two
spectroscopic methods in order to decouple biochemical changes from
morphological changes in the target tissue and determines a
condition of the target tissue based on the combined determined
characteristics.
49. The system of claim 48, wherein the at least two spectroscopic
methods comprise fluorescence measurement methods and scattering or
reflectance measurement methods.
50. The system of claim 48, wherein the at least two spectroscopic
methods are selected from the group consisting of absorption
measurements, scattering measurements, reflectance measurements,
polarization anisotropy measurements, steady state fluorescence
measurements and time resolved fluorescence measurements.
51. The system of claim 48, wherein the device that senses returned
electromagnetic radiation is configured to simultaneously sense
fluorescent radiation emitted by endogenous fluorophores in
response to the excitation radiation and excitation electromagnetic
radiation that is scattered from the target tissue.
52. The system of claim 48, wherein the device that senses
electromagnetic radiation is configured to sense electromagnetic
radiation returned from a plurality of interrogation points
distributed over the target tissue.
53. The system according to claim 52, wherein the processor divides
the target tissue into a first set of field areas, determines
characteristics of the returned electromagnetic radiation in each
of said first set of field areas using said at least two
spectroscopic methods, combines the characteristics determined by
each of said at least two spectroscopic methods for each of said
first set of field areas and determines a condition of the target
tissue in each of said first set of field areas based on the
combined determined characteristics of the respective field
areas.
54. The system of claim 53, wherein the processor is further
configured to, after the processor determines a condition of the
target tissue in each of the first set of field areas based on the
combined determined characteristics of the respective field areas,
divide the target tissue into a second set of field areas,
different from the first set of field areas; determine
characteristics of the returned electromagnetic radiation in each
of said second set of field areas using said at least two
spectroscopic methods, combine the characteristics determined by
each of said at least two spectroscopic methods for each of said
second set of field areas and determine a condition of the target
tissue in each of the second set of field areas based on the
combined determined characteristics of the respective field
areas.
55. A system for determining a condition of a target tissue in a
human or animal, comprising: an electromagnetic radiation source
for providing excitation electromagnetic radiation; a device that
couples the excitation electromagnetic radiation to a target
tissue; a device that senses electromagnetic radiation returned
from the target tissue; and a processor configured to determine
characteristics of the returned electromagnetic radiation using at
least two spectroscopic methods, thereby decoupling biochemical
changes from morphological changes in the target tissue occurring
due to disease and determine a condition of the target tissue based
on the determined characteristics.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The invention relates to apparatus and methods for
determining tissue characteristics of, for example, a human or
animal.
[0003] 2. Background of the Related Art
[0004] Spectroscopic methods for determining tissue characteristics
are known and have been widely used to interrogate changes in
tissue. A number of these distinct spectroscopic techniques are
available that provide specific information depending on the nature
of the interaction of light with cells and the natural chromophores
present in tissue. These interactions include the absorption of
light at a particular wavelength, the reemission of absorbed light
as fluorescence, the scattering (redirection) of light at a
particular wavelength and the change in polarization between the
absorbed or scattered light and the reemitted light.
[0005] For example, it is known to irradiate a target tissue with
electromagnetic radiation and to detect returned electromagnetic
radiation to determine characteristics of the target tissue. In
known methods, the amplitudes and wavelengths of the returned
radiation are analyzed to determine characteristics of the target
tissue. For instance, U.S. Pat. No. 4,718,417 to Kittrell et al.
discloses a method for diagnosing the type of tissue within an
artery, wherein a catheter is inserted into an artery and
excitation light at particular wavelengths is used to illuminate
the interior wall of the artery. Material or tissue within the
artery wall emits fluorescent radiation in response to the
excitation light. A detector detects the fluorescent radiation and
analyzes the amplitudes and wavelengths of the emitted fluorescent
radiation to determine whether the illuminated portion of the
artery wall is normal, or covered with plaque. The contents of U.S.
Pat. No. 4,718,417 are hereby incorporated by reference.
[0006] U.S. Pat. No. 4,930,516 to Alfano et al. discloses a method
for detecting cancerous tissue, wherein a tissue sample is
illuminated with excitation light at a first wavelength, and
fluorescent radiation emitted in response to the excitation light
is detected. The wavelength and amplitude of the emitted
fluorescent radiation are then examined to determine whether the
tissue sample is cancerous or normal. Normal tissue will typically
have amplitude peaks at certain known wavelengths, whereas
cancerous tissue will have amplitude peaks at different
wavelengths. Alternatively the spectral amplitude of normal tissue
will differ from cancerous tissue at the same wavelength. The
disclosure of U.S. Pat. No. 4,930,516 is hereby incorporated by
reference. The above described methods are referred to as
fluorescence spectroscopy.
[0007] Still other patents, such as U.S. Pat. No. 5,369,496 to
Alfano et al., disclose methods for determining characteristics of
biological materials, wherein a target tissue is illuminated with
light, and backscattered or reflected light is analyzed to
determine the tissue characteristics. The contents of U.S. Pat. No.
5,369,496 are hereby incorporated by reference. This type of method
is referred to as absorption spectroscopy.
[0008] It is also known to look at the decay time of fluorescent
emissions to determine the type or condition of an illuminated
tissue. These methods are referred to as time resolved
spectroscopy. Generally, apparatus for detection of the lifetime of
fluorescent emissions have concentrated on directly measuring the
lifetime of the fluorescent emissions. Typically, a very short
burst of excitation light is directed at a target tissue, and
fluorescent emissions from the target tissue are then sensed with a
detector. The amplitude of the fluorescent emissions are recorded,
over time, as the fluorescent emissions decay. The fluorescent
emissions may be sensed at specific wavelengths, or over a range of
wavelengths. The amplitude decay profile, as a function of time, is
then examined to determine a property or condition of the target
tissue.
[0009] For instance, U.S. Pat. No. 5,562,100 to Kittrell et al.
discloses a method of determining tissue characteristics that
includes illuminating a target tissue with a short pulse of
excitation radiation at a particular wavelength, and detecting
fluorescent radiation emitted by the target tissue in response to
the excitation radiation. In this method, the amplitude of the
emitted radiation is recorded, over time, as the emission decays.
The amplitude profile is then used to determine characteristics of
the target tissue. Similarly, U.S. Parent No. 5,467,767 to Alfano
et al. also discloses a method of determining whether a tissue
sample includes cancerous cells, wherein the amplitude decay
profile of fluorescent emissions are examined. The contents of U.S.
Pat. Nos. 5,562,100 and 5,467,767 are hereby incorporated by
reference.
[0010] Other U.S. patents have explained that the decay time of
fluorescent emissions can be indirectly measured utilizing phase
shift or polarization anisotropy measurements. For instance, U.S.
Pat. No. 5,624,847 to Lakowicz et al. discloses a method for
determining the presence or concentration of various substances
using a phase shift method. U.S. Pat. No. 5,515,864 to Zuckerman
discloses a method for measuring the concentration of oxygen in
blood utilizing a polarization anisotropy measurement technique.
Each of these methods indirectly measure the lifetime of
fluorescent emissions generated in response to excitation
radiation. The contents of U.S. Pat. Nos. 5,624,847 and 5,515,864
are hereby incorporated by reference.
[0011] None of the prior art methods discussed above alone is
sufficient to accurately measure changes in tissue characteristics.
That is, as more fully discussed below, as tissue undergoes changes
from normal to, for example, cancerous tissue, fluorescence
spectroscopy becomes less effective in determining tissue
characteristics because it is less sensitive to the morphological
changes occurring, as compared to absorption spectroscopy.
Likewise, absorption spectroscopy alone is insufficient to assess
changes in tissue characteristics because it is less sensitive to
biochemical changes in tissue, as compared to fluorescence
spectroscopy.
[0012] It is known to combine two or more measurement techniques to
arrive at a more accurate ultimate determination. For example, U.S.
Pat. No. 5,582,168 to Samuels et al., the contents of which are
hereby incorporated by reference, discloses an apparatus and method
for detecting changes in the lens of an eye. Samuels et al. teach
measuring both transmission or Raman or fluorescence emission, as
well as scattering, reflection or similar effects. The material
under examination is then normalized using a ratio of the
fluorescence emission intensity to the scattering or reflected
intensity. However, while this method addresses biochemical changes
due to disease, it does not address morphological changes due to
disease.
[0013] Further, generally, prior art spectroscopic methods focus on
tissue characteristics at a single point or minium number of points
on the tissue. Taking measurements at just one point or a minimum
number of points can be misleading as it does not provide a
sufficient sampling of tissue area to accurately reflect the
tissue's condition.
SUMMARY OF THE INVENTION
[0014] The invention focuses on providing methods and apparatus
that provide accurate measurements of changes in characteristics of
tissues. The methods and apparatus according to the invention
combine more than one optical modality (spectroscopic method),
including but not limited to fluorescence, absorption, reflectance,
polarization anisotropy, and phase modulation to decouple
morphological and biochemical changes associated with tissue
changes, and thus to provide an accurate diagnosis of the tissue's
condition. The measurements taken according to the various
spectroscopic methods can be equally weighted for diagnostic
purposes, or can be weighted in various manners to produce the best
diagnostic results. For example, the results may be weighted based
on characteristics particular to the tissue subject, such as, for
example, patient ages, hormonal metabolism, mucosal viscosity,
circulatory and nervous system differences.
[0015] The invention encompasses apparatus and methods for
determining characteristics of target tissues, wherein excitation
electromagnetic radiation is used to illuminate a target tissue and
electromagnetic radiation returned from the target tissue is
analyzed to determine the characteristics of the target tissue.
Some apparatus and methods embodying the invention can be used to
perform a diagnosis at or slightly below the tissue surface of, for
example, a human or animal. For instance, methods and apparatus
embodying the invention could be used to diagnose the condition of
skin, the lining of natural body lumens such as the
gastrointestinal tract, or the surfaces of organs or blood vessels.
Other apparatus and methods embodying the invention can be used to
perform a diagnosis deep within tissues of, for example, a human or
animal, where the excitation radiation has to pass through several
centimeters of tissue before it interacts with the target tissue,
such as in diagnosis of tumors and lesions deep in a breast of a
human or animal.
[0016] According to a preferred embodiment of the invention, an
apparatus and method are provided which utilize fluorescence in
combination with reflectance in order to decouple the biochemical
changes from the morphological changes. The fluorescence and
reflectance information may be separately analyzed and compared, or
alternatively, can be calibrated to take into account the
attenuation due to absorption and scattering. Other combinations of
spectroscopic methods besides fluorescence and reflectance may also
be appropriate.
[0017] Measurements using the various spectroscopic methods may be
taken simultaneously, or may be taken one after the other provided
that a critical timing window, defined as the time period between
the measurements, is maintained below a certain time interval.
[0018] The above described techniques are preferably used to
determine characteristics of multiple portions of a target tissue.
The target tissue may be analyzed as a whole by simultaneously
taking measurements at a plurality of interrogation points covering
substantially the entire tissue surface, or by taking measurements
at only a portion of the plurality of interrogation points covering
substantially the entire tissue surface at timing intervals until
measurements have been taken at all of the plurality of
interrogation points.
[0019] Further, the target tissue can be divided into a plurality
of field areas to create a field pattern. Measurements may then be
taken at a plurality of interrogation points within each of the
field areas. The field areas may be then separately analyzed and
compared in order to diagnose a condition of the target tissue. The
target tissue can then be redivided into a different set of field
areas and the field areas analyzed and compared in order to
diagnose the condition of the tissue. The field areas may be all
identically sized and/or shaped, or may have varied sizes and/or
shapes. Further, the target tissue may be redivided into field
areas of the same size and shape as the original field areas, which
then are merely repositioned, or it may be redivided into field
areas of a different size and/or shape, or of varied sizes and/or
shapes.
[0020] As discussed above, techniques embodying the invention can
be used to determine the conditions of multiple portions of a
target tissue, and the determined conditions can be used to create
a map of the target tissue. Such a map could then be either
displayed on a display screen, or presented in hard copy
format.
[0021] Further, the techniques can be used to feed information into
a pattern recognition algorithm, or neural network.
[0022] Additional advantages, objects, and features of the
invention will be set forth in part in the description which
follows and in part will become apparent to those having ordinary
skill in the art upon examination of the following or may be
learned from practice of the invention. The objects and advantages
of the invention may be realized and attained as particularly
pointed out in the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Preferred embodiments of the invention will now be described
with reference to the following drawing figures, wherein like
elements are referred to with like reference numerals, and
wherein:
[0024] FIG. 1 is a schematic diagram showing an apparatus embodying
the invention capable of performing a phase shift measurement;
[0025] FIG. 2 is a schematic diagram of an endoscope embodying the
invention;
[0026] FIGS. 3A and 3B show another embodiment of the
invention;
[0027] FIGS. 4A, 4B and 4C show the end portions of various
embodiments of the invention;
[0028] FIG. 5 is a cross-sectional view of another embodiment of
the invention;
[0029] FIGS. 6A and 6B are alternative cross-sectional views of the
apparatus of FIG. 5 taken along section line 10-10;
[0030] FIGS. 7A-7D, 8 and 9 show various arrangements of optical
fibers;
[0031] FIG. 10 shows another embodiment of the invention;
[0032] FIG. 11A is a schematic diagram showing another embodiment
of the invention;
[0033] FIGS. 11B-11D show how target tissue can be divided into a
plurality of field areas;
[0034] FIG. 12 shows the steps of a method embodying the invention;
and
[0035] FIGS. 13-51 are graphs illustrating the results of various
tests conducted utilizing the invention.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0036] In the prior art methods in the Background of the Invention
Section, the information content of the interaction of light (and
consequently the spectroscopic method used) is, generally speaking,
specific to the type of change in tissue. That is, tumorous tissue
differs from normal tissue in several ways. Tumorous tissue is
generally derived from normal tissue after the latter has undergone
several changes. These changes can be induced by various intrinsic
and extrinsic factors. These include the presence of certain
inherited traits, chromosomal mutation, virus induced malignant
transformation of cells and the mutagenic effects of UV and X-ray
irradiation, to name a few.
[0037] The earliest changes that occur in the course of normal
cells becoming malignant are biochemical. One of the first changes
noted is that of increased glycolytic activity which allows tumors
to grow to a large size with decreased oxygen requirements.
Invasive tumor cells secrete type IV collegenase destroying the
basement membrane barrier, a principle component of which it
Collagen IV. This allows the invading tumor cells to pervade into
the underlying stroma or connective tissue. A number of other
enzymes (e.g. cathepsins, hyaluronidases, proteoglycans and type I,
II and III collagenases weaken the extracellular matrix and
contribute to further tumor invasion. As tumors enlarge in size to
beyond 1-2 mm.sup.3, the supply of oxygen and other nutrients
becomes limiting. A number of tumors have been shown to secrete
tumor angiogenesis factors, which induce the formation of blood
vessels within the tumor to supply the necessary oxygen and
nutrients for sustained tumor growth.
[0038] Morphological changes appear later in the course of tumor
progression. Such changes are defined as any change in average cell
size, cell appearance, cell arrangement and the presence of
non-native cells. In addition, increased perfusion due to the
effects of angiogenesis results in an overall difference in tissue
appearance. Normal tissue is highly differentiated in cell type and
arrangement. In addition, normal cells are highly tissue specific.
Tumor cells lose this tissue specificity, as well as cell
differentiation and arrangement. A marked difference between tumor
and normal cells is the change in the cytoskeleton, the network of
microtubules and microfilaments in the cytoplasm. The cytoskeleton
in normal cells is highly organized whereas that in tumor cells is
disorganized. Moreover because tumor cells are rapidly dividing,
the chromatin content in the nucleus and the nuclear size are both
higher than in normal cells.
[0039] Absorption spectroscopy is more sensitive to the
morphological changes that occur later in tumor progression.
Measurements are made either in a transmission geometry where the
sample is placed between the light source and detector, or in a
reflectance geometry where the source and detector, are on the same
side. In any configuration, changes in tissue absorption that occur
between tumors and normal tissue can be measured. For example, the
increased vascularization due to angiogenesis causes increased
blood absorption. Light propagating through and reemitted from
tissue is, however, strongly affected by light scattering
interactions and does not simply depend on the absorption spectrum
of tissue chromophores. Therefore, in addition to reporting changes
in absorption, such techniques are sensitive to changes in size,
structure and arrangement of cells and cellular organelles, all of
which contribute to a change in the scattering properties of
tissue. Tumor cells have enlarged nuclei and since nuclei have a
different refractive index from that of the cell cytoplasm, they
serve as efficient light scatterers. A similar behavior is observed
from other cell organelles such as, for example, mitochondria and
endoplasmic reticuli.
[0040] In absorption spectroscopy therefore, two effects,
absorption and scattering, dictate the amount of radiation measured
at the detector. Simply stated, these effects can either be
additive or may tend to cancel out each other. It is necessary,
therefore, to in some way to decouple these effects to provide an
accurate measurement of tissue properties. A number of techniques
have been described in the prior art to accomplish this. See, for
example, U.S. Pat. No. 5,630,423 to Wang, et al. and the references
cited therein, which are hereby incorporated by reference. It is
now possible to obtain within reasonable accuracy the coefficients
for scattering and absorption.
[0041] A different approach to absorption spectroscopy is the use
of reflectance depolarization techniques. In this approach linearly
polarized light is directed on the tissue and the returned
reflective image is viewed through polarizers parallel and
perpendicular to the direction of polarization of the incident
light. The parallel component has sampled the surface tissue and
the perpendicular component, after sampling deeper tissue, is
scattered multiple times and is consequently depolarized. By
analyzing photons that have sampled surface tissue the absorption
spectrum of this tissue independent of scattering effects can be
generated. Additionally, by modulating the extent of depolarization
in the returned radiation used for analysis, the depth of tissue
interrogated can be controlled.
[0042] Early biochemical changes are best detected by the change in
fluorescence properties of native chromophores. The principle
fluorophores present in tissue are the aromatic amino acids
tyrosine, phenylalanine and typtophan, the metabolites NAD(H) and
FAD(H) and structural proteins collagen and elastin. All of these
fluorophores possess characteristic absorption and fluorescence
spectra. The fluorescence properties of these molecules depends
upon their physicochemical environment including pH, solvation and
oxidation state. For example, the reduced form NAD(H) fluoresces
while the oxidized form does not. The reverse is true for FAD (H).
The action of various proteases secreted by tumor cells as
described above, on structural proteins, causes the fluorescent
moieties (tryptophan, phenylalanine etc.) to be exposed to a
different local environment (different salvation, viscosity and
hydrophobicity) thus changing their fluorescent
characteristics.
[0043] Although biochemical changes precede the morphological
changes that occur as a result of the former, it is unrealistic to
think of diseased tissue that differs from surrounding normal
tissue only in its intrinsic biochemistry. If this were true then
by simply measuring the fluorescence one could identify and locate
disease. In reality varying degrees of morphological change
accompany the biological changes. These changes appear later in the
course of tumor progression and are defined as any change in
average cell nuclei, cell size, cell appearance, cell arrangement
and the presence of non native cells. In addition, effects of the
host response such as, for example, increased perfusion from
angiogenesis results in an overall difference in tissue appearance.
The morphological changes add more complexity to the measurements
by absorbing and scattering both excitation and fluorescent light
thereby altering the true fluorescence signal. If the tumor is
early, the possibility of measurable morphological changes having
occurred are low and consequently fluorescence alone may be able to
identify early tumors from nearby normal tissue. However, once
significant changes in morphology have occurred the measurement now
involves the added complication of deconvolving or decoupling the
effects of fluorescence spectral changes from changes in
fluorescence signal due to scattering and reabsorption. For
example, in the diagnosis of hyperplasia and adenomatous polyps
from normal colonic tissue, a decrease in 390 nm fluorescence (337
nm excitation) is seen as the tissue types change from normal as
taught by Shoemacher et. al. at pages 63-78 of Lasers in Surg. Med
(12) 1992, which is hereby incorporated by reference. This could be
interpreted as a decrease in collagen fluorescence or an increase
in hemoglobin absorption. In fact, the authors show that the effect
is due to a screening of fluorescence from collagen (itself
unchanged) in the submucosal layer by the thickening mucosa in an
adenoma.
[0044] Clearly, therefore simply measuring the change in
fluorescence spectral shifts or intensity changes will not be
sufficient for accurately measuring changes in tissue
characteristics, and making, for example, a fluorescence based
diagnosis. It is difficult to make a fluorescence measurement that
is truly independent of the effect of scattering and
absorbance.
[0045] In order to decouple the effects of biochemical and
morphological changes, the relative degrees of which vary depending
upon the extent of tumor progression, a multimodal approach is
required. Such an approach requires a device capable of measuring
both fluorescence and absorption spectra of the area of interest.
Both measurements must be made on the same site at preferably the
same time so as to ensure identical condition.
[0046] The decoupling can be carried out in a variety of ways,
which are later discussed.
[0047] Time resolved fluorescence methods are largely independent
of the effects of scattering and absorbance. This is especially
true for diagnosis of epithelial cancer and similar conditions
where the distance traversed by light is small. Time resolved
measurement measures the fluorescence lifetime of a fluorophore.
This is an intrinsic molecular property and as such is independent
of extraneous interferences such as fluorosphore concentration
(provided a measurable signal with adequate signal to noise is
present) or light source fluctuations. Such methods have been
demonstrated for transcutaneous measurements from fluorescent
implants and have been shown to be superior to steady state
fluorescence measurements. See Bambot, et al., Biosens and
Bioelectronics (10) 1995 at pages 643-652 and U.S. Pat. No.
5,628,310 to Rao, et al. which are hereby incorporated by
reference. The same tissue biochemical changes that result in
fluorescence spectral shifts and intensity changes also generally
change fluorescence lifetimes. It is commonly known that non
radiative processes that depopulate the excited state of
fluorophore cause large changes in fluorescence lifetime. Such non
radiative processes are likely the result of a changing
physicochemical environment surrounding intrinsic fluorophores in
an emerging tumor.
[0048] Time resolved methods are accomplished in either the time
domain or frequency domain, the latter is also known as phase
modulation fluorimetry. Phase modulation measurements can be
accomplished with cheaper and less complex instrumentation than is
used to directly measure the decay time of fluorescence. For
example, an intensity modulated light beam may be directed upon the
sample. The fluorescence returned from the sample is also intensity
modulated at the same frequency. However, because of the finite
fluorescence lifetime of the fluorophore in tissue, the returned
fluorescence signal is phase shifted and this phase shift is
related to the fluorescence lifetime.
[0049] The biggest impediment to using time resolved methods
presently is cost. This cost is proportional to the magnitude of
both the modulation frequency and the frequency of light. The
modulation frequency used is nominally the inverse of the lifetime
of the fluorosphore being interrogated. Given the short (few
nanoseconds) fluorescence lifetimes of intrinsic chromophores in
tissue that serve as markers for disease, high modulation
frequencies (several hundred megahertz) are required, necessitating
the need for RF equipment and techniques. In addition, most
intrinsic chromophores have absorption maxima at low wavelengths
(high frequencies). Solid state light sources and detectors that
operate at these wavelengths and that are capable of being
intrinsically modulated at the requisite modulation frequencies are
expensive and rare. Having said this, both areas, low wavelength
light sources/detectors and RF frequency digital electronics are an
active area of research and development and significant cost
reductions are expected in the future.
[0050] An alternative to the phase change method discussed above to
determine fluorescence lifetime is the measurement of fluorescence
depolarization or anisotropy. The instrumentation used is similar
to that used for reflectance depolarization. Indeed the same
instrument can readily be used for measurement based on both
principles. In clear solution (where photons are not depolarized
due to scattering the measurement of fluorescence polarization
anisotropy provides an estimate of the fluorescence lifetime of the
fluorophores being interrogated. This is represented by the Perrin
Equation (Perrin et. al.) which relates fluorescence Anisotropy (r)
to Lifetime (.tau.) r o r = 1 + r .PHI. Equation .times. .times. 1
##EQU1## where r.sub.o, is the anisotropy of the molecule when
Brownian motion is absent, i.e. in the frozen state or in a highly
viscous medium, r is the time averaged anisotropy observed, .tau.
is the fluorescence lifetime of the molecule and .phi. is the
Brownian rotation correlation time.
[0051] Strictly speaking the above equation is valid only for a
single exponential decay in both fluorescence lifetime and
anisotropy. The anisotropy decay is single exponential only for a
spherical molecule (isotropic depolarization). The rotational
correlation time is defined, for simplicity, for a sphere to be;
.PHI. = .eta. .times. .times. V RT Equation .times. .times. 2
##EQU2## where .eta. is the viscosity, V the volume, R the
universal gas constant and T the absolute temperature.
[0052] As illustrated in Equation 1, the anisotropy reflects
changes from both fluorescence lifetime and rotational correlation
time. The fluorescence lifetime of intrinsic fluorophores change
with tumor progression. Similarly a change in local physical
properties such as microviscosity, temperature or membrane fluidity
will change the rotational correlation time and resulting in a
change in an isotropy. U.S. Pat. Nos. 4,115,699, 4,122,348 and
4,131,800, which are hereby incorporated by reference, disclose the
measurement of changes in local microviscosity and fluidity due to
malignancy using exogenous lipophilic dyes and the method of
fluorescence depolarization.
[0053] The principle drawback with this technique when applied to
in vivo tissue measurements is the depolarization caused by
multiple scattering events in tissue. It has been shown, however,
that a significant portion of the polarized excitation remains
polarized before exciting the fluorophore and the resulting
fluorescence is also substantially polarized when it reaches the
detector. Nevertheless, it is necessary to decouple depolarization
due to scattering from depolarization due to fluorescence lifetime
and rotation correlation time.
[0054] The techniques according to the invention are designed to
discriminate normal tissue from various cancerous tissue stages
based on spectroscopic data alone. Additional factors, such as, for
example, patient age, menopausal status, menstrual state, and/or
previous history of disease can be added to the spectroscopic
inputs in achieving better discrimination.
[0055] The multimodal approach according to the invention may be
carried out in an imaging mode. In other words the multiple
spectroscopic methods are used in interrogating tissue at several
interrogation points at high spatial resolution concurrently. The
reasoning behind this approach is the variability in spectroscopic
signature of known normal tissue between patients and the fact that
in 99% of the patients the entire organ is not diseased. The best
way to do this without an a prior knowledge of what is normal is to
measure both the normal and abnormal tissue, that is, the entire
organ.
[0056] Visually normal areas on an organ with precancers or cancers
are always suspect and a conclusive analysis is almost always the
result of a biopsy and histology. This observation is consistent
with the phenomenon of field cancerization, as discussed by D. P.
Slaughter, et al., in "Field Cancerization in Oral Squamour
Epithelium: Clinical Implication of Multicentric Origin" in Cancer
6, 1953, at pages 963-968, which is hereby incorporated by
reference. A considerable body of evidence exists, particulary for
breast cancer, which shows that supposedly normal breast epithelium
derived from patient's breast cancer is "condemned" in that it is
precancerous. This explains the relatively high rate of second
breast cancer incident in women treated for the disease, as
discussed by G. F. Schwartz, et al. in "The Prevalence of carcinoma
In Situ In Normal and Cancer Associated Breasts", Hum Pathol 16,
1985, at pages 796-807, which is hereby incorporated by reference.
The inventors believe a similar pattern exists with cervical cancer
and may explain the high number of women (50%) with a history of
negative pap tests who develop cervical cancer, as discussed in
Cancer Diagnostics, The World Market, Clinica Reports, PJB
Publications, 1997, at page 72, which is hereby incorporated by
reference. Such a pattern further warrants the use of imaging modes
in cancer detection.
[0057] The invention will now be further discussed with reference
to the drawings.
[0058] FIG. 1 is a schematic diagram of an apparatus according to a
preferred embodiment of the invention. The apparatus includes a
source 20, which produces electromagnetic radiation that is
conducted to a target tissue 50, preferably by one or more emission
optical fibers 52. The apparatus may also include a filter 22 for
selectively controlling the electromagnetic radiation emitted from
the radiation source 20. The source 20 could comprise, for example,
a laser, a light emitting diode, a fluorescent tube, an
incandescent bulb, or any other type of device that is capable of
emitting electromagnetic radiation, as is well known to those
skilled in the art.
[0059] Electromagnetic radiation returned from target tissue 50, is
sensed by a detector 56. As discussed below, the detector may
employ any of the known methods for determining tissue
characteristics, including but not limited to fluorescence,
absorption, reflectance, anisotropy, phase change, and any other
know spectroscopic methods including those methods discussed in the
Background of the Invention section of this disclosure. Preferably,
the detector employs two or more spectroscopic methods which
provides for a better or more accurate measure of target tissue
characteristics than one measurement alone, and thus a more
complete diagnosis of the tissue's condition.
[0060] The returned electromagnetic radiation comprises both
fluorescent emissions from fluorophores in the target tissue that
have been excited by the excitation radiation and the excitation
electromagnetic radiation that is scattered or reflected from the
target tissue. In a preferred embodiment of the invention, as later
discussed, the detector 56 makes intensity based measurements on
both forms of said electromagnetic radiation. These measurements
are combined to decouple the morphological changes from the
biochemical changes. The detector may comprise, for example, a
photomultiplier tube, a photosensitive diode, a charge coupled
device, or any other type of electromagnetic radiation sensor, as
is also well known to those skilled in the art.
[0061] If the detector is a small charge coupled device, it could
be located at a distal end of an endoscope or catheter instrument.
In this instance, the charge coupled device would already be
located adjacent the target tissue such that the detector could
directly sense the return radiation. The charge coupled device
would then need some means for communicating its information to a
processor 44.
[0062] If the detector is not a charge coupled device located at a
distal end of an instrument, the returned electromagnetic radiation
may be conducted to the detector 56 through one or more return
optical fibers 54. The return optical fibers 54 and the excitation
optical fibers 52 may be co-located within the same instrument, or
they may be located in separate instruments. Alternately, the same
optical fibers within an instrument may be used to perform both
excitation and return functions.
[0063] The processor device 44 may include a memory 45 and a
display 47. In fact, the processor device may comprise a typical
personal computer.
[0064] In the preferred embodiments of the invention, the detector
56 may detect the fluorescent emissions from fluorophores in the
target tissue simultaneously with the excitation electromagnetic
radiation that is scattered or reflected from the target tissue to
provide a complete analysis of the subject tissue. Alternatively,
the device may be configured to first detect the fluorescent
emissions from fluorophores in the target tissue, and then
subsequently, the excitation electromagnetic radiation that is
scattered or reflected from the target tissue. In the later case,
the time period between detections, hereinafter referred to as the
"critical timing window," must be minimized to avoid motion
artifacts and/or significant tissue changes that will denigrate the
overall results. The time period between detections is preferably
less than approximately 0.25 seconds; however, the smaller the time
period, the more accurate the results will be.
[0065] FIG. 2 shows an endoscope that could be used to practice the
measuring techniques according to the invention. The endoscope 60
includes a transmit optical fiber bundle 52, which can convey
excitation electromagnetic radiation from a radiation source 20 to
a target tissue. The endoscope 60 also includes a return optical
fiber bundle 54 for communicating fluorescent emissions and/or
reflected/scattered electromagnetic radiation from a target tissue
to a detector 56. In alternative embodiments, the transmit and
return optical fibers may be co-located, may be the same fibers, or
may be a double set of fibers, as discussed below.
[0066] That is, it is preferable to make simultaneous detections at
a plurality of interrogation points rather than at just one point
or a minimum number of points. This allows evaluation of the field
effect changes over an area of the tissue or substantially the
entire tissue, as will be more fully discussed below. Taking
measurements at just one interrogation point or a minimum number of
interrogation points can be misleading as it does not provide a
sufficient sampling of tissue area to accurately reflect the
tissue's condition.
[0067] For example, the detector could be configured to make
detections at a large number of interrogation points distributed
over substantially the entire surface area of the subject tissue.
That is, return optical fibers 54 could include a large number of
optical fibers distributed to allow detections to be made at a
corresponding large number of interrogation points on the tissue,
preferably covering substantially the entire surface of the subject
tissue. Each of the optical fibers could transmit excitation
electromagnetic radiation to the subject tissue and then return the
return electromagnetic radiation to the detector 56. The tissue
could be analyzed as a whole, or divided into a plurality of field
areas.
[0068] Alternatively, a transmitting optical fiber and a return
optical fiber could be located at each of the interrogation points
(see, for example, FIG. 7B). Further, each interrogation point
could include a double set of optical fibers, a transmitting
optical fiber and a return optical fiber for detecting
fluorescence, and a transmitting optical fiber and a return optical
fiber for detecting scattering or reflectance (see, for example,
FIG. 7C). In such a case, the optical fibers could be arranged to
focus on the same point on the subject tissue (see, for example,
FIG. 7D).
[0069] Additionally, the apparatus may include a rotatable core
114, as discussed with respect to the embodiment of FIG. 5, or
alternatively, the tissue may be mounted on a rotatable table (not
shown), so that the detector 56 would make detections at just a
portion of the multiple interrogation points. Then, either the
rotatable head or the rotatable table could be rotated and the
detector would make detections at the next set of interrogation
points. The process would continue to complete, for example, six
rotations in order to cover substantially the entire surface of the
subject tissue.
[0070] The endoscope 60 may also include a handle 62 for
positioning the endoscope, or for operating a device 64 on a distal
end of the endoscope 60 intended to remove tissue samples from a
patient. The endoscope may also include a device 66 for introducing
a dose of medication to a target tissue. Also, the source of
electromagnetic radiation 20 may be configured to emit a burst of
therapeutic radiation that could be delivered to a target tissue by
the endoscope.
[0071] FIGS. 3A and 3B show the structure of an endoscope or
catheter which may embody the invention. The apparatus includes a
long body portion 70 which is intended to be inserted into a body
of a human or animal. The body portion 70 must have a diameter
sufficiently small to be inserted into blood vessels or other
natural lumens of the human or animal.
[0072] The device includes a proximal end 80, which holds proximal
ends of optical fibers 72a-72c. The optical fibers extend down the
length of the device and terminate at a distal holding portion 74.
The distal holding portion 74 holds the optical fibers in a
predetermined orientation. The optical fibers are held such that
they can illuminate selected portions of the distal end 76 of the
device. This orientation also allows the distal end of the optical
fibers to receive radiation from selected areas outside the distal
end 76 of the device.
[0073] As best seen in FIG. 3B, the optical fibers are arranged
such that there is a single central optical fiber 72a surrounded by
a first ring of optical fibers 72b, which is in turn surrounded by
a second ring of optical fibers 72c. Of course, other orientations
of the optical fibers are possible.
[0074] By applying excitation electromagnetic radiation to selected
ones of the optical fibers, and monitoring the returned
electromagnetic radiation through selected ones of the optical
fibers, it is possible to determine characteristics of target
tissues at selected locations outside the distal end of the device.
For instance, if the central optical fiber 72a emits
electromagnetic radiation 90 toward a target tissue, and returned
electromagnetic radiation is sensed through the same optical fiber,
the returned electromagnetic radiation can be analyzed using any of
the above methods to determine characteristics of a target tissue
located adjacent the center of the distal end of the device. The
same process can be used to determine the condition of a target
tissue at different locations around the distal end of the
device.
[0075] FIGS. 4A-4C show various different distal ends of the
device.
[0076] In FIG. 4A, the distal ends of the optical fibers are held
by a holding portion 98 that aims the distal ends of the optical
fibers 97 in a particular direction. A flexible wire or bar 96 is
attached to the holding portion 98 and extends to the proximal end
of the device. By rotating the flexible wire or bar 96, the holding
portion 98 can also be rotated. This allows the distal ends of the
optical fibers to be aimed at different portions of the distal end
of the device.
[0077] FIG. 4B shows another embodiment of the invention that
includes one or more inflatable balloon portions 92a, 92b. An
optical fiber 72 is located in the center of the device by a
holding portion 94. Each of the inflatable balloons 92a, 92b is
also held by the holding portion 94. By selectively inflating or
deflating the different balloon portions, the optical fiber 72 may
be aimed to illuminate different portions of the distal end of the
device or to receive return radiation from selected locations
adjacent the distal end of the device.
[0078] FIG. 4C shows an embodiment of the device similar to the
embodiment shown in FIGS. 3A and 3B. This figure shows how
electromagnetic radiation passing down through the optical fibers
72a-72c can be used to selectively illuminate material or tissue
adjacent selected portions of the distal end of the device. In FIG.
4C, only the upper optical fibers are emitting electromagnetic
radiation outside the device. This electromagnetic radiation is
being used to destroy or atomize plaque which has formed on an
inner wall of a blood vessel. By applying electromagnetic radiation
to selected ones of the optical fibers, a doctor can carefully
remove or correct problems with target tissues or materials.
[0079] Another device embodying the invention that can be used to
determine tissue characteristics is shown, in longitudinal
cross-section, in FIG. 5. The instrument 110 includes a cylindrical
outer housing 112 with a circular end cap 120 configured to abut
the target tissue. A rotating cylindrical inner core 114 is mounted
in the outer housing 112. A bundle of optical fibers 116 are
located inside the inner core 114.
[0080] The optical fibers 116 pass down the length of the inner
core 114 and are arranged in a specific pattern at the end adjacent
the end cap 120 of the outer housing 112. The end of the inner core
114 adjacent the end cap 120 is mounted within the outer housing
112 with a rotating bearing 122. The end cap 120 is at least
partially transparent or transmissive so that electromagnetic
radiation can pass from the optical fibers, through the end cap, to
illuminate a target tissue adjacent the end cap 120. Light
scattered from or generated by the target tissue would then pass
back through the end cap 120 and back down the optical fibers
116.
[0081] The inner core 114 is also mounted inside the outer housing
112 by a detent mechanism 118. The detent mechanism is intended to
support the inner core 114, and ensure that the inner core is
rotatable within the outer housing 112 by predetermined angular
amounts.
[0082] A cross sectional view of an embodiment of the instrument,
taken along section line 10-10 of FIG. 5, is shown in FIG. 6A. The
inner core 114 is supported within the outer housing 112 by the
detent mechanism. In this embodiment, the detent mechanism includes
two mounts 134 with spring loaded fingers 136 that are biased away
from the inner core 114. The detent mechanism also includes four
stoppers 130, each of which has a central depression 132. The
spring loaded fingers 136 are configured to engage the central
depressions 132 of the stoppers 130 to cause the rotatable inner
core to come to rest at predetermined angular rotational positions.
In the embodiment shown in FIG. 6A, four stoppers are provided in
the inner surface of the outer housing 112. Thus, the inner core
114 will be rotatable in increments of approximately 90.degree.. In
alternate embodiments similar to the one shown in FIG. 6A, four
mounts 134, each having its own spring loaded finger 136, could be
attached to the inner core 114. The provision of four such mounts
would serve to keep the inner core 114 better centered inside the
outer housing 112.
[0083] An alternate embodiment of the detent mechanism is shown in
FIG. 6B. In this embodiment, six stoppers 130 are spaced around the
inside of the outer housing 112. Three mounts 134, each having its
own spring loaded finger 136, are mounted on the inner core 114.
The three mounts 134 are spaced around the exterior of the inner
core 114 approximately 120.degree. apart. This embodiment will
allow the inner core to be rotated to predetermined positions in
increments of 60.degree.. In addition, the location of the three
mounts, 120.degree. apart, helps to keep the inner core 114
supported in the center of the outer housing 112.
[0084] With reference to FIG. 5, the ends of the optical fibers may
be mounted on a circular end plate 121 that holds the optical
fibers in a predetermined pattern. The circular end plate 121 would
be rigidly attached to the end of the cylindrical inner core 114.
In addition, an index matching agent 123 may be located between the
end plate 121 and the end cap 120 on the outer housing 112. The
index matching agent 123 can serve as both an optical index
matching agent, and as a lubricant to allow free rotation of the
end plate 121 relative to the end cap 120.
[0085] A diagram showing how the optical fibers are positioned on
the face of an embodiment of the instrument is shown in FIG. 7A.
The face of the instrument, which would be the end cap 120 of the
device shown in FIG. 5, is indicated by reference number 140 in
FIG. 7A. The black circles 142 represent the locations of optical
fibers behind the end cap 120. The hollow circles 144 represent the
positions that the optical fibers will move to if the inner core
114 of the instrument is rotated approximately 90.degree.. Thus,
each of the circles represent positions that can be interrogated
with the optical fibers.
[0086] In some embodiments of the device, a single optical fiber
will be located at each of the positions shown by the black circles
142 in FIG. 7A. In this instance, excitation light would travel
down the fiber and be emitted at each interrogation position
indicated by a black circle 142. Light scattered from or produced
by the target tissue would travel back up the same fibers to a
detector or detector array, such as detector 56 shown in FIG.
1.
[0087] In alternate embodiments, pairs of optical fibers could be
located at each position indicated by the black circles 142A, 142B,
as shown in FIG. 7B. In the alternate embodiments, one optical
fiber of each pair would conduct excitation light to the target
tissue, and the second optical fiber of each pair would conduct
light scattered from or generated by the target tissue to a
detector. In still other alternate embodiments, multiple fibers for
carrying excitation light and/or multiple fibers for carrying light
scattered from or generated by the target tissue could be located
at each interrogation position indicated by a black circles 142A,
142B, 142C, 142D to allow simultaneous detection of, for example,
both fluorescence and reflectance, as shown in FIG. 7C. In this
latter case, the optical fibers could be arranged to focus on the
same point of subject tissue, as shown in FIG. 7D.
[0088] To use an instrument having the optical fiber pattern shown
in FIG. 7A, the instrument would first be positioned so that the
end cap 120 is adjacent the target tissue. The end cap 120 may be
in contact with the target tissue, or it might be spaced from the
surface of the target tissue. Also, an index matching material may
be interposed between the end cap 120 and the target tissue. Then,
the optical fibers would be used during a first measurement cycle
to simultaneously measure tissue characteristics at each of the
interrogation positions in FIG. 7A having a black circle 142. The
tissue characteristics could be measured using any of the
measurement techniques discussed above. Then, the inner core 114
would be rotated approximately 90.degree. within the outer housing
112, and the optical fibers would be used during a second
measurement cycle to simultaneously measure tissue characteristics
at each of the interrogation positions in FIG. 7 having a hollow
circle 144.
[0089] The instrument may include markings (not shown) on the end
cap 120 or elsewhere, which acts as a locator tool to allow a user
to determine how many rotations have been made, and thus how much
of the tissue has been analyzed.
[0090] Constructing an instrument as shown in FIGS. 5, 6A or 6B,
and having any of the optical fiber patterns shown in FIGS. 7A-7D,
has many important advantages. For example, constructing an
instrument in this manner allows the instrument to interrogate many
more points in the target tissue than would have been possible if
the inner core did not rotate. The ability to rotate the inner core
114, and take a second series of measurements at different
locations on the target tissue, essentially increases the
resolution of the device.
[0091] In addition, when a large number of optical fibers are
packed into the tissue contacting face of an instrument, cross-talk
between the optical fibers can occur. The cross-talk can occur when
excitation light from one interrogation position scatters from the
target tissue and enters an adjacent interrogation position.
Cross-talk can also occur if excitation light from a first
interrogation position travels through the target tissue and enters
an adjacent interrogation position. One of the easiest ways to
reduce or eliminate cross-talk is to space the interrogation
positions farther apart. However, increasing the spacing between
interrogation positions will reduce the resolution of the
device.
[0092] An instrument embodying the invention, with a rotatable
inner core, allows the interrogation positions during any single
measurement cycle to be spaced far enough apart to reduce or
substantially eliminate cross-talk. Because multiple measurement
cycles are used, the device is able to obtain excellent resolution.
Thus, good resolution is obtained without the negative impact to
sensitivity or selectivity caused by cross-talk. In addition, fewer
optical fibers and fewer corresponding detectors are required to
obtain a given resolution.
[0093] In addition, the ability to obtain a plurality of tissue
measurements simultaneously from positions spaced across the entire
target tissue has other benefits. If the instrument is intended to
detect cancerous growths or other tissue maladies, the target
tissue area interrogated by the instrument is likely to have both
normal tissue, and diseased tissue. As noted above, tissue
characteristics can vary significantly from person to person, and
the tissue characteristics can vary significantly over relatively
short periods of time. For these reasons, one way to determine the
locations of diseased areas is to establish a baseline for normal
tissue, then compare the measurement results for each interrogation
point to the baseline measurement. The easiest way to determine the
location of a diseased area is to simply look for a measurement
aberration or variance.
[0094] Because tissue characteristics can change relatively
quickly, in order to establish accurate, clearly defined variances
between tissue characteristics, it is desirable to take a plurality
of readings simultaneously over as large an area as possible. In
the preferred method according to the invention, this could include
taking fluorescence measurements at a plurality of interrogation
points, and then subsequently taking reflectance measurements, at
the same plurality of interrogation points. Alternately, the
fluorescence and reflectance measurements could be taken
simultaneously. Ideally, all measurements should be conducted
during the same time period. In a preferred embodiment, the
apparatus and method conduct measurements at least within this
critical time window. The critical time window is defined as the
maximum duration of time between two spectroscopic measurements
which yields the benefits described herein. Although this value may
vary depending on a variety of factors including those described
below, it has been determined that the critical timing window
between subsequent measurements should be less than approximately
0.25 seconds and more preferably less than approximately 0.1
second, as further discussed below.
[0095] There are several effects which make it desirable to conduct
fluorescent and reflectance measurements of the interrogated points
either simultaneously, or as nearly simultaneously as possible.
First, changes in blood pressure, which occur during each heart
beat cycle can have a large affect on blood content in the tissue.
Because blood strongly absorbs certain wavelengths of light, the
varying amount of blood present at an interrogated point during
different parts of the heart beat cycle can cause significantly
varying measurement results.
[0096] To eliminate this potential error source, both fluorescent
and reflectance measurements should be taken within a small enough
time window that the blood content remains the same. Time periods
of less than approximately 0.25 seconds should be sufficient.
Another way to eliminate the potential error is to take multiple
measurements of the same interrogation point during different
portions of the heart beat cycle, then average the results.
[0097] Another factor to consider is patient movement. If the
patient moves, even slightly, during a measurement cycle, the
contact pressure between the measurement instrument and the
interrogated tissue can change. This can also affect the
measurement results. Thus, obtaining measurements simultaneously,
or as nearly simultaneously as possible, also helps to prevent
measurement errors caused by patient movement.
[0098] Also, because tissue tumors can be as small as approximately
1 mm, the resolution of the device is preferably approximately 1
mm. In other words, to obtain the requisite resolution, the spacing
between interrogation positions should be approximately 1 mm.
Unfortunately, when the interrogation positions are approximately 1
mm apart during a single measurement cycle, significant cross-talk
can occur, and the accuracy of the measurement results is poor.
[0099] An instrument embodying the invention allows the
interrogation positions to be spaced sufficiently far apart to
essentially eliminate cross-talk, while still obtaining the
requisite 1 mm resolution. Although not all measurements are
obtained at exactly the same time, during each measurement cycle,
simultaneous measurements are made at positions spaced across the
entire target tissue, which should include both normal and diseased
areas. Thus, the results from each measurement cycle can be used to
detect variances in tissue characteristics that help to localize
diseased areas. For these reasons, an instrument embodying the
invention balances the competing design requirements of resolution,
elimination of cross-talk, and the desire to make all measurements
simultaneously to ensure that time-varying tissue characteristics
are taken into account.
[0100] A second arrangement for the optical fibers of a device as
shown in FIG. 5 is depicted in FIG. 8. In this embodiment, the
interrogation positions are arranged in a hexagonal honeycomb
pattern. The black circles 142 indicate the positions that would be
occupied by optical fibers during a first measurement cycle, and
the hollow circles 144 indicate positions that would be occupied by
the optical fibers during a second measurement cycle after the
inner core 112 has been rotated by approximately 60.degree.. This
pattern achieves maximum spacing between adjacent interrogation
positions during each measurement cycle, and essentially doubles
the resolution of the instrument.
[0101] A third arrangement for the optical fibers of a device shown
in FIG. 5 is depicted in FIG. 9. In this embodiment, the optical
fibers are again arranged according to a hexagonal honeycomb
pattern. However, far fewer optical fibers are used in this
embodiment. This third embodiment is intended for use in a
measurement process that calls for six measurement cycles. The
inner core of the device would be rotated approximately 60.degree.
between each measurement cycle. Over the course of the six
measurement cycles, the device would ultimately interrogate all the
black circled 142 and hollow circled 144 interrogation positions
shown in FIG. 9. This embodiment allows for even greater separation
distances between the interrogation positions during a single
measurement cycle (to reduce or substantially eliminate
cross-talk), while still achieving excellent measurement
resolution. In addition, far fewer optical fibers and corresponding
detectors would be required to achieve a given measurement
resolution.
[0102] Experimental studies were conducted by the applicants to
determine the spacing between interrogation positions that is
needed to substantially eliminate cross-talk. The studies were
conducted using a pair of optical fibers at each interrogation
position, wherein one fiber in each pair provides excitation light,
and the other fiber in each pair is used to detect light. The
excitation optical fibers had a diameter of approximately 200
.mu.m, the detection fibers had a diameter of approximately 100
.mu.m. Measurements were made on optical reference standards, and
tissue. Under these conditions, it was necessary to space the
interrogation positions approximately 3 mm apart to substantially
eliminate cross-talk. Thus, if an instrument were not designed as
described above, so that the inner core can rotate the
interrogation positions to different locations on the target
tissue, the device would only be capable of achieving a resolution
of approximately 3 mm.
[0103] The presently preferred embodiment of the invention utilizes
an optical fiber pattern similar to the one shown in FIG. 9. Thus,
the device is designed to conduct six measurement cycles to
complete all measurements within the target tissue. The inner core
114 is rotated 60.degree. between each measurement cycle. The
presently preferred embodiment utilizes optical fiber pairs at each
interrogation position. Each optical fiber pair includes an
excitation fiber having an approximately 200 .mu.m diameter, and a
detection optical fiber having an approximately 100 .mu.m diameter.
The arrangement of the optical fibers allows the interrogation
positions to be spaced approximately 3.0-3.5 mm apart, while still
achieving a resolution of approximately 1 mm.
[0104] To determine the locations of diseased areas within a target
tissue it is necessary to take measurements at a plurality of
different locations in the target tissue spaced in at least two
dimensions. Each measurement may require multiple excitation
wavelengths, and detection of multiple wavelengths of scattered or
generated light. Thus, the measurements involve three measurement
dimensions, two dimensions for the area of the target tissue, and a
third dimension comprising the spectral information. A device
capable of conducting measurements in these three dimensions is
shown in FIG. 10.
[0105] The instrument includes a light source 20, and a filter
assembly 22. A plurality of excitation optical fibers 116a lead
from the filter assembly 22 to the target tissue 50. A plurality of
detection fibers 116b lead away from the target tissue 50. The
excitation optical fibers 116a and the detection optical fibers
116b are arranged in pairs as described above.
[0106] The light source 20 and filter assembly 22 allow specific
wavelengths of light to be used to illuminate the target tissue 50
via the excitation optical fibers 116a. The filter assembly 22
could be a single band pass optical filter, or multiple optical
filters that can be selectively placed between the light source 20
and the excitation optical fibers 116a. Alternatively, the light
source 20 and filter assembly 22 could be replaced with a
wavelength tunable light source. In yet other alternate
embodiments, a plurality of light sources, such as lasers, could be
used to selectively output specific wavelengths or wavelength bands
of excitation light. Other sources may also be appropriate.
[0107] The detection fibers lead to an optical system 55. The light
from the detection fibers 116b passes through the optical system
and into a detector array 56. The detector array may comprise a
plurality of photosensitive detectors, or a plurality of
spectrophotometers. The detector array 56 is preferably able to
obtain measurement results for each of the detection fibers 116b
simultaneously.
[0108] The optical system 55 can include a plurality of optical
filters that allow the detector array 56 to determine the intensity
of light at certain predetermined wavelengths. In a preferred
embodiment, the detector array would be a two dimensional array of
photosensitive detectors, such as a charge coupled device (CCD).
The optical system would comprise a spectrograph that is configured
to separate the light from each detection optical fiber 116b into a
plurality of different wavelengths, and to focus the different
wavelengths across a line of pixels on the CCD. Thus, each line of
pixels on the CCD would correspond to a single detection fiber. The
intensities of the different wavelengths of light carried by a
single detection fiber 116b could be determined based on the
outputs of a line pixels of the CCD. The greater the output of a
particular pixel, the greater the intensity at a particular
wavelength.
[0109] The preferred embodiment is able to achieve excellent
flexibility. Because all wavelengths of light are always detected,
the instrument software can simply select the pixels of interest
for each measurement, and thereby determine the intensity at
particular wavelengths. During a first measurement, certain pixels
representative of fluorescent characteristics could be examined.
During a subsequent measurement, different pixels representative of
scattering characteristics could be examined. Also, the device
could be essentially re-configured to take completely different
measurements by simply changing the control software. Thus, a
single device could be used for a wide variety of different kinds
of measurements.
[0110] In preferred methods of the invention, one of the structures
described above would be used to conduct a series of measurements
cycles. Where the embodiment having the rotatable core is employed,
the inner core of the device would be rotated between measurement
cycles. Once all measurements of a measurement cycle are completed,
the inner core would be rotated, and additional measurement cycles
would be conducted.
[0111] In the preferred methods, however, measurements are
conducted using two or more spectroscopic methods during each
measurement cycle. For instance, during a single measurement cycle
the device may conduct a measurement of fluorescent
characteristics, and a measurement of reflectance characteristics.
However, other measurements and combinations of spectroscopic
methods may also be appropriate. Then, the fluorescence and
reflectance measurements can be compared and analyzed to decouple
the effects due to biochemical and morphological tissue changes to
provide for a more accurate diagnosis of the tissue's
conditions.
[0112] As previously discussed, the measurements can be taken over
substantially the entire surface area of the subject tissue,
simultaneously or in intervals, and the results analyzed.
Alternatively, the subject tissue can be divided into field areas
to create a field pattern. Dividing the subject tissue into field
areas allows analysis of particular areas of the tissue, for
example, particular areas of the tissue where changes are likely to
occur.
[0113] For example, the apparatus of FIG. 1 could further include a
field area adjusting unit 560 and field area processing unit 570,
as shown in FIG. 11A. The field area adjusting unit 560 would
divide the target tissue into a plurality of field areas 580, as
shown in FIGS. 11B-11D, to create a field pattern 500. The field
areas 580 could be any desired shape and size (see, for example,
the different sized and shaped field areas shown in FIGS. 11B-11D).
Further, the divisions could be based on visual inspection of the
target tissue, or on results of previous testing performed on the
target tissue, and could be preprogramed into the apparatus, or
input by a user. Measurements would then be taken by the detector
54 at each of a plurality of interrogation points 542 within the
respective field area and the field area processing unit 570 would
then analyze the measurements for each of the respective field
areas 580. The field area processing unit 570 could further compare
the results for each respective field area 580 to the results for
other field areas 580.
[0114] FIGS. 11B and 11C show 4 and 8 "pie-shaped" field areas,
respectively. In each case, after measurements were taken by the
detector 54 at each of a plurality of interrogation points 542
within the respective field areas and the results analyzed by the
field area processing unit 570 for each of the respective field
areas 580, the field area adjusting unit 560 could reset the field
areas 580 by rotating the field area to group different sets of
interrogation points (see arrow in FIG. 11C), or could set field
areas having a different size and shape, such as the field areas
shown in FIG. 11D. As shown in FIG. 11 D, these field areas do not
need to be identical in size and/or shape.
[0115] Alternatively, the field area adjusting unit 560 and field
area processing unit 570 could be incorporated into the processor
44 and the divisions could be preprogramed into the processor or
accompanying software.
[0116] FIG. 12 shows steps of a preferred method according to the
invention. In a first step S1000, a target tissue is illuminated
with electromagnetic radiation at predetermined wavelengths,
preferably one wavelength for detecting fluorescence
characteristics and one wavelength for detecting reflectance
characteristics. In a second step S1010, the detector 56,
preferably utilizing one of the optical fiber arrangements
discussed above, detects returned electromagnetic radiation. In
step S1020, the fluorescence and reflectance intensities are
calculated, and in step S1030, the fluorescence and reflectance
intensities are compared and analyzed using a preferred method
discussed below. In step S1040, the tissue characteristics are
determined.
[0117] The deconvolution, or decoupling can be carried out in a
variety of ways as described below. Any or all of the discriminant
parameters can be combined together in order to improve the overall
discrimination.
[0118] 1. Using a linear combination of fluorescence and
reflectance measured intensities as the discriminant parameter.
P=aF.sub..lamda.m+bR.sub..lamda.x+cR.sub..lamda.m Equation 1 Where
.lamda.x is the fluorescence excitation wavelength, .lamda.m is the
fluorescence emission wavelength, F is the fluorescence intensity
and R is the reflectance intensity. The factors a, b and c are
weighing factors that are empirically selected to give the best
discrimination.
[0119] 2. Using a linear combination for fluorescence and
reflectance ratios as the discriminant parameter. P = a .times. F _
.lamda. .times. .times. m + b .times. F _ .lamda. .times. .times. m
.times. R .lamda. .times. .times. m R .lamda. .times. .times. x
Equation .times. .times. 2 ##EQU3##
[0120] 3. Using a linear combination for fluorescence and
reflectance ratios at multiple fluorescence emission wavelengths as
the discriminant parameter. P = a .times. F _ .lamda. .times.
.times. 1 .times. m + b .times. R _ .lamda. .times. .times. 1
.times. m + c .times. R _ .lamda. .times. .times. 2 .times. m
.times. F .lamda. .times. .times. 2 .times. m R .lamda. .times.
.times. 1 .times. x R .lamda. .times. .times. 2 .times. x Equation
.times. .times. 3 ##EQU4##
[0121] Where .lamda.1m and .lamda.2m are two distinct fluorescence
emission wavelengths, .lamda.1x and .lamda.2x are the corresponding
excitation wavelengths, F is the fluorescence intensity and R is
the reflectance intensity. The factors a, b and c are weighing
factors that are empirically selected to give the best
discrimination.
[0122] 4. Using quantum yield (also known as quantum efficiency)
measurement as the discriminant parameter. The quantum yield
defines the true fluorescence yield in terms of the number of
fluorescence photons generated by the fluorophore per photon of
light absorbed. P = aF _ .lamda. .times. .times. m .times. 1 - bR
.lamda. .times. .times. x Equation .times. .times. 4 ##EQU5## The
fluorescence and reflectance intensities are corrected for
background light and normalized to the intensities measured off a
calibration target. The factors a and b are weighing factors that
are empirically selected to give the best discrimination.
[0123] 5. Blood has broadband absorbance with three distinct
visible peaks at around 410 nm, 545 nm and 575 nm. On the one hand,
blood absorbance changes from increased vascularization in cancer
tissue, and is an important marker for disease. On the other hand,
blood absorbance related artifacts occur in the measured spectra
from local bleeding and inflammation. The spectral discriminant
factor described above must therefore be corrected for blood
absorbance. This can either be done by normalizing the discriminant
factor to blood reflectance. P corr = P _ R blood Equation .times.
.times. 5 ##EQU6## Or by subtracting the blood reflectance.
P.sub.corr=P-d.R.sub.blood Equation 6 Where d is an empirical
correction factor and R.sub.blood is the reflectance of blood at an
empirically selected wavelength.
[0124] 6. Alternatively the intensity set, F.sub..lamda.m,
R.sub..lamda.m and R.sub..lamda.x, where .lamda. is selected for
each fluorophore are collectively modulated against the pathology
results in a principle component analysis or a logistic regression.
These can then form the basis of pattern recognition techniques,
such as, for example, classification and regression trees (CART),
as taught by L. Brieman, et al. in Classification and Regression
Trees, Monterey Calif.: Wadsworth & Brooks/Cole, 1984, which is
hereby incorporated by reference, normal networks and hybrids
thereof.
[0125] The techniques according to the invention are designed to
discriminate normal tissue from various cancerous tissue stages
based on spectroscopic data alone. Additional factors, such, as for
example, patient age, menopausal status, menstrual state, previous
history of disease can be added to the spectroscopic input in
achieving better discrimination.
[0126] In each of the embodiments described above, in which a
plurality of measurement cycles are conducted on a target tissue,
and an inner core having optical fibers arranged in a predetermined
pattern is rotated between measurement cycles to make a plurality
of measurements on a target tissue, alternate embodiments could use
some other movement mechanism other than a rotating one. The
invention encompasses other types of movement or translational
devices that allow a plurality of measurements to be taken on a
target tissue with a limited number of detectors that are spaced
far enough apart to avoid cross-talk. Also, as previously
discussed, the measurements could be taken over the entire area of
the subject tissue simultaneously, or the target tissue could be
divided into field areas and measurements could be taken in each
field area.
[0127] Further, the apparatus and methods embodying the invention
make it possible to conduct in vivo measurements of tissues on the
inside of body passages or lumens. An endoscope embodying the
invention can be inserted into a natural body lumen of a human or
animal to search for the presence of cancerous or diseased tissue.
This means that no surgery would be required to locate and examine
tissues inside the body of the human or animal under study.
[0128] The use together of fluorescence measurements along with
reflectance measurements provides a more accurate determination of
target tissue characteristics than one of the measurements
alone.
[0129] The techniques described above can be used to map the
conditions of an area of target tissue. For instance, the
above-described techniques can be used to determine a condition of
a target tissue adjacent a distal end of a measuring device. The
measuring device could then be moved adjacent a different portion
of the target tissue, and the measurements could be repeated. This
process could be repeated numerous times to determine the
conditions of different portions of a target tissue area. The
determined conditions could then be used to create a map of the
target tissue area, which could be printed or displayed on a
monitor.
[0130] One of the most difficult problems with in vivo tissue
diagnostics and disease measurement is the biological diversity of
normal tissue properties between different patients, or even within
the same patient. Furthermore, this diversity is time variant both
in the long term and in the short term. Long term variations may be
due to patient age, hormonal milieu, metabolism, mucosal viscosity,
and circulatory and nervous system differences. Short term
variations may be from blood perfusion changes due to heart beat,
physical movement, local temperature changes etc.
[0131] Because of the variability of tissue characteristics, to
accurately determine whether a target tissue is diseased, one needs
to compare measurements of the target tissue to measurements of
normal tissues from the same patient. The measurements of the known
normal tissue should be made concurrently or simultaneously with
the measurements of the target tissue. The normal tissue
measurements then serve as a baseline for normalcy, variations from
which may be interpreted as disease. To arrive at a baseline
measurement, a number of strategies can be used.
[0132] First, visual characteristics such as pigmentations (nevi)
in skin, or polyps in the colon, can be used to identify
potentially abnormal regions. Normalized or averaged spectra of
multiple regions surrounding these potentially abnormal, visually
distinct regions can be used to establish baseline measurements.
The baseline measurements can then be compared to measurements
taken on the abnormal, visually distinct regions.
[0133] Measurements of normal and abnormal regions based on visual
characteristics could be automated using imaging capabilities of
the measurement device itself.
[0134] In an alternate strategy, measurements can be taken on
spaced apart regions along a portion of a lumen or tissue. The
spacing between the regions would be dependent on the type of
tissue being diagnosed. Then, differentials between individual
measurements taken at different regions would be calculated. If
differentials are greater than a preset amount, the tissue between
the excessively high differentials would be diagnosed as diseased.
In yet another alternate strategy, a gradient in spectral response
as one moves away from a visually suspicious site could also be
used as a marker for disease. This is easily automated and can be
implemented effectively in any imaging modality.
[0135] In addition, pattern recognition algorithms (e.g. neural
nets) could also be used to analyze differences in readings taken
from various sites in the same patient or from multiple readings
from different patients.
[0136] Preliminary testing was completed utilizing the above taught
apparatus and methods to determine the effectiveness of the
invention in determining tissue changes in the cervix. The results
are set forth below. The testing compared the results obtained by
the invention to cytology, colopscopy and histology results.
[0137] The study involved 27 human enrollees; however, data from
one patient could not be collected due to an equipment error. Five
of the patients were measured using a first generation probe having
a rigid transparent window at the device/cervix interface and a
monochrometer capable of producing excitation electromagnetic
radiation at wavelengths of 290 and 460 mm. However,
signal-to-noise analyses indicated that stray light and other
problems rendered much of the data unusable, especially at higher
wavelengths (460 nm) with respect to the fluorescence measurements
and at all wavelengths with respect to the reflectance
measurements. For the remaining patients, a second generation probe
was used having a flexible window as well as a new monochrometer,
which allowed an additional excitation electromagnetic wavelength
of 350 nm to be used.
[0138] Cytology, colopscopy and histology results of the twenty-one
patients are compared in Table 1 below. The histopathology results
for the twenty-one patients revealed that six had moderate/high
grade dysplasia or above, including one cancer. Of the fifteen
sub-high grade cases tested, seven had low grade dysplasia, three
had inflammation, two appeared normal at colposcopy but had a
history of cervical disease and were treated with a topical
therapeutic ninety days prior under a separate experimental
protocol, two had abnormal Pap results but were not biopsied due to
normal colposcopy, and one had both normal Pap test and colposcopy
results and therefore was not biopsied. TABLE-US-00001 TABLE 1
Patient Number Cytology from Pap Test Coposcopy Diagnosis Histology
from Biopsy 101-001 ASCUS Low Grade Dysplasia Low Grade Dysplasia
101-002 Low Grade Dysplasia Low Grade Dysplasia No dysplasia seen
101-003 High Grade Dysplasia High Grade Dysplasia High Grade
Dysplasia 101-004 Cancer Cancer Invasive Cancer 101-005 Low Grade
Dysplasia No Lesion Seen No Biopsy 101-006 ASCUS No Lesion Seen
Inflammation 101-007 Reactive Changes High Grade Dysplasia High
Grade Dysplasia 101-008 ASCUS Low Grade Dysplasia Low Grade
Dysplasia 101-009 ASCUS Low Grade Dysplasia Low Grade Dysplasia
101-010 Normal Normal No Biopsy (Normal) 101-011* N/A N/A N/A
101-012 ASCUS Metaplasia Low Grade Dysplasia 101-013 Reactive
Changes High Grade Dysplasia Inflammation 101-014 Not available yet
Low Grade Dysplasia Inflammation 101-015 ASCUS No Lesion Seen No
Biopsy 101-016 ASCUS Metaplasia High Grade Dysplasia 101-017 High
Grade Dysplasia No Lesion Seen High Grade Dysplasia 102-001 Normal
Low Grade Dysplasia Low Grade Dysplasia 102-002 High Grade
Dysplasia Parakeratosis No lesion seen** 102-003 Normal Low Grade
Dysplasia Low Grade Dysplasia 102-004 Inflammation High Grade
Dysplasia Low Grade Dysplasia 102-005 Normal Metaplasia High Grade
Dysplasia *Patient enrolled but not measured. **There was no lesion
seen on colposcopy, but there might be changes inside the
canal.
[0139] Of the six high grade dysplasias/cancer, the Pap test
mis-classified three as being either normal, reactive or ASCUS
(sensitivity=50%). Of the ten sub-high grade cases for which both
Pap test and biopsy results were available, the Pap test classified
all ten as sub-high grade (specificity=100%). Colposcopy also
classified only three of six high grade/cancer cases accurately
(50% sensitivity) but correctly classified eight of ten sub-high
grade lesions correctly (specificity=80%).
[0140] Intensities were examined at specific wavelengths which
correspond to the presence and activity of known biomolecules in
cervical tissue. Fluorescence measurements were taken at
wavelengths of approximately 290 nm (Tryptophan), 350 nm (NADH) and
460 nm (FAD). Reflectance measurements were taken at wavelengths of
approximately 320 nm, 420 nm (Hemoglobin) and a range of 540-580 nm
(Hemoglobin). These reference the dominant biomolecules for these
wavelengths; however, secondary biomolecules, such as, for example,
collagen and elastin may also be excited. A reflectance peak was
found at about 320 nm which appears to represent a point in the
spectrum where the above discussed and other biomolecules do not
absorb, thus producing the observed reflectance peak.
[0141] The measurements were made using a fiber optic system, which
acquired fluorescence and reflectance intensity data as a series of
CCD images. In order to extract meaningful data, the tissue spectra
underwent a series of correction and calibration operations prior
to tissue measurements. Wavelength calibration was performed, which
involves assigning a wavelength to each spectral data point.
Background subtraction was performed, which involves removing the
"dark" signal present on the CCD and any ambient light signal (e.g.
from room lights) acquired during tissue measurements. Intensity
calibration was performed, which involves normalizing tissue
spectral intensities by the intensities measured of a
fluorescence/reflectance standard. Stray light correction was
performed, which involves correcting tissue fluorescence spectra
for excitation monochromator stray light. Also, a system response
correction was performed, which involves correcting for the
non-uniform spectral response of the collection system (optical
fibers, filter, spectrograph, CCD).
[0142] In order to assess data quality, signal-to-noise ratio
(SNR), cross talk and variability were examined. Signal-to-noise
ratio is electronic noise from the CCD as well as optical "noise"
and artifacts superimposed on the fluorescence signal. If the SNR
is greater than needed, the exposure time can be reduced. If the
SNR is too low, the exposure time may need to be increased and
other options explored. Cross talk occurs when the amount of light
collected by a given collection optical fiber is influenced by
other surrounding excitation fibers. This parameter is strongly
dependent on the stand off between the fibers and the tissue.
Variability involves the amount of inter- and intra-patient
variability in spectra (e.g., intensity and/or location of peaks)
measured from epithelium in a given state (normal, dysplastic,
etc.) which would influence the diagnostic capability of
fluorescence/reflectance-based discrimination.
[0143] During this testing, three general types of data analysis
were performed: mean, standard deviation and coefficient of
variance. The mean, standard deviation and coefficient of variance
of the intensity at each of the wavelengths for all the spectra
from a patient were calculated. Analyses were performed using
measurements taken at 252 data points distributed over the whole
surface of the cervix. The cervix was also divided into quadrants
and measurements were made for each quadrant. Then, quadrants
containing normal tissue biopsy results were compared with
quadrants containing tissue having abnormal biopsy results.
[0144] Inspection of the spectra for each patient from the 252
points on the cervix would be the most straightforward means of
estimating SNR. For assessing crosstalk and variability, an
effective means is to produce false-color maps of the cervix based
on the spectral data. A key parameter of each spectrum (e.g.
intensity at a signal wavelength, intensity ratio between two
wavelengths) can be color coded and mapped to the location at which
it was measured on the cervix. Maps of this type allow the large
volume of data acquired from each patient to be condensed to a more
manageable form for obtaining qualitative insight into spatial
relationships in the data.
[0145] The fluorescence and reflectance intensity measurements, at
each of the respective wavelengths, for all 252 data points on the
cervix, was then analyzed in several different ways. First, the
mean, standard deviation and the coefficient of variance was
calculated using the measurement results from all 252 data points.
Graphs depicting these calculated values appear in FIGS. 13-26. The
data points are characterized as normal, low grade
dysplasia/inflammation, or high grade displasia based on a
histological examination that was performed subsequent to the
spectroscopic measurements.
[0146] Next, the cervix was divided into four zones, or field
areas, and mean, standard deviation and coefficient of variance
values were calculated on a zone-by-zone basis. The results for
each zone can then be compared to one another to attempt to
localize potentially abnormal zones on the cervix. The calculated
values for each zone were then examined to determine if a
sufficient signal-to-noise ration had been obtained. If the
signal-to-noise ratio for a particular zone was too low, the data
for that zone was discarded.
[0147] Also, as mentioned above, a subsequent histological
examination was performed on the tissue samples collected from each
patient. If a tissue sample was taken and analyzed for a particular
zone having the requisite signal-to-noise ratio, the result is
plotted in FIGS. 27-38. However, if a tissue sample for a
particular zone of a patient was not obtained and analyzed, there
was no way to characterize the data point, and the results were not
plotted in FIGS. 27-38. Thus, the data points in FIGS. 27-38 only
represent quadrants that had a sufficiently high signal-to-noise
ratio, and that were subsequently histologically analyzed to
determine their actual condition.
[0148] Finally, the data was analyzed on a "by-rotation basis." As
described above, the device used to collect the data has forty-two
(42) optical fiber interrogation points distributed over the face
that contacts the cervix. A first measurement cycle is conducted to
collect 42 measurement results. Then, the optical fibers are
rotated 60.degree.. During a second measurement cycle, an
additional 42 measurement results are obtained at the new
locations. This process of rotation and measurement is repeated
until measurements have been conducted at all 252 points across the
cervix.
[0149] The obtained measurements were analyzed on a by-rotation
basis. In other words, the mean, standard deviation and coefficient
of variance was calculated for the 42 measurements taken during the
first measurement cycle, the new values were calculated using the
42 measurements taken during each subsequent measurement cycle.
Note, that the measurement results from each cycle are
substantially evenly distributed over the entire cervix. All the
calculated values for each by-rotation measurement cycle are shown
in FIGS. 39-50.
[0150] FIGS. 13 and 14 show biparameter plots of the means versus
the coefficient of variance (CV) and standard deviation (SD),
respectively, for all twenty-one cases. The first five cases were
standardized to take into account the differences in window type
between these and the other sixteen cases. In FIG. 13, the six high
grade/cancer cases appear to be clustered in the upper right hand
corner of the graph (above the diagonal line and to the right of
the three low grade lesions above the line).
[0151] The remaining analyses involved the latter sixteen patients
tested with the second generation probe and new monochrometer.
FIGS. 15-26 show the calculated values for the whole cervix. Table
2 below summarizes the degree of overlap between high grade cases
and low grade/inflammation/normal cases for each of the three
fluorescence and reflectance measurements, showing the percentage
of correct negative predictions where n=12 using the threshold
below the lowest level measure for the four high grade cases (i.e.,
at 100% sensitivity). Table 2 includes all sub-high grade dysplasia
case, including low grade dysplasia (n=7), inflammation (n=3),
abnormal Pap results but no biopsy (n=2), symptomless patient with
history of disease who underwent an experimental treatment (n=2),
and normals (n=1). As can be seen in Table 2, wavelengths of 290 nm
for fluorescence measurements and 320 nm for reflectance
measurements show the least amount of overlap between high grade
and sub-high grade cases. TABLE-US-00002 TABLE 2 Standard
Coefficient of Variable Mean Deviation Variance 290 FL Excitation
58% 75% 75% 350 FL Excitation 42% 17% 58% 460 FL Excitation 67% 0%
33% 320 Reflectance 25% 67% 75% 420 Reflectance 8% 67% 58% 540-580
Reflectance 25% 58% 67%
[0152] FIGS. 27-38 show the calculated measurements for quadrants
that had a sufficiently high signal-to-noise ration, and that were
subsequently analyzed to determine their condition. The objective
of the by quadrant analysis was to indicate whether spatial
information down to the quadrant level was available and to
determine whether normal quadrants could be differentiated from
abnormal quadrants.
[0153] A total of nineteen quadrants could be reliably identified
as containing either a diseased or normal biopsied site. Of these,
none were normal quadrants, eight contained low grade/inflammatory
disease and two contained high grade disease. Use of a wavelength
of 290 nm for fluorescence measurements appeared to separate the
data by virtue of within quadrant measures of variability (SD and
CV). Mean fluorescence appears to be discriminative at 350 and 420
nm. There appears to one high grade lesion, which was diagnosed by
Pap test as normal and by colposcopy as metaplasia, which can be
misdiagnosed as high grade disease at biopsy. Thus, while it was
seen previously that whole cervix measurements were of little
diagnostic value, when taken down to the quadrant level, i.e., a
smaller field area, the measurements become more diagnosticly
"meaningful".
[0154] FIGS. 39-50 show the by rotation calculated measurements.
This analysis was done in order to determine whether individual
rotation data, from a single measurement cycle, provided any clue
as to whether all or a subset for the six rotation positions are
necessary. In general, the single rotation data mirror that of the
integrated data set, with a bit more overlap. Based on the results,
it appears that the by-rotation data is similar to the entire
cervix data. This suggests that the resolution obtained from 42
interrogation points may be sufficient to accurately predict the
condition of the cervix.
[0155] FIG. 51 shows a biparameter plot of the calculated standard
deviation of the reflectance measurements using a wavelength of 320
nm against the calculated means of the fluorescence measurements
using a wavelength of 460 nm. As can be seen, this plot show a
differentiation between normal and high grade lesions.
[0156] Although it is premature to draw definitive conclusions
regarding this small data set, the result are encouraging. There
were high grade lesions misclassified by both Pap tests and
colposcopy which could be discriminated by the spectroscopic
methods of the invention. Moreover, the results of these
preliminary cases are consistent with known biologic phenomena and
field effects due to carcinogenesis. Of note is that both
fluorescence and reflectance measurements provide discriminative
information.
[0157] Having looked at overall means, standard deviation and
coefficient of variation at individual wavelengths, spatial and
spectral information can then be exploited. Those spectra measured
from points on the tissue for which histopathology is available
(e.g., at/near a biopsy site) can be examined specifically by
category, for example, normal versus abnormal. To further utilize
spectral information, the preferred method involves taking various
intensity ratios at the key wavelengths discussed above. Beyond
that approach, advanced statistical analysis techniques (for
example, principal component analysis, Bayesian Classification,
Classification Trees, Artificial Neural Networks,) may be used to
help to identify other wavelengths which can be effective for
discriminating and modeling a pattern recognition.
[0158] The foregoing embodiments are merely exemplary and are not
to be construed as limiting the invention. The present teaching can
be readily applied to other types of apparatuses. The description
of the invention is intended to be illustrative, and not to limit
the scope of the claims. Many alternatives, modifications, and
variations will be apparent to those skilled in the art.
* * * * *