U.S. patent application number 10/164450 was filed with the patent office on 2003-03-20 for detection of cancer using cellular autofluorescence.
Invention is credited to Banerjee, Bhaskar.
Application Number | 20030055341 10/164450 |
Document ID | / |
Family ID | 24081338 |
Filed Date | 2003-03-20 |
United States Patent
Application |
20030055341 |
Kind Code |
A1 |
Banerjee, Bhaskar |
March 20, 2003 |
Detection of cancer using cellular autofluorescence
Abstract
Apparatus and methods especially useful for detection of cancer
using cellular, Tryptophan-associated autofluorescence are
described. The apparatus includes a light source to produce a beam
of light transmitted to a tissue via a two-way fiber optic bundle
which, in one embodiment, is passed through a conventional
endoscope. The light beam excites the tissue, resulting in an
emission of primarily cellular autofluorescence at a wavelength of
about 330 nm. Light from the tissue is directed back through the
fiber optic bundle and passes through a photodetector. The
photodetector produces a signal, representative of the intensity of
the Tryptophan-associated autofluorescence.
Inventors: |
Banerjee, Bhaskar; (St.
Louis, MO) |
Correspondence
Address: |
Donald R. Holand
Suite 400
7700 Bonhomme
St. Louis
MO
63105
US
|
Family ID: |
24081338 |
Appl. No.: |
10/164450 |
Filed: |
June 6, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10164450 |
Jun 6, 2002 |
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09522557 |
Mar 10, 2000 |
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6405070 |
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09522557 |
Mar 10, 2000 |
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09097931 |
Jun 16, 1998 |
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6405074 |
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Current U.S.
Class: |
600/476 |
Current CPC
Class: |
A61B 5/4331 20130101;
G01N 33/533 20130101; A61B 5/0084 20130101; G01N 33/5005 20130101;
A61B 5/0071 20130101; A61B 5/4375 20130101; A61B 5/4233 20130101;
G01N 21/6486 20130101; A61B 5/441 20130101; A61B 5/0075 20130101;
G01N 21/6456 20130101 |
Class at
Publication: |
600/476 |
International
Class: |
A61B 006/00 |
Claims
1. A method for detection of cancerous cells, said method
comprising the step of measuring an intensity of
Tryptophan-associated autofluorescence emitted from a tissue
sample.
2. A method in accordance with claim 1 wherein measuring an
intensity of Tryptophan-associated autofluorescence emitted from
the tissue sample comprises the step of measuring an intensity of
autofluorescence emitted at a wavelength of about 300-400 nm.
3. A method in accordance with claim 1 wherein measuring an
intensity of Tryptophan-associated autofluorescence emitted from
the tissue sample comprises the step of measuring an intensity of
autofluorescence emitted at a wavelength of about 320-340 nm.
4. A method in accordance with claim 1 wherein measuring an
intensity of Tryptophan-associated autofluorescence emitted from
the tissue sample comprises the step of measuring an intensity of
autofluorescence emitted at a wavelength of about 330 nm.
5. A method in accordance with claim 1, said method further
comprising the step of exposing the tissue sample to a beam of
light.
6. A method in accordance with claim 5 wherein said step of
exposing the tissue sample to a beam of light comprises the step of
exposing the tissue sample to a beam of light having a wavelength
in a range of about 200 nm to about 400 nm.
7. A method in accordance with claim 5 wherein said step of
exposing the tissue sample to a beam of light comprises the step of
exposing the tissue sample to a beam of light having a wavelength
in a range of about 270 nm to about 310 nm.
8. A method in accordance with claim 5 wherein said step of
exposing the tissue sample to a beam of light comprises the step of
exposing the tissue sample to a beam of light having a wavelength
of about 290 nm.
9. A method in accordance with claim 5 wherein said step of
exposing the tissue sample to a beam of light comprises the step of
exposing the tissue sample to a beam of pulsed light having a
wavelength equal to a whole multiple of a wavelength in the range
about 200 nm to about 400 nm.
10. A method in accordance with claim 5 wherein exposing the tissue
sample to a beam of ultraviolet light comprises the step of
delivering the beam of light to the tissue sample using a two-way
fiber optic bundle.
11. A method in accordance with claim 10 wherein delivering the
beam of light to the tissue sample using a two-way fiber optic
bundle comprises the step of passing the two-way fiber optic bundle
through a biopsy channel of an endoscope.
12. A method in accordance with claim 1 wherein measuring the
intensity of Tryptophan-associated autofluorescence comprises the
step of visually representing the Tryptophan-associated
autofluorescence using a charge-coupled device.
13. A method in accordance with claim 1 further comprising the step
of applying a contrast agent to the tissue sample.
14. A method in accordance with claim 13 wherein applying a
contrast agent to the tissue sample comprises the step of applying
a contrast agent comprising an exogenous fluorescent chemical.
15. A method in accordance with claim 13 wherein applying a
contrast agent to the tissue sample comprises the step of applying
methylene blue to the tissue sample.
16. A method comprising the steps of: exposing a first tissue to a
beam of ultraviolet light; measuring an intensity of
Tryptophan-associated autofluorescence emitted in the first tissue;
exposing a second tissue whose condition is known to a beam of
ultraviolet light; measuring an intensity of Tryptophan-associated
autofluorescence emitted in the second tissue; and comparing the
intensity measurements of the first tissue sample to the intensity
measurements for the second tissue sample whose condition is known
to determine the degree of cancer in the first tissue.
17. A method in accordance with claim 16 wherein comparing the
intensity measurements of the first tissue with the intensity
measurements of the second tissue further comprises the step of
comparing the intensity measurements at an emission wavelength of
about 300-400 nm.
18. A method in accordance with claim 16 wherein comparing the
intensity measurements of the first tissue with the intensity
measurements of the second tissue further comprises the step of
comparing the intensity measurements at an emission wavelength of
about 320-340 nm.
19. A method in accordance with claim 16 wherein comparing the
intensity measurements of the first tissue with the intensity
measurements of the second tissue further comprises the step of
comparing the intensity measurements at an emission wavelength of
about 330 nm.
20. A method in accordance with claim 16 wherein said steps of
exposing a first tissue to a beam of ultraviolet light and exposing
a second tissue whose condition is known to a beam of ultraviolet
light comprise exposing the first tissue and the second tissue to
ultraviolet light having a wavelength in a range of about 200 nm to
about 400 nm.
21. A method in accordance with claim 16 wherein said steps of
exposing a first tissue to a beam of ultraviolet light and exposing
a second tissue whose condition is known to a beam of ultraviolet
light comprise exposing the first tissue and the second tissue to
ultraviolet light having a wavelength in a range of about 270 nm to
about 310 nm.
22. A method in accordance with claim 16 wherein said steps of
exposing a first tissue to a beam of ultraviolet light and exposing
a second tissue whose condition is known to a beam of ultraviolet
light comprise the steps of exposing the first tissue and the
second tissue to ultraviolet light having a wavelength of about 290
nm.
23. A method in accordance with claim 16 further comprising the
step of producing a signal representative of the difference between
the intensity measurements of the first tissue and the intensity
measurements of the second tissue.
24. A method in accordance with claim 16 further comprising the
step of obtaining a cytological sample representative of normal
tissue.
25. A method in accordance with claim 24 further comprising the
step of using the cytological sample as the first tissue.
26. A method in accordance with claim 24 further comprising the
step of using the cytological sample as the second tissue whose
condition is known.
Description
[0001] This application is a continuation-in-part application of
prior application Ser. No. 09/097,931, filed Jun. 16, 1998, and
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] This invention relates generally to detection of cancerous
cells and more particularly, to detecting cancerous cells using
cellular autofluorescence.
[0003] The survival rate for cancer patients increases with early
detection of cancer. Known methods of gaining early detection of
cancer are limited to techniques such as surveillance endoscopy and
random tissue biopsies, both of which are costly and inefficient.
In addition, methods which employ relatively high levels of
radiation which cause tissue damage generally are not preferred.
Autofluorescence has been used in attempts to detect cancerous
tissue. Particularly, fluorescence occurs when certain substances
called fluorophores emit light of a longer wavelength after being
excited by light of another, shorter wavelength. The fluorescence
which occurs in human and animal tissues is commonly referred to as
autofluorescence because the fluorescence results from fluorophores
occurring naturally in the tissues. The intensity of
autofluorescence differs in normal and cancerous tissues, and
autofluorescence can be used to detect cancerous tissue in
different organs, including the colon, esophagus, breast, skin, and
cervix.
[0004] In many medical and laboratory applications, the use of
autofluorescence often is preferred for detecting cancerous tissue
because autofluorescence avoids the introduction of exogenous
fluorophores or any other exogenous agent. The use of exogenous
agents increases costs and results in time delays due to lag in
incorporating the exogenous agents into the examined tissue.
Exogenous agents also introduce the risk of adverse reaction.
[0005] Known work on the use of autofluorescence to detect cancer
has been limited to examinations of whole tissue in which a
decrease in tissue autofluorescence indicates the presence of
cancer. However, such work is limited because it relies on
measurement of autofluorescence which includes in large part
non-specific autofluorescence which is emitted from certain but
varied extracellular components of whole tissue. Such extracellular
components include blood, blood vessels, collagen and elastin,
which all emit autofluorescence. While these extracellular
components may change during the progression from normal to
cancerous tissue, the changes are not specific to the cells which
constitute the actual cancerous tissue. Thus, known uses of
autofluorescence to detect cancerous tissue cannot distinguish
between specifically cellular changes and non-specific
extracellular changes in the progression from normal to cancerous
tissue.
[0006] It would therefore be desirable to provide apparatus and
methods which facilitate the early detection of cancerous cells
using autofluorescence. It would also be desirable to provide such
autofluorescence apparatus and methods which exclude extracellular
changes which are non-specific to cancer. It would further be
desirable to provide an objective method for early detection of
cancer which is simple to practice and avoids the need for complex,
subjective comparisons. It would be yet still further desirable to
provide a method for the early detection of cancer which exhibits a
reliability which is unaffected by tissue inflammation.
SUMMARY OF THE INVENTION
[0007] These and other objects may be attained by apparatus and
methods for measuring cellular, Tryptophan-associated
autofluorescence to enable the early detection of cancerous cells.
In one embodiment, the apparatus includes a light source for
producing a beam of light to excite a tissue to emit cellular
autofluorescence. The beam of light is first filtered through a
narrow-band optical filter configured to pass light at a wavelength
of about 200 to about 400 nm, which is optimal for producing
cellular autofluorescence. The beam of light is then transmitted to
the tissue via a two-way fiber optic bundle having a sampling end
positioned at or near the tissue being examined. A lens-system is
positioned between the sampling end of the two-way fiber optic
bundle and the tissue, and the lens system is configured to collect
a light sample from the tissue. The light sample is transmitted
back through the two-way fiber optic bundle and passes through a
narrow-band optical filter configured, in one embodiment, to pass
light at wavelengths of about 320-340 nm. A photodetector
positioned at the output end of the two-way fiber optic bundle
measures the intensity of cellular autofluorescence emitted from
the tissue.
[0008] In another aspect the present invention relates to a method
for detecting pre-cancerous and cancerous cells in a tissue and in
one embodiment, the method includes the steps of exciting the
tissue with a beam of light delivered through a two-way fiber optic
bundle, and measuring the intensity of cellular
Tryptophan-associated autofluorescence emitted from the tissue. The
two-way fiber optic bundle may be inserted through the biopsy
channel of an endoscope, through a laparoscope, or through a needle
inserted into the tissue. The light beam has a wavelength of about
200 to about 400 nm, and the light sample is transmitted back
through the two-way fiber optic bundle and through a narrow-band
optical filter configured to pass light at wavelengths of about
300-400 nm. In an exemplary embodiment the optical filter is
configured to pass light at wavelengths of about 320-340 nm, and a
filter passing light at wavelengths of about 330 nm is especially
suitable.
[0009] Measuring the intensity of the light sample at an emission
wavelength of about 300-400 nm, and particularly at about 330 nm,
enables detection of pre-cancerous and cancerous cells.
Specifically, the intensity of the light sample at about 330 nm
increases systematically with the progression of cancer from normal
to cancerous tissue. Although Tryptophan is believed to be present
in some extracellular proteins, it is predominantly present in
cells. It is believed that the cell specific fluorescence
originates from membranous cellular structures which contain the
amino acid Tryptophan. Thus, at the wavelengths identified above,
extracellular changes which are non-specific to cancer are largely
excluded and therefore, primarily the cellular changes are
detected.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a schematic illustration of an apparatus for
detection of cancer using cellular autofluorescence in accordance
with one embodiment of the present invention.
[0011] FIG. 2 is a schematic illustration of an apparatus for
detection of cancer using cellular autofluorescence in accordance
with another embodiment of the present invention.
[0012] FIG. 3 is a flow chart illustrating a method for detection
of cancer using cellular autofluorescence in accordance with an
embodiment of the present invention.
[0013] FIG. 4 is a schematic illustration of an apparatus for
detection of cancer using cellular autofluorescence in accordance
with yet another embodiment of the present invention.
[0014] FIG. 5 is an exemplary autofluorescence emission spectrum of
a tissue sample.
[0015] FIG. 6 is an emission spectrum of cultured cells.
[0016] FIG. 7 is an emission spectrum of Tryptophan in aqueous
solution.
[0017] FIG. 8 shows emission spectra of normal colon cells and
cancerous colon cells.
[0018] FIG. 9 shows emission spectra of membranous and cytosolic
fractions derived from cells obtained from normal colonic
tissue.
[0019] FIG. 10 shows autofluorescence ratios of a
Tryptophan-associated emission peak for different types of
esophageal tissue.
[0020] FIG. 11 shows autofluorescence ratios of the
Tryptophan-associated peak for different types of colonic tissue
studied.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention is directed to apparatus and methods
for detecting cancer in vitro and in vivo using cellular
autofluorescence. Although specific embodiments of the apparatus
and methods are described below, many variations and alternatives
are possible. Also, the term tissue as used herein refers to both
in vitro and in vivo tissues. In addition, the term tissue as used
herein refers to tissue, organs (in vivo or in live animals or
humans), as well as samples of cells, such as in cytology
(examination of a film of cells on a glass slide). Further, the
cancer detection apparatus and methods can be used in connection
with the detection of early cancer, or pre-cancer, or
dysplasia.
[0022] Referring specifically to the drawings, FIG. 1 is a
schematic view of an apparatus 10 for detecting cancer in vitro or
in vivo using cellular autofluorescence. Apparatus 10 includes a
light source 12, such as a Xenon arc lamp or a laser, powered by a
conventional power source. A first optical filter 14 with a narrow
bandwidth of about 10 nm to about 50 nm, configured to pass light
at a wavelength in a range of about 200 to about 400 nm is
positioned in the path of the light beam produced by light source
12. In an exemplary embodiment, first optical filter 14 has a
narrow bandwidth of about 10 nm and is configured to pass light at
a wavelength in a range of about 270-310 nm. In an alternative
embodiment, first optical filter 14 has a band width of about 35
nm. The light beam emerging from first optical filter 14 passes
through an optical chopper 16 which removes wavelengths of any
background light. The light beam then passes through a two-way
fiber optic bundle 22, sometimes referred to herein as a probe,
which is positioned to catch the light beam as it emerges from
optical chopper 16. The two-way fiber optic bundle 22 has a
sampling end 28, and comprises two groups of optic fibers. A first
group of optic fibers 18 transmits light from source of light 12 to
a tissue T. A second group of optic fibers 32 transmits a light
sample back from tissue T for analysis.
[0023] The two optical fiber groups of two-way fiber optic probe 22
are intermeshed. Two-way fiber optic probe 22 is less than about
2.5 mm in diameter and is long enough to pass through the biopsy
channel of an endoscope or laparoscope, e.g., about 1-2 m in
length. Specifically, probe 22 is configured to pass through the
biopsy channel of a conventional endoscope 24, such as the
endoscopes commonly used to examine the gastrointestinal tract or
the lungs. In an alternate embodiment, two-way fiber optic bundle
22 may be passed through a needle or trocar to obtain measurements
of cellular autofluorescence intensity from solid masses or organs
such as breast, liver or pancreas.
[0024] In one embodiment, a lens system 30 is positioned between
sampling end 28 of two-way fiber optic bundle 22 and tissue T. Lens
system 30 is provided to avoid direct contact between the tissue
and probe 22. Light emerging from tissue T, including emissions of
cellular autofluorescence and reflected and scattered light, is
transmitted down fiber optic bundle 22 and collected by lens system
30 to form a light sample. In an alternative embodiment, probe 22
does not include a lens system and probe 22 makes direct contact
with the tissue. The light emerging from tissue T is transmitted
down fiber optic bundle 22.
[0025] The light sample is directed to sampling end 28 of two-way
fiber optic bundle 22. The light sample is then transmitted back
through two-way fiber optic bundle 22, along second group of optic
fibers 32, from sampling end 28 to a second optical filter 34. In
one embodiment, second optical filter 34 has a narrow bandwidth of
about 20 nm, configured to pass light at a wavelength of about 320
to about 340 nm, and is positioned in the path of the light sample
transmitted back from tissue T. In an alternative embodiment,
optical filter 34 is configured to pass light at a wavelength of
about 330 nm. In another embodiment, optical filter 34 has a
broader bandwidth and passes light at wavelengths of about 300 to
400 nm. A photodetector 36 is positioned to collect the light
sample as it emerges from second optical filter 34. Photodetector
36 is configured to measure the intensity of the light sample
across wavelengths varying from about 300 nm to about 400 nm. In an
alternative exemplary embodiment, photodetector 36 is configured to
measure the intensity of the light sample across wavelengths
varying from about 320 nm to about 340 nm.
[0026] Photodetector 36 generates an electrical output signal e
whose magnitude is proportional to the intensity of the light
sample at a wavelength of about 330 nm. Electrical output signal e
is amplified and displayed on a monitor 38 as a wave form or meter
response. The intensity of cellular autofluorescence in tissue T
may thus be noted and compared to the intensity of cellular
autofluorescence at about 330 nm in a tissue whose condition is
known, such as a cancerous, pre-cancerous or normal tissue. The
presence of cancerous cells is indicated by an increase, relative
to normal tissue, in intensity of cellular autofluorescence at an
emission wavelength of about 330 nm. A ratio of the intensity of
cellular autofluorescence in the tissue F.sub.t to the intensity of
cellular autofluorescence in a known normal sample F.sub.n may be
constructed. The greater the value of F.sub.t/F.sub.n, the more
severe the degree of cancer or malignancy.
[0027] FIG. 2 is a schematic view of an apparatus 100 for real time
detection of cancer in vitro or in vivo using cellular
autofluorescence and video imaging technology. Apparatus 100
includes a source of white light 102, such as a Xenon arc lamp or a
laser, is powered by a conventional power source and produces a
beam of light. The light beam then passes through a first group of
optic fibers 104 of a two-way fiber optic bundle 108 which is
positioned to catch the light beam as it emerges from white light
source 102. The first group of optic fibers 104 transmits the light
beam to a tissue T. Two-way optic fiber bundle 108 passes through a
conventional endoscope 109. In alternate embodiments, the two-way
fiber optic bundle may pass through a large-bore needle, trocar or
laparoscope. A lens system 110 is part of the endoscope 109 and
interposed between tissue T and two-way fiber optic bundle 108. It
is positioned to catch reflected and scattered light from tissue T,
as well as emissions of cellular autofluorescence, to form a light
sample from tissue T. A second group of optic fibers 106 in two-way
fiber optic bundle 108 transmits the light sample back from tissue
T.
[0028] The light sample transmitted along second group of optic
fibers 106 of two-way fiber optic bundle 108 is directed into an
image acquisition module 114 by a lens 112. Image acquisition
module 114 uses a standard optical device such as a prism or
dichromatic mirror to split the light sample into two beams of
light b.sub.1 and b.sub.2, each comprising identical wavelengths.
Light beam b1 is transmitted to a conventional video detector 116
which produces a video signal c1 representative of the standard
visual image obtained from tissue T with endoscope 109 and lens
system 110. Light beam b2 is transmitted to an optical filter 118
with a bandwidth of about 20 nm at about 330 nm. Light beam b2 then
impinges on an image intensifier 120, and then a charge-coupled
device or CCD 122 which produces a second video signal c2. Video
signal c2 is representative of the intensity of cellular
autofluorescence emitted from tissue T. Video signal c2 is
color-coded according to the intensity of cellular autofluorescence
to visually represent different stages of malignancy of the lesion.
Video signals c1 and c2 are then directed via conventional cable
means to a computerized image controller 124 which combines the two
video signals c1 and c2 into a single signal which represents the
superimposition of the image represented by c2 onto the image
represented by c1. The combined signal is then directed to a
standard color video monitor 126 for display of the combined
images.
[0029] FIG. 3 is a flow chart illustrating a method 150 for
utilizing autofluorescence to detect dysplasia, early cancer and
cancer. Method 150 includes exposing a first tissue to a light beam
152 which excites the tissue and results in an emission of cellular
autofluorescence at a wavelength of about 330 nm. In this
embodiment, the first tissue is being examined for the detection of
cancer. After exposure of the tissue to the beam of light, the
intensity of cellular autofluorescence emitted from the tissue is
measured, at a wavelength of about 330 nm, using a standard
photodetector 154.
[0030] In parallel, or in series, with steps 152 and 154, a second
tissue whose condition is known as normal, pre-cancerous, or
cancerous also is examined. Particularly, the second tissue is
exposed to a light beam 156 which excites the tissue and results in
an emission of cellular autofluorescence at a wavelength of about
330 nm. After exposure of the tissue to the beam of light, the
intensity of cellular autofluorescence emitted from the tissue is
measured, at a wavelength of about 330 nm, using a standard
photodetector 158.
[0031] The intensity measurements from the first and second tissues
are then compared 160. The intensity measurements obtained from the
second tissue, which is of known condition, serves as a standard.
Using the results of the comparison, the condition of the first
tissue can be determined 162.
[0032] Method 150 may be practiced in vivo using a two-way fiber
optic bundle passed through the biopsy channel of a conventional
endoscope, as described above in connection with FIGS. 1 and 2.
Alternatively, the first and second tissues may be collected tissue
samples and method 150 may be practiced in a laboratory. In
addition, method 150 could be practiced in connection with the use
of a charge-coupled device and video imaging equipment. With such
devices and equipment, and at steps 154 and 158, the intensity of
the autofluorescence could be visually represented in a real time
video image. Real time video scanning of cellular autofluorescence
would allow large areas of tissue to be scanned both in vitro and
in vivo. In alternative embodiments, method 150 is practiced on
samples of cells obtained from brushings or aspirations.
[0033] FIG. 4 is a schematic illustration of an apparatus 200 for
detection of cancer using cellular autofluorescence in accordance
with yet another embodiment of the present invention. Apparatus 200
includes a light source 202 which may be a component of a
conventional endoscopic illumination system. Light source is, for
example, a Xenon lamp, a source of laser energy or other light
source. Source 202 is coupled to a lens system 204 by a optical
fiber bundle 206. Lens system 204 is focused on a tissue T, such as
a tissue, a tissue sample, an organ, or cells. A lens system 208 is
positioned to collect light from tissue T, and lens system 208 is
coupled to an image acquisition module 210 by an optical fiber
bundle 212. At image module 210, the light received from bundle 212
is split using a splitter such as a dichromatic mirror or a prism
to produce two identical beams B1 and B2.
[0034] Light beam BI is transmitted to a conventional video
detector 214 which produces a video signal S1 representative of the
standard visual image obtained from tissue T. Light beam B2 is
transmitted to an optical filter 216 with a narrow band width of
about 20 nm which allows wavelengths in the range of about 300 to
about 400 nm to pass through. In one embodiment, optical filter 216
allows wavelengths in the range of about 320 nm to about 340 nm to
pass through. Especially suitable are for optical filter 216 are
optical filters having a narrow band width and allowing primarily
wavelengths of about 330 nm to pass through. While a whole range of
other, broader band filters can be used, narrow band filters
maximize the signal. Light beam B2 then impinges on an image
intensifier 218, and then a charge-coupled device or CCD 220 which
produces a second video signal S2. Video signal S2 is
representative of the intensity of cellular autofluorescence
emitted from tissue T.
[0035] Signals S1 and S2 are supplied to a computerized image
controller 222 coupled to a display 224. The autofluorescence image
from signal S2 could be color coded (i.e., different colors
represent different grades of fluorescence intensities, and hence
stages of malignancy) and superimposed on the standard endoscopic
image from signal S1. The intensity of cellular fluorescence would
be stronger in malignant tissues than in normal tissue of the same
organ, for example. The intensity of malignant areas also would be
greater than that in dysplastic areas, which should be stronger
than that in normal areas. If a laser source is used as light
source 202, a gating mechanism could be utilized to rapidly and
alternately illuminate the sample with white light (for routine
video endoscopy) and the laser (for fluorescence imaging).
[0036] Without being bound to a particular theory, experimental
evidence supports the theory that the cell specific fluorescence
originates from proteins or other molecules which are present
largely in membranous structures in cells and contain the amino
acid Tryptophan. The intensity of autofluorescence from Tryptophan
alone appears to be directly correlated to cellular changes
associated with cancer, early (pre-cancer or dysplasia. A stepwise
increase in Tryptophan-induced autofluorescence is observed when
normal tissue progresses to pre-cancer and cancer. Experiments
using established techniques and standard equipment establish this
correlation and are described below.
[0037] All fluorescence scans were performed using a
spectrofluorometer from Shimatzu Inc., Columbia, Md., with a Xenon
lamp and two spectrometers. Emission scans were performed with
excitation from 230-350 nm, at 10 nm intervals. The
autofluorescence intensity was measured in arbitrary units at 1 nm
increments, from 10 nm above the excitation wavelength to 10 less
than twice the excitation wavelength. The excitation scans were
performed with emission at 350 nm and 400 nm, with excitation from
220 nm to 10 nm less than the emission wavelength.
EXAMPLE 1
[0038] The autofluorescence of whole tissue samples was studied.
More specifically, samples of normal, dysplastic and malignant
colonic tissue were studied. The tissue samples included
hyperplastic and adenomatous polyps of the colon, and paired tissue
samples from colon, each pair including a sample of normal mucosa
and a sample of adenocarcinoma or polyp from the same colon. All
tissue samples were immediately frozen in liquid Nitrogen and
stored at -70.degree. C. after harvesting. Just prior to
spectroscopy, the tissue sample was thawed at room temperature and
moistened with phosphate buffered saline (PBS) at a pH of 7.4.
Solid tissue of similar shape and size was mounted on a specially
constructed sample holder having a black matte surface and placed
inside the spectrofluorometer. Spectra from the tissue samples were
digitally recorded and later compared to spectra obtained from
cells.
[0039] Emission spectra obtained from adenocarcinoma, polyps of the
colon (both hyperplastic and adenomatous), and normal colon tissue
samples revealed four major emission peaks or maxima, one at about
330 nm (A), one at about 365 nm (B), one at about 385 nm (C), and
one at about 450 nm (D). FIG. 5 shows an exemplary emission
spectrum, from an adenomatous polyp with excitation at 310 nm.
Three of the four major emission peaks A-D were observed, at 330 nm
(A), 365 nm (B), and 450 nm (D). Different emission peaks appeared
as the excitation wavelength was varied, but the four major
emission peaks A-D were observed across the range of excitation
wavelengths studied. Esophageal, gastric and small intestinal
tissue gave similar results. Peak A will be discussed in more
detail below. As also will be discussed in more detail below, at
least the relatively broad peak D at about 450 nm is likely to be
of extracellular origin and therefore not indicative of the
cell-specific changes associated with the development of cancer
from normal tissue.
[0040] Table 1 summarizes the distribution of the major emission
maxima A-D for normal (n), adenomatous (a) and cancerous (t)
colonic tissue. Table 1 lists the mean wavelength, with standard
error (SE), and the range of wavelengths at which each maximum
occurred in each tissue type. A one way analysis of variance
(ANOVA) was performed on the wavelength distribution of each
maximum for each tissue type (e.g. A.sub.n, A.sub.a and A.sub.t),
giving the P values as listed in Table 1. For normal tissue
samples, N=20; for adenomatous polyps, N=20; for malignant tissue,
N=20.
1TABLE 1 Tissue Mean Range Maxima type Wavelength SE P value (nm) A
n 331.7 0.5 0.857 328-336 a 331.8 0.5 329-336 t 331.4 0.6 326-336 B
n 365.9 0.6 0.303 360-370 a 365.1 0.6 360-370 t 364.7 0.5 361-368 C
n 385.4 1.0 0.463 380-391 a 385.3 0.8 380-390 t 386.6 0.6 384-392 D
n 454.5 1.5 0.472 442-460 a 452.1 1.4 443-463 t 452.9 1.3
448-463
EXAMPLE 2
[0041] Cell specific autofluorescence was studied in cultured
cells. Specifically, cultured cells were grown to isolate
cell-specific autofluorescence from tissue autofluorescence which
includes several nonspecific, extracellular sources such as, for
example, collagen and elastin among others. Cultured cells do not
contain any extracellular matrix. Cells derived from human colon
adenocarcinoma (HT29-18N2) were grown on glass coverslips, in
single and multi-layers until seen to be confluent. Cells were
washed in PBS prior to spectroscopy to remove growth media.
[0042] FIG. 6 shows the emission spectrum of a monolayer of human
colon adenocarcinoma (HT29-18N2) cells. Excitation of the cultured
cells from 280 nm to 330 nm revealed only one major peak S at about
330 nm. Despite excitation at numerous wavelengths, S was the only
major peak observed across the range from 280 nm to 700 nm. Thus S,
a cellular autofluorescence peak, coincided with peak A, a major
tissue autofluorescence peak as shown in FIG. 5. A similar peak at
about 330 nm was observed in another cell culture derived from
human breast tissue (MCF7). No peaks were observed in cells to
match peaks B-D as described in Example 1 and FIG. 5 above.
[0043] To see if any other emission maxima might be present under
different excitation wavelengths, excitation scans measuring light
absorption at different wavelengths were performed. The excitation
scans revealed maxima, or strongest absorption of light at 240 nm
and 290 nm. Excitation of the cells from 235 nm to 270 nm revealed
the S peak at about 330 nm, and an ill defined emission at 260 nm.
However, because such low excitation wavelengths are potentially
harmful, the 260 nm emission was not studied further. Excitation of
the cells at the higher wavelength of 290 nm again revealed only
the S peak.
[0044] All cultured cells, as well as cells separated from normal
and malignant solid tissue including human colon, esophagus and
stomach, which were studied at an excitation of 290 nm showed the S
peak. No other emission peaks were seen.
[0045] Previously, it has been proposed that the broad D peak
observed in tissue spectra at about 450 nm is due to the molecule
NADH which is present in cells. However, none of the
autofluorescence spectra from the cultured or extracted cells
studied showed an emission peak matching D, thus indicating that
the D fluorescence peak is not due to a cellular source.
EXAMPLE 3
[0046] To identify the origin of the S peak, the emission and
excitation spectra of several known fluorophores were investigated
at an excitation wavelength of 290 nm. The fluorophores included
Phenylalanine, Tryptophan, Tyrosine, Collagen Type IV, Elastin,
NADH, and FAD. Only the spectrum of Tryptophan, in aqueous
solution, produced a peak matching the cellular autofluorescence S
peak at about 330 nm, and the tissue autofluorescence peak A at
about 330 nm. FIG. 7 shows the emission spectrum of Tryptophan in
aqueous solution, at an excitation wavelength of 290 nm. Thus,
Tryptophan is highly likely to be the predominant source of the
cellular autofluroescence S peak and matching tissue
autofluorescence A peak.
[0047] Further, the emission spectrum of NADH (not shown) showed a
peak at 460 nm, not 450 nm. These results further support the idea
that the broad D peak observed in tissue spectra at about 450 nm is
not likely to be due to NADH. Thus, known methods which use the
broad D peak to detect cancer are based on the likely false
assumption that the D peak is correlated with the cellular marker
NADH. Instead, the D peak is likely to be of extracellular origin
and thus non-specific to the cellular changes associated with
cancer. Further, the magnitude of peak D appears to fall with
malignancy, probably due to a higher ratio of cells to
extracellular tissue in cancer.
EXAMPLE 4
[0048] To demonstrate that increased autofluorescence at about 330
nm in cancerous cells is due to intracellular changes and not
explained by greater cell density in cancerous tissue, cell samples
were prepared and studied. Specifically, cells were separated from
the extracellular matrix of normal and malignant tissue from colon
and other organs. Cells separated from tissue and suspended in
non-fluorescent solution were confirmed by light microscopy and
then placed in a quartz cuvette for spectroscopy. A portion of each
cell sample was stained with Trypan Blue and the number of viable
cells and total number of cells (cells per cubic millimeter)
estimated using known microscopic techniques. The intensity of
autofluorescence emitted from the samples was measured at 330 nm,
with an excitation wavelength of 290 nm. The fluorescence intensity
in each sample at 330 nm was divided by the estimated number of
cells. The results from normal and malignant cells were then
compared.
[0049] FIG. 8 shows the emission spectra of cells obtained from
normal colon tissue (normal cells) and cells from adenocarcinoma of
the colon (cancer cells). The spectra show the S peak for both cell
types at about 330 nm. No other emission peaks were observed, and
the spectra were identical to those obtained with the same
excitation wavelength from cultured cells of the same type. FIG. 8
also shows that the intensity of the autofluorescence represented
by the peak at about 330 nm in spectra of cancerous cells was
substantially higher than that observed in the spectra of normal
cells.
[0050] Table 2 shows the mean autofluorescence per cell in normal
and malignant cells extracted from colon and esophagus. Table 2
shows that the mean autofluorescence per cell was greater in
malignant cells than in normal cells.
2 TABLE 2 Mean autofluorescence per cell/mm.sup.3 Tissue Colon
Esophagus Normal 4.5 4.9 Malignant 13.4 18.2 Ratio:
malignant/normal 3 3.7
EXAMPLE 5
[0051] To investigate the effect of fixative on cellular
autofluorescence, cells fixed in a solution of 10% formalin were
studied. Specifically, cultured human colon adenocarcinoma cells
(HT29-18N2) were grown on a coverslip and then fixed. Cells were
then kept at room temperature in a closed box. Emission spectra
were obtained at several post-fixation time points, at an
excitation wavelength of 290 nm, and the peak autofluorescence
intensity at about 330 nm was measured. Spectroscopy was performed
at the following times after fixation: 50 minutes, 1 day, 8 days,
14 days and 75 days.
[0052] Table 3 shows the peak intensity at about 330 nm of the
cultured cells, as measured at the different time periods after
fixation. The results show cellular autofluroescence, and more
specifically, the cellular Tryptophan-associated peak at about 330
nm is maintained for many days at room temperature after cells have
been fixed in a standard fixative.
3 TABLE 3 Time post fixation Peak intensity at about 330 nm
Baseline (no formalin) 5.3 50 min 4.5 1 day 6.4 8 days 7.0 14 days
5.8 75 days 4.9
[0053] A similar experiment was performed on cells extracted from
tissue. The cells were also fixed in a 10% formalin solution and
spectroscopy performed as described above. Although the absolute
intensities of the cellular Tryptophan-associated peak was changed,
the difference in intensity between normal and malignant cells was
maintained. Before fixation, the intensity of malignant cells at
about 330 nm was 71% greater than that of normal cells. After
fixation, the intensity of the same malignant cells was 125%
greater than that of normal cells. Thus, it appears that fixation
on formalin not only preserves the Tryptophan-associated
autofluorescence, but for cell samples amplifies the difference in
the autofluorescence intensity between normal and malignant cells.
This suggests that the Tryptophan-associated autofluorescence can
be used for automated cytology. For example, cell smears obtained
from organs can be fixed in formalin and transported at room
temperature to a facility where cellular autofluorescence is
measured and a figure for autofluorescence per cell obtained.
EXAMPLE 6
[0054] To investigate the cellular source of the
Tryptophan-associated autofluorescence, cells were separated from
colonic tissue, homogenized and sonicated to rupture the cell wall,
and centrifuged to produce a supernatant of cytosol and a
membranous sample. The membranous sample was separated and
dissolved, and then both the cytosolic supernatant and membranous
sample were subjected to spectroscopy. The Tryptophan-associated
peak at about 330 nm was observed in both fractions, but at much
greater intensity in the membranous fraction.
[0055] FIG. 9 shows the emission spectrum of membranous and
cytosolic fractions derived from cells obtained from normal colonic
tissue. The peak observed at about 330 nm is most likely due to
Tryptophan as discussed in more detail above. Thus it appears that
the Tryptophan-associated peak originates primarily from a source
in the membranous constituents of cells. Such a source is likely to
be a membrane-associated protein, group of proteins, or other
Tryptophan-containing molecules. It is believed that the such a
molecule or molecules is present in increased amounts in cancerous
and pre-cancerous cells, thus accounting for the increase in
intensity of the Tryptophan-associated autofluorescence in such
cells.
[0056] This increase in cellular Tryptophan-associated
autofluorescence is observable with excitation wavelengths from
about 200 nm to about 400 nm. However, excitation with light having
a wavelength from about 280 nm to about 300 nm is especially
suitable. At excitation wavelengths outside of the range of 280 nm
to 300 nm, for example with excitation at 310 nm to 320 nm, other
emission peaks appear in addition to the cellular
Tryptophan-associated peak at about 330 nm, although the peak is
still detectable and allows intensity measurements to be made. Even
when whole tissue is studied with a suitable excitation wavelength,
only autofluorescence from the cells within the tissue is observed.
This creates a selective optical window through which cellular
autofluorescnce can be observed without interference from
extracellular fluorophores. The increase in cellular
Tryptophan-associated autofluorescence with a peak at about 330 nm,
observed with excitation in the wavelength range of about 230 nm to
about 350, is thus distinguishable from a reported decrease in
tissue autofluorescence in malignant tissue, with excitation in the
same wavelength range, at an emission wavelength of about 450 nm to
460 nm.
EXAMPLE 7
[0057] To demonstrate that single intensity measurements of the
cellular Tryptophan-associated peak at about 330 nm can identify
dysplasia and cancer of the esophagus, tissue samples were obtained
from the esophagus, stomach, colon and small intestine. Normal,
pre-malignant (dysplastic), malignant and inflamed tissue samples
were obtained and the intensity of the Tryptophan-associated peak
at about 330 nm measured using an excitation wavelength of 290 nm.
A normalized intensity ratio of diseased tissue over normal tissue,
(where normal tissue was assigned an intensity value of 1), was
then calculated.
[0058] Table 4 shows the mean intensity ratio for esophageal tissue
.+-. the standard error of the mean (SE). FIG. 10 shows the
autofluorescence ratio of the Tryptophan-associated peak for the
different types of esophageal tissue studied. Barrett's esophagus
is an intestinal metaplasia with a potential for malignant
transformation. Esophagitis is an inflammatory condition.
Dysplasia, an abnormal sate that progresses to malignancy, and
carcinoma are neoplastic conditions of increasing malignancy. The
results indicate that inflammation does not enhance, but instead
slightly reduces cellular autofluorescence at about 330 nm. In
contrast, the intensity ratio increases for low grade dysplasia and
carcinoma. Thus, the single intensity measurement distinguishes
inflamed tissue and Barrett's metaplasia (with a reduction in the
intensity ratio), and dysplastic and malignant tissue (with an
increase in the intensity ratio) from normal tissue. This will
avoid false positive results during cancer surveillance in patients
with inflammatory conditions.
4 TABLE 4 Intensity ratio: Diagnosis (*) Number */normal .+-. SE
Barrett's (intestinal 17 0.62 .+-. 0.67 metaplasia) Dysplasia (low
grade) 8 2.01 .+-. 0.29 Carcinoma 7 3.36 .+-. 1.03 Esophagitis 4
0.63 .+-. 0.06 (inflammation)
[0059] Table 5 shows the mean intensity ratio for colonic tissue
.+-. the standard error of the mean (SE). FIG. 11 shows the
autofluorescence ratio of the Tryptophan-associated peak for the
different types of colonic tissue studied. Hyperplastic polyps are
growths lacking malignant potential. They are polyploid, but
contain normal cells. Adenomatous polyps are benign growths with
malignant potential and include cells which are "atypical". If
detected, adenomatous polyps should be removed, but they are not
cancerous. However, if left unremoved, adenomatous polyps can
develop into cancer. Inflammatory Bowel Disease (IBD), including
Ulcerative Colitis and Crohn's Disease, are chronic inflammatory
conditions with an increased risk of cancer.
[0060] Referring to Table 5, the results indicate that inflammation
(IBD) does not enhance, but instead slightly reduces cellular
autofluorescence intensity at about 330 nm, similar to the results
obtained with inflamed esophageal tissue as described above. In
contrast, the intensity ratio is higher for hyperplastic tissue
than for normal mucosa. The ratio increases stepwise for adenomas
and cancer. Thus, the single intensity measurement distinguishes
inflamed colonic tissue (with a reduction in the intensity ratio),
and hyperplastic, dysplastic and malignant tissue (with an increase
in the intensity ratio) from normal tissue. This will avoid false
positive results during cancer surveillance in patients with
IBD.
5 TABLE 5: Intensity ratio: Diagnosis (*) Number */normal .+-. SE
Hyperplastic 9 1.48 .+-. 0.16 Adenomatous 22 2.13 .+-. 0.16
Carcinoma 11 3.81 .+-. 0.71 IBD 15 0.9 .+-. 0.04
EXAMPLE 8
[0061] The use of excitation scans to detect cancer was also
investigated. In excitation scans, as opposed to the emission scans
described above, the emission wavelength is kept constant and the
excitation wavelength varied. The excitation scans for Tryptophan,
cultured cells, and cells extracted from tissue all reveal a major
excitation peak at 290 nm. This peak is also observed in whole
tissue, and reveals the presence of cancer in a manner similar to
that using emission scans as described above.
[0062] Specifically, excitation spectra of normal, dysplastic and
cancerous and esophageal tissue were obtained by varying the
excitation wavelength from 220 nm to 340 nm. A single intensity
measurement of the major Tryptophan-associated excitation peak was
taken at 290 nm for each tissue type. The mean intensity
measurement for each tissue type was normalized to the intensity
measurement for normal tissue at 290 nm (mean intensity=1). The
ratios of mean emission intensities were: 1.42.+-.0.35 (SE) for low
grade dysplasia of Barrett's, (N=6); and 4.03.+-.1.17 for
adenocarcinoma (N=9). Thus, the results indicate that single
intensity measurements of cellular Tryptophan-associated excitation
spectra distinguishes cancerous tissue from dysplastic and normal
tissue.
[0063] The methods thus embody the first application of
cell-specific autofluorescence in the tissue diagnosis of
malignancy. When whole tissue is excited at a wavelength in the
range of about 230 nm to about 350 nm, for example at about 290 nm,
the autofluorescence emitted from the tissue and measured at an
emission wavelength of about 330 nm comes predominantly from cells,
and is most likely due to the amino acid Tryptophan. The intensity
of cellular Tryptophan-associated autofluorescence is
distinguishable in normal, pre-cancerous, and cancerous cells,
increasing with an increase in malignancy. The methods use the
Tryptophan-associated cellular autofluorescence to detect the
specifically cellular changes indicative of cancer and early
cancer. The methods employ the optical techniques of obtaining
either excitation spectra or emission spectra of tissue or cell
samples to reveal the change in cellular Tryptophan-associated
autofluorescence which is indicative of early cancer or cancer. The
methods have the clear advantage of involving only a single
intensity measurement from a peak in a spectrum, instead of
multiple point analysis of a complex waveform. Further, the methods
provide rapid optical detection of malignancy.
[0064] Cellular Tryptophan-associated autofluroescence is not
affected by the presence of inflammatory conditions in the same way
as it is affected by the presence of malignancy. Inflammation
causes a decrease in cellular Tryptophan-associated
autofluorescence. Therefore, when screening for cancer in patients
with inflammatory conditions, a decreased risk exists of obtaining
false positive results due to the inflamed tissue in such
patients.
[0065] In alternative embodiments of the method, whole multiples of
the excitation wavelengths are used to obtain the same cellular,
Tryptophan-associated autofluorescence with multi-photon
excitation. The multi-photon excitation approach is especially
suitable for penetrating deep tissue, but is also suitable for
examining surface tissue. To obtain emission spectra using
multi-photon excitation, pulsed excitation at multiples of 290 nm,
or an other suitable excitation wavelength in the range of about
200 nm to about 400 nm as described above, can be used. For
example, to penetrate deep tissue, two photon pulsed excitation at
580 nm, or three photon pulsed excitation at 870 nm is used to
obtain the same cellular, Tryptophan-associated
autofluorescence.
[0066] The methods are applicable to cell or tissue samples from a
wide range of organs. For example, the methods are applicable to
direct examination of organs such as skin, or by using two way
optic fiber probes passed through endoscopes to examine internal
organs such as the esophagus, stomach, colon, lung, bladder,
cervix, bile and pancreatic ducts. Breast tissue and other solid
organs are accessible to the method by passing a fiber optic bundle
through a needle or trocar. Alternatively, deep tissue or
sub-surface spectroscopy is accomplished with the methods using
multi-photon excitation.
[0067] Further, the method is useful for defining a safe margin in
real time as a malignancy is being resected by a surgeon, thus
avoiding the need for frozen sections to be examined by a
pathologist during the surgery as is typically done. The methods
are also applicable to automated cell measurements, or cytometry,
wherein cell samples of normal or suspected malignant tissue,
obtained via tissue brushings, smears or fluid aspirations, are
examined, fixed or unfixed, in an automated cytometer for the
Tryptophan-associated autofluorescence. Thus, any tissue sample
being used to practice the method can be a cytological sample
obtained through such cytological sampling methods.
[0068] Still further, the methods are suitable for use in
combination with the use of known dyes, stains and contrast agents
because the cellular, Tryptophan-associated autofluorescence peak
remains unaffected by such agents. These agents include, for
example, methylene blue and other dyes or stains commonly used by
physicians as contrast agents during, for example, endoscopy
procedures or the like. The contrast agents help locate probable
areas of pathology. In addition, the methods described herein are
consistent with the use of exogenous dyes and fluorescent agents,
which also do not affect the Tryptophan-associated autofluorescence
peak. Therefore, alternative embodiments of the method include the
step of applying a suitable contrast agent to a sample of tissue or
tissue site to be examined, and then using the contrast agent to
identify likely areas of pathology which are then further examined
for Tryptophan-associated autofluorescence.
[0069] Even further, a charge-coupled device (CCD) can be used in
combination with the methods to construct visual images of tissue
being examined, wherein the intensity measurements are calibrated
to, for example, a color coded scale and displayed on a video
monitor. Such an application of the methods allows simultaneous
scanning of large areas of tissue.
[0070] From the preceding description of various embodiments of the
present invention, it is evident that the objects of the invention
are attained. Although the invention has been described and
illustrated in detail, it is to be clearly understood that the same
is intended by way of illustration and example only and is not to
be taken by way of limitation. Accordingly, the spirit and scope of
the invention are to be limited only by the terms of the appended
claims.
* * * * *