U.S. patent number RE39,672 [Application Number 10/056,335] was granted by the patent office on 2007-06-05 for method and devices for laser induced fluorescence attenuation spectroscopy.
This patent grant is currently assigned to Cedars-Sinai Medical Center. Invention is credited to Warren S. Grundfest, Vasilis Z. Marmarelis, Ramez E. N. Shehada.
United States Patent |
RE39,672 |
Shehada , et al. |
June 5, 2007 |
Method and devices for laser induced fluorescence attenuation
spectroscopy
Abstract
The Laser Induced Fluorescence Attenuation Spectroscopy (LIFAS)
method and apparatus preferably include a source adapted to emit
radiation that is directed at a sample volume in a sample to
produce return light from the sample, such return light including
modulated return light resulting from modulation by the sample, a
first sensor, displaced by a first distance from the sample volume
for monitoring the return light and generating a first signal
indicative of the intensity of return light, a second sensor,
displaced by a second distance from the sample volume, for
monitoring the return light and generating a second signal
indicative of the intensity of return light, and a processor
associated with the first sensor and the second sensor and adapted
to process the first and second signals so as to determine the
modulation of the sample. The methods and devices of the inventions
are particularly well-suited for determining the
wavelength-dependent attenuation of a sample and using the
attenuation to restore the intrinsic laser induced fluorescence of
the sample. In turn, the attenuation and intrinsic laser induced
fluorescence can be used to determined a characteristic of
interest, such as the ischemic or hypoxic condition of biological
tissue.
Inventors: |
Shehada; Ramez E. N. (La
Mirada, CA), Marmarelis; Vasilis Z. (Irvine, CA),
Grundfest; Warren S. (Calabasas, CA) |
Assignee: |
Cedars-Sinai Medical Center
(Los Angeles, CA)
|
Family
ID: |
25394356 |
Appl.
No.: |
10/056,335 |
Filed: |
January 23, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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Reissue of: |
08889017 |
Jul 7, 1997 |
06124597 |
Sep 26, 2000 |
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Current U.S.
Class: |
250/461.2;
250/458.1; 356/342; 600/320; 600/321; 600/323 |
Current CPC
Class: |
A61B
5/0075 (20130101); A61B 5/0084 (20130101); G01N
21/49 (20130101); G01N 21/6402 (20130101); G01N
21/6428 (20130101); G01N 21/6486 (20130101); A61B
5/0071 (20130101); A61B 5/0086 (20130101); A61B
5/1455 (20130101); G01N 2021/6484 (20130101); G01N
2201/06193 (20130101); A61B 5/7267 (20130101) |
Current International
Class: |
G01N
21/64 (20060101) |
Field of
Search: |
;250/461.2,458.1
;600/321,320,323 ;356/342 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0 703 445 |
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Sep 1995 |
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EP |
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WO 89/12223 |
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Jun 1989 |
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WO |
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WO 97/08538 |
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Aug 1996 |
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WO |
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WO 99/02956 |
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Jan 1999 |
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WO |
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WO 99/02956 |
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Apr 1999 |
|
WO |
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Primary Examiner: Gabor; Otilia
Attorney, Agent or Firm: Knobbe Martens Olsen & Bear
LLP
Claims
We claim:
1. A spectroscopic method of analyzing a sample, comprising:
irradiating a sample with radiation to produce fluorescence from
the sample, wherein the fluorescence is modulated by the sample;
monitoring a first portion of the modulated fluorescence at a first
distance from the sample; monitoring a second portion of the
modulated fluorescence at a second distance from the sample, the
second distance being different from the first distance; .Iadd.and
.Iaddend. comparing the first and second portions of the modulated
fluorescence .Iadd.to each other .Iaddend.to determine a modulation
characteristic of the sample.
2. The method of claim 1, wherein the radiation comprises
substantially monochromatic light.
3. The method of claim 1, wherein the radiation comprises laser
light.
4. The method of claim 1, wherein irradiating the sample comprises
directing radiation at the sample using a waveguide.
5. The method of claim 4, wherein the waveguide is an optical
fiber.
6. The method of claim 4, wherein the waveguide is an optical fiber
bundle.
7. The method of claim 1, wherein monitoring of the modulated
fluorescence comprises: collecting a portion of the modulated
fluorescence; and determining the intensity of the collected
portion of modulated fluorescence.
8. The method of claim 7, wherein the first portion of the
modulated fluorescence is collected with a first waveguide and the
second portion of the modulated fluorescence is collected with a
second waveguide.
9. The method of claim 8, wherein the first waveguide is an optical
fiber.
10. The method of claim 8, wherein the first waveguide is an
optical fiber bundle.
11. The method of claim 8, wherein the second waveguide is an
optical fiber.
12. The method of claim 8, wherein the second waveguide is an
optical fiber bundle.
13. The method of claim 1, wherein irradiating the sample comprises
directing radiation to the sample using a first waveguide and
wherein the fluorescence is monitored using the first
waveguide.
14. The method of claim 7, wherein the intensity of the collected
portion of the fluorescence is determined with a sensor.
15. The method of claim 7, wherein the intensity of the first
portion of the modulated fluorescence is determined with a
sensor.
16. The method of claim 7, wherein the intensity of the second
portion of the modulated fluorescence is determined with a
sensor.
17. The method of claim 7, wherein the intensity of the first
portion of the modulated fluorescence is determined with a first
sensor and the intensity of the second portion of the modulated
fluorescence is determined with a second sensor.
18. The method of claim 7, wherein the first and second portions of
the modulated fluorescence are measured consecutively.
19. The method of claim 7, wherein the first and second portions of
the modulated fluorescence are measured simultaneously.
20. The method of claim .[.11.]. .Iadd.1.Iaddend., wherein the
method further includes determining the intrinsic fluorescence of
the sample.
21. The method of claim 1, wherein the sample is biological
material.
22. The method of claim 21, wherein the biological material is
living tissue.
23. The method of claim 21, wherein the method further includes
determining a physiological property of the biological material
using the modulation characteristic.
24. The method of claim 21, wherein the method further includes
determining a pathological property of the biological material
using the modulation characteristic.
25. The method of claim 22, wherein the method further includes
determining a physiological property of the living tissue using the
modulation characteristic.
26. The method of claim 25, wherein the physiological property of
the tissue is tissue oxygenation.
27. The method of claim 22, wherein the method further includes
determining a pathological property of the tissue using the
modulation characteristic.
28. The method of claim 27, wherein the pathological property of
the tissue is the malignant condition of the tissue.
29. The method of claim 1, wherein either but not both of the
distances is substantially zero.
30. A spectroscopic method of analyzing a sample, comprising:
irradiating a sample with radiation to produce return radiation
from the sample, wherein the return radiation is modulated by the
sample; monitoring a first portion of the modulated return
radiation at a first distance from the sample; monitoring a second
portion of the modulated return radiation at a second distance from
the sample; processing the first and second portions of the
modulated return radiation to determine a modulation characteristic
of the sample, wherein the return radiation is modulated by
attenuation.
31. The method of claims 30, wherein the return radiation is
attenuated by scattering.
32. The method of claim 30, wherein the return radiation is
attenuated by adsorption.
33. The method of claim 30, wherein the modulation characteristic
of the sample is attenuation.
34. The method of claim 30, wherein the modulation characteristic
of the sample is absorption.
35. The method of claim 34, wherein the method further includes
determining transmittance.
36. The method of claim 30, wherein the modulation characteristic
of the sample is optical rotation.
37. A spectroscopic method of analyzing a sample, comprising:
irradiating a sample with radiation to produce return radiation
from the sample, wherein the return radiation is modulated by the
sample; monitoring a first portion of the modulated return
radiation at a first distance from the sample; monitoring a second
portion of the modulated return radiation at a second distance from
the sample; processing the first and second portions of the
modulated return radiation to determine a modulation characteristic
of the sample; wherein the sample is biological material; wherein
the method further includes determining a physiological property of
the tissue using the modulation characteristic; and wherein the
physiological property of the tissue is hypoxia.
38. A spectroscopic method for determining the oxygenation of a
biological material, comprising: irradiating a sample of a
biological material with radiation to produce fluorescence from the
sample, wherein the fluorescence is modulated by attenuation of the
sample; monitoring a first portion of the modulated fluorescence at
a first distance from the sample; monitoring a second portion of
the modulated fluorescence at a second distance from the sample,
the second distance being different from the first distance;
comparing the first and second portions of the modulated
fluorescence .Iadd.to each other .Iaddend.to determine the
attenuation of the sample; .Iadd.and.Iaddend. determining
oxygenation of the sample using the attenuation of the sample.
39. A spectroscopic method for determining the oxygenation of a
biological material, comprising: irradiating a sample of a
biological material with radiation to produce return radiation from
the sample, wherein the return radiation is modulated by
attenuation of the sample; monitoring a first portion of the
modulated return radiation at a first distance from the sample;
monitoring a second portion of the modulated return radiation at a
second distance from the sample; processing the first and second
portions of the modulated return radiation to determine the
attenuation of the sample; determining oxygenation of the sample
using the attenuation of the sample; wherein the oxygenation of the
sample is determined by comparing the attenuation of the sample to
the attenuation of a sample having a known level of
oxygenation.
40. A spectroscopic method for determining the concentration of
hemoglobin in a biological material, comprising: irradiating a
sample of biological material with radiation to produce
fluorescence from the sample, wherein the fluorescence is modulated
by attenuation of the sample; monitoring a first portion of the
modulated fluorescence at a first distance from the sample;
monitoring a second portion of the modulated fluorescence at a
second distance from the sample, the second distance being
different from the first distance; comparing the first and second
portions of the modulated fluorescence .Iadd.to each other
.Iaddend.to determine the attenuation of the sample; .Iadd.and
.Iaddend. determining the concentration of hemoglobin in the sample
using the attenuation of the sample.
41. A spectroscopic method for determining the concentration of
hemoglobin in a biological material, comprising: irradiating a
sample of a biological material with radiation to produce return
radiation from the sample, wherein the return radiation is
modulated by attenuation of the sample; monitoring a first portion
of the modulated return radiation at a first distance from the
sample; monitoring a second portion of the modulated return
radiation at a second distance from the sample; determining the
concentration hemoglobin in the sample using the attenuation of the
sample; wherein the concentration of hemoglobin is determined by
comparing the attenuation of the sample to the attenuation of a
sample having a known concentration of hemoglobin.
42. A method for determining a physiological characteristic of a
biological material, comprising: irradiating a sample of biological
material with radiation to produce fluorescence from the sample,
wherein the fluorescence is modulated by the sample; monitoring a
first portion of the modulated fluorescence at a first distance
from the sample; monitoring a second portion of the modulated
fluorescence at a second distance from the sample, the second
distance being different from the first distance;
.Iadd.and.Iaddend. comparing the first and second portions of the
modulated fluorescence .Iadd.to each other.Iaddend., using a
predictive model, to determine a physiological characteristic of
the sample.
43. A method for determining a physiological characteristic of a
biological material, comprising: irradiating a sample of a
biological material with radiation to produce return radiation from
the sample, wherein the return radiation is modulated by the
sample; monitoring a first portion of the modulated return
radiation at a first distance from the sample; monitoring a second
portion of the modulated return radiation at a second distance from
the sample; processing the first and second portions of the
modulated return radiation, using a predictive model, to determine
a physiological characteristic of the sample; wherein the
predictive model is a multivariate linear regression.
44. A method for determining a physiological characteristic of
biological material, comprising: irradiating a sample of biological
material with radiation to produce fluorescence from the sample,
wherein the fluorescence is modulated by the sample; monitoring a
first portion of the modulated fluorescence at a first distance
from the sample; monitoring a second portion of the modulated
fluorescence at a second distance from the sample, the second
distance being different from the first distance; comparing the
first and second portions of the modulated fluorescence .Iadd.to
each other .Iaddend.to determine a modulation characteristic of the
sample; .Iadd.and.Iaddend. processing the modulation
characteristic.Iadd., .Iaddend.using a predictive model.Iadd.,
.Iaddend.to determine a physiological characteristic of the
sample.
45. A method for determining a physiological characteristic of a
biological material, comprising: irradiating a sample of a
biological material with radiation to produce return radiation for
the sample, wherein the return radiation is modulated by the
sample; monitoring a first portion of the modulated return
radiation at a first distance from the sample; monitoring a second
portion of the modulated return radiation at a second distance from
the sample; processing the first and second portions of the
modulated return radiation, using a predictive model, to determine
a physiological characteristic of the sample; wherein the
predictive model is a multicriteria associative memory
classifier.
46. Apparatus for analyzing a sample, comprising: a source adapted
to emit radiation that is directed at a sample to produce
fluorescence from the sample, wherein the fluorescence is modulated
by the sample; a first sensor adapted to monitor the fluorescence
at a first distance from the sample and generate a first signal
indicative of the intensity of the fluorescence; a second sensor
adapted to monitor the fluorescence at a second distance from the
sample and generate a second signal indicative of the intensity of
the fluorescence, the second distance being different from the
first distance; and a processor associated with the first sensor
and the second sensor and adapted to compare the first and second
signals .Iadd.to each other .Iaddend.to determine a modulation
characteristic of the sample.
47. The apparatus of claim 46, wherein fiber optics transmit the
fluorescence to the sensors.
48. Apparatus for analyzing a sample, comprising: a source adapted
to emit radiation that is directed at a sample volume in a sample
to produce fluorescence from the sample, such fluorescence
including modulated fluorescence resulting from modulation by the
sample; a first sensor adapted to monitor the fluorescence at a
first distance from the sample volume and generate a first signal
indicative of the intensity of the fluorescence; a second sensor
adapted to monitor the fluorescence at a second distance from the
sample volume and generate a second signal indicative of the
intensity of the fluorescence, the second distance being different
from the first distance; .Iadd.and.Iaddend. a processor associated
with the first sensor and the second sensor and adapted to compare
the first and second signals .Iadd.to each other .Iaddend.to
determine a modulation characteristic of the sample.
49. Apparatus for determining a modulation characteristic of a
biological material, comprising: a source adapted to emit
excitation light; a first waveguide disposed .Iadd.at .Iaddend.a
first distance from the sample adapted to transmit the excitation
light from the light source to the biological material to cause the
biological material to produce fluorescence.Iadd., .Iaddend.and
adapted to collect a first portion of the fluorescence; a first
sensor, associated with the first waveguide, adapted to measure the
intensity of the first portion of the fluorescence and generate a
first signal indicative of the intensity of the first portion of
the fluorescence; a second waveguide disposed at a second distance
from the sample adapted to collect a second portion of the
fluorescence, the second distance being different from the first
distance; a second sensor, associated with the second waveguide,
adapted to measure the intensity of the second portion of the
fluorescence and generate a second signal indicative of the
intensity of the second portion of the fluorescence;
.Iadd.and.Iaddend. a processor adapted to compare the first and
second signals .Iadd.to each other .Iaddend.to determine a
modulation characteristic of the biological material.
50. Apparatus for analyzing a sample, comprising: a source adapted
to emit radiation that is directed at a sample volume in a sample
to produce fluorescence from the sample, such fluorescence
including modulated fluorescence resulting from modulation by the
sample; a first sensor, displaced by a first distance from the
sample volume adapted to monitor the fluorescence and generate a
first signal indicative of the intensity of the fluorescence;
.[.and.]. a second sensor, displaced by a second distance from the
sample volume adapted to monitor the fluorescence and generate a
second signal indicative of the intensity of fluorescence, the
second distance being different from the first distance;
.Iadd.and.Iaddend. a processor associated with the first sensor and
the second sensor and adapted to compare the first and second
signals .Iadd.to each other .Iaddend.to determine a physiological
property of the sample.
51. Apparatus for determining a physiological property of .Iadd.a
.Iaddend.biological material, comprising: a source adapted to emit
excitation light; a first waveguide disposed .Iadd.at .Iaddend.a
first distance from the sample.Iadd., and .Iaddend.adapted to
transmit the excitation light from the light source to the
biological material to cause the biological material to produce
fluorescence.Iadd., .Iaddend.and .Iadd.further .Iaddend.adapted to
collect a first portion of the fluorescence; a first sensor,
associated with the first waveguide, for measuring the intensity of
the first portion of the fluorescence and generating a first signal
representative of the intensity of the first portion; a second
waveguide disposed at a second distance from the sample.Iadd., and
.Iaddend.adapted to collect a second portion of the fluorescence,
the second distance being different from the first distance; a
second sensor, associated with the first waveguide, for measuring
the intensity of the second portion of the fluorescence and
generating a second signal representative of the intensity of the
second portion; .Iadd.and.Iaddend. a processor adapted to compare
the first and second signals .Iadd.to each other .Iaddend.to
determine a physiological property of the biological material.
52. A spectroscopic method of analyzing a sample, comprising:
irradiating a sample with radiation to produce fluorescence from
the sample, wherein the fluorescence is modulated by the sample;
monitoring a first portion of the modulated fluorescence at a first
distance from the sample; monitoring a second portion of the
modulated fluorescence at a second distance from the sample, the
second distance being different from the first distance; comparing
the first and second portions of the modulated fluorescence
.Iadd.to each other .Iaddend.to determine a modulation
characteristic of the sample; wherein the sample is .Iadd.a
.Iaddend.biological .[.material.]. .Iadd.tissue.Iaddend.; wherein
the method further includes determining a physiological property of
the tissue using the modulation characteristic; and wherein the
physiological property of the tissue is ischemia.
53. A method for determining a physiological characteristic of a
biological material, comprising: irradiating a sample of a
biological material with radiation to produce fluorescence from the
sample, wherein the fluorescence is modulated by the sample;
monitoring a first portion of the modulated fluorescence at a first
distance from the sample; monitoring a second portion of the
modulated fluorescence at a second distance from the sample, the
second distance being different from the first distance;
.Iadd.and.Iaddend. comparing the first and second portions of the
modulated fluorescence .Iadd.to each other.Iaddend., using a
predictive model, to determine a physiological characteristic of
the sample.[.;.]. .Iadd.,.Iaddend. wherein the predictive model is
multivariate.
54. A spectroscopic method of analyzing a sample, comprising:
irradiating a sample with radiation to produce fluorescence from
the sample, wherein the fluorescence is modulated by the sample;
monitoring a first portion of the modulated fluorescence at a first
angle from the sample.Iadd.;.Iaddend. monitoring a second portion
of the modulated fluorescence at a second angle from the
sample.Iadd.; and.Iaddend. comparing the first and second portions
of the modulated fluorescence .Iadd.to each other .Iaddend.to
determine a modulation characteristic of the sample.
Description
BACKGROUND OF THE INVENTION
The present invention is directed to methods and devices for
determining a spectroscopic characteristic of a sample utilizing
laser induced fluorescence attenuation spectroscopy ("LIFAS"). More
particularly, the invention is directed to methods and devices for
measurement of the wavelength-dependent attenuation of the sample
and subsequent restoration of the intrinsic laser induced
fluorescence ("LIF") for physiological monitoring, biological
tissue characterization and biochemical analysis.
Conventionally, samples have been characterized by determining the
attenuation and laser induced fluorescence ("LIF"). Once the
attenuation and LIF of a sample have been determined, these
spectroscopic properties can be utilized to determine a physical or
physiological property of the sample. For example, the attenuation
of a sample can be used to determine the concentrations of mixture
components or turbidity of a fluid. Similarly, the LIF of a sample
has been used in fields such as analytical chemistry, environmental
monitoring, industrial inspection and medical diagnosis. In the
medical field, for example, LIF spectroscopic techniques have been
used for tissue characterization, malignant tumor identification,
atherosclerotic plaque diagnosis, metabolism evaluation, and the
like.
Conventionally, the attenuation or the absorption of a sample is
determined by placing the sample between a light source and a
detector and measuring any reduction in the intensity of the light
as it passes through the sample. In order to obtain a measurement
having an acceptable signal-to-noise ratio using these conventional
techniques, it is important to transmit the incident light with
sufficient intensity. Thus, the thickness of the sample and the
wavelength of the incident light are important factors affecting
the reliability of the resulting measurement. Moreover, because it
is necessary to place the sample between the light source and the
detector, it is difficult to perform attenuation measurements on
certain types of samples, such as living tissue.
More recently, fiber optic techniques have been developed for the
measurement of attenuation in which an optical fiber is used to
guide the incident light to illuminate a sample inside a small
chamber at the tip of a probe. A reflector is placed on the
opposite side of the chamber to reflect the incident light into a
second fiber which is associated with a detector. Unfortunately,
these techniques find limited application with materials, such as
fluids, that can readily pass into the chamber in the tip of the
probe.
Conventionally, laser induced fluorescence ("LIFS") spectroscopic
techniques utilize various optical configurations in which a laser
is directed at a sample using an optical fiber and the LIF from the
sample is collected using a second optical fiber. Alternatively,
the same fiber can be used for excitation of the sample and the
collection of LIF. In either case, the LIF collected by the fiber
is modulated by the sample, e.g. by the wavelength-dependent
absorption and scattering of constituents of the sample. Therefore,
existing LIFS methods are limited by the fact that the "intrinsic"
or "true" fluorescence of the sample's fluorophores cannot be
determined. Recent reports have suggested measuring the diffuse
reflectance spectrum of the tissue as a means for correcting the
intrinsic LIF using Monte-Carlo mathematical formulations. However,
such correction methods are critically dependent on the
backscattering characteristics of the tissue. Furthermore,
backscattering does not account for the effects of absorption and
scattering suffered by the intrinsic fluorescence prior to its
measurement.
In biological LIF techniques, lasers are used to cause fluorophores
in the sample to emit fluorescence. The main fluorophores in normal
biological tissue are tryptophan, collagen and elastin. Other
fluorophores, such as NAD (nicotinamide adenine dinucleotide) and
FAD (flavin adenine dinucleotide), are also normally present, but
at much lower concentrations. Under certain conditions, the
contribution of the various fluorophores to the LIF can change. For
example, during an ischemic or hypoxic event, the tissue is
deprived of oxygen and anerobic respiration takes place.
Consequently, the weak fluorophore NAD will be converted into the
strong fluorophore reduced nicotinamide adenine dinucleotide
("NADH"). As a result, the LIF collected from the sample will
reflect an increased contribution from NADH. This change is
typically observed as a rise in the intensity of the LIF spectrum
in the region of peak NADH emission, from about 470 to 490 nm.
Thus, the metabolic state of the tissue can be determined by
measuring the relative change in LIF intensity at the wavelength of
peak NADH emission as compared to the relative change of the LIF
intensity of fluorophores that are normally present in the tissue,
such as elastin or collagen.
Unfortunately, the LIF of biological tissue is heavily modulated in
the 390-450 nm range by the peak absorption of the main tissue
chromophore, hemoglobin. Thus, although the intrinsic LIF spectra
of normal tissue should approximately resemble that of the pure
fluorophore components of the tissue, the measured LIF spectra has
a valley in the 400-450 nm region that is associated with the
hemoglobin absorption. As a result, the measured LIF spectrum
tissue appears to have a double peak instead of the single peak
spectrum associated with the pure fluorophores of the tissue.
Hence, the LIF spectrum of normal tissue begins to resemble the
spectrum associated with tissue suffering hypoxia or ischemia,
which makes it more difficult to identify tissue abnormalities.
Furthermore, since the optical properties of biological tissue are
influenced by its hemoglobin concentration, the measured LIF will
vary with the level of blood perfusion throughout the cardiac
cycle. The LIF of contractile tissue, such as the myocardium, is
highly dependent on its state of contraction. Contraction increases
the concentration of the fluorophore NADH and, hence, its
contribution to tissue fluorescence. During contraction, however,
blood is pumped out of the tissue, thereby reducing its hemoglobin
concentration and, hence, light attenuation. Thus, a contracted
myocardium retains less blood (i.e., a lower hemoglobin
concentration) and, therefore, exhibits lower light absorption,
than when relaxed.
Organs such as the brain, heart and kidney are the most sensitive
to oxygen deficiency and can suffer permanent damage following an
ischemic or hypoxic event. During open-heart surgery, for example,
continuous monitoring of kidney perfusion, i.e., ischemia, is
required. Similarly, since the success of a transplantation surgery
is highly dependent on the level of organ perfusion at harvest and
during preservation of the tissue, continuous monitoring of the
organ is typically required.
Ischemia and hypoxia are both conditions that deprive tissue of
oxygen, leading to anaerobic metabolism and the accumulation of the
metabolic coenzyme NADH. The coenzyme NADH is a fluorescent
molecule. Therefore, ischemia and hypoxia can be indirectly
detected using LIF techniques by sensing increased concentrations
of NADH and interpreting its elevation as a sign of oxygen
deficiency. A common indicator of oxygen deficiency is the ratio
between the LIF intensity at wavelengths associated with the peak
fluorescence emission of NADH, collagen and elastin. However, such
methods have not been practically applied for the detection of
ischemia because of several complications. First, these methods
cannot determine whether the elevated NADH concentration is caused
by ischemia, hypoxia or hypermetabolism. Second, scarred or
fibrosed tissue would be detected as normal because of their low
NADH concentration. Finally, the indicator ratios are calculated by
normalizing the intensity of NADH peak fluorescence by that of
collagen or elastin. Although the fluoresence of the structural
proteins elastin and collagen does not vary with tissue
oxygenation, their fluorescence .[.vary.]. .Iadd.varies
.Iaddend.with the site of measurement.
The intrinsic LIF of biological tissue has a single peak spectral
profile resembling that of the pure structural proteins which are
primarily elastin and collagen. However, this single peak profile
is modulated by the attenuation of hemoglobin, especially about 410
nm, to yield a distorted, double-peak spectrum. The second spurious
peak at about 470 nm results from the spectral valley created by
heavy hemoglobin absorption at about 410 nm. Unfortunately, this
second peak overlaps and obscures the peak fluorescence emission of
NADH at about 470-490 nm, which may impede ischemia detection
techniques that are based on NADH concentration.
The methods and devices of the present invention will enable
measurement of several LIF parameters that directly relate to the
state of tissue perfusion and/or hemoglobin oxygenation. This
offers superior accuracy over the currently available LIF
techniques which detect ischemia and hypoxia indirectly by sensing
an upsurge in NADH fluorescence. In particular, the methods and
devices of the present invention can directly indicate the presence
and the level of hypoxia or ischemia. Furthermore, the methods of
the present invention utilize novel criteria for discrimination
between normal and ischemic or hypoxic tissue.
It is readily apparent that existing LIFS techniques in medicine
and industry are affected by modulations of the intrinsic LIF.
Thus, there is need for methods and devices for determining the
intrinsic laser induced fluorescence while eliminating or
minimizing modulating influences in order to provide more accurate
and complete characterization of a sample. In particular, there is
a need for method and devices for characterization of biological
tissue. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION
The present invention is embodied in a system and related method
for laser-induced fluorescence attenuation spectroscopy ("LIFAS")
in which the attenuation and intrinsic LIF of a sample can be
determined. The LIFAS system employs a source, a first sensor, a
second sensor and a processor. The source, preferably a laser,
emits light to irradiate a sample volume in a sample so that the
sample volume produces return light, which preferably includes
laser induced fluorescence. The first sensor monitors the return
light at a first distance from the sample volume and generates a
plurality of signals representing the intensity of the return light
in predetermined wavelength bands. The second sensor monitors the
return light at a second distance from the sample volume and
generates a plurality of signals representing the intensity of the
return light, preferably over the same wavelength bands. Where the
first and second distances are different, the processor can be used
to determine the wavelength-dependent attenuation of the sample
using the signals of both sensors. The measured attenuation will
typically reflect the effects of both absorption and scattering by
the sample. For maximum signal-to-noise ratio, it is preferred that
the first and second detectors monitor return light from the sample
volume at a location in proximity to the sample volume.
Once the wavelength-dependent light attenuation is determined, the
attenuation can be used to restore the intrinsic LIF of the
fluorophores of the sample. Once the attenuation and intrinsic
fluorescence have been determined, these factors can be used in a
variety of applications, including medical diagnosis. In the
preferred embodiment, the attenuation and fluorescence are used for
the detection of ischemia and hypoxia of biological tissue.
Ischemia and hypoxia are detected by monitoring spectral changes in
the intrinsic LIF to detect a rise in the concentration of NADH in
the tissue. Since ischemia is caused by a decrease in the blood
content of the tissue, ischemic tissue will have lower
concentrations of hemoglobin. Consequently, in ischemic tissue,
absorption by hemoglobin, especially in the 390-450 nm spectral
band, will be reduced. Changes in the wavelength of peak light
transmission, especially in the 450-500 nm band, can also be used
to detect hemoglobin oxygenation and, hence, the presence of
hypoxia or ischemia. Finally, multicriteria associative memories
(MAM) can be used as an attenuation or fluorescence spectral
classifier.
Other features and advantages of the present invention will be
apparent from the following description of the preferred
embodiments, taken in conjunction with the accompanying drawings,
which illustrate, by way of example, the principles of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic diagram of a spectroscopic system in
accordance with the invention;
FIG. 2(a) is a schematic diagram of a Laser Induced Fluorescence
Attenuation Spectroscopy system ("LIFAS") in accordance with the
present invention having an excitation waveguide and a first and
second collection waveguides;
FIG. 2(b) is a schematic diagram of an alternative configuration of
the collection waveguides of FIG. 2(a);
FIG. 2(c) is a schematic diagram of yet another alternative
configuration of the collection waveguides of FIG. 2(a);
FIG. 3(a) is a schematic diagram of a LIFAS system in accordance
with the present invention having a first excitation-collection
waveguide and a second collection only waveguide;
FIG. 3(b) is a schematic diagram of an alternative configuration of
the collection waveguides of FIG. 3(a);
FIG. 4 is schematic diagram of a LIFAS system for biomedical
applications in which excitation radiation is used to induce
fluorescence of tissue;
FIGS. 5(a) and (b) are end perspective views of a LIFAS probe
having an optical fiber and an optical fiber bundle as the central
waveguide;
FIGS. 6(a) and (b) are partial perspective views of an electrical
subsystem used to synchronize a LIFAS system with the electrical
activity of tissue using electrodes integrated into the optical
probe;
FIGS. 7(a) and (b) are schematic diagrams showing the relative
positions of the action potential and electrodes integrated into
the optical probe; and
FIGS. 7(c) and (d) are the corresponding action potential timing
diagrams;
FIG. 8 is a perspective view of a probe incorporating a visible
light source for probe positioning and a circular LED array for
indicating the direction of contraction propagation;
FIGS. 9(a)-(c) are partial perspective views of three LIFAS probes
having waveguides arranged in different geometrical configurations
adapted for different applications .Iadd.(top row is an elevation
view; middle row is a side view; bottom row is a bottom
view).Iaddend.;
FIG. 10 is a schematic diagram of a biomedical LIFAS system in
accordance with the invention in which a single optical detector is
used to measure the return light from two collection fibers;
FIG. .[.11.]. .Iadd.18 .Iaddend.is a block diagram of the method
for determining the attenuation and the intrinsic fluorescence of a
sample;
FIG. .[.12.]. .Iadd.19 .Iaddend.is a graph of the attenuation
spectrum measured from normal and ischemic renal cortex of rabbit
kidney using LIFAS devices and methods in accordance with the
invention;
FIG. .[.13.]. .Iadd.20 .Iaddend.is a scatter plot of the
fluorescence intensity at 480 nm acquired from hypoxic (+), normal
(o) and hyperoxic (x) tissue through excitation-collection and
collection-only waveguides;
FIG. .[.14.]. .Iadd.21 .Iaddend.is a graph of the intensity
I.sub.co(.lamda.).sup.c of the signal measured by a collection-only
waveguide from normal, hyperoxic and hypoxic tissue;
FIG. .[.15(a).]. .Iadd.22(a) .Iaddend.is a graph of the modulated
LIF spectra of normal and ischemic kidney, and
FIG. .[.15(b).]. .Iadd.22(b) .Iaddend.is a graph of the intrinsic
LIF spectra of normal and ischemic kidney;
FIGS. .[.16(a) and (b).]. .Iadd.23(a) and (b) .Iaddend.are graphs
of typical modulated LIF spectra of normal and ischemic kidney,
respectively, depicting the difference in the symmetry of the main
lobe; and
FIGS. 17(a)-(b) are graphs of the mean LIF intensity of live kidney
and heart tissue and
FIGS. 17(c)-(d) are the corresponding LIFAS-derived attenuation
spectra.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
As shown in the exemplary drawings and, in particular, FIG. 1
thereof, the present invention is embodied in a spectroscopic
system 110 and related method for measuring the attenuation and/or
optical rotation caused by a sample 114. The spectroscopic system
110 shown in FIG. 1, includes a source 111 that produces radiation
112, which is directed at a sample volume 113 within the sample
114. The source 111 is preferably a laser and the radiation 112 is
preferably monochromatic ultraviolet (UV), visible or infrared (IR)
radiation 112. Nevertheless, the source 111 can be selected to
produce other types of radiation, such as broadband or polarized
radiation, useful for a particular application. The spectroscopic
system 110 further includes a first sensor 116 displaced by a
distance Z1 from the sample volume 113, and a second sensor 118
displaced by a distance Z2 from the sample volume 113, which are
used to monitor return radiation 120 from the sample 114. In the
preferred embodiment, the return radiation 120 will include
fluorescence of the fluorophores of the sample 114.
The sensors 116 and 118 each include an aperture 116a and 118a,
respectively, through which the return radiation 120 is observed
and collected. The sensors 116 and 118 typically can only observe
the events occurring within the volume defined by a solid angle
emerging from the aperture of the sensor. This volume is known as
the numerical aperture or the field of view of the sensor. Thus,
first and second sensors 116 and 118 include numerical apertures
117 and 119, respectively, that are adapted to receive a portion of
the return radiation 120 from the sample 114. The sensors 116 and
118 can be tilted at different angles .THETA..sub.1 and
.THETA..sub.2 and selected to have different numerical apertures
117 and 119 so that the sensors 116 and 118 can selectively monitor
return radiation 120 from all or part of the sample volume 113.
The sensors 116 and 118 each generate a signal 124a and 125a,
respectively, representing the intensity I.sub.c1(.lamda.) and
I.sub.c2(.lamda.) and/or polarization of the return radiation 120.
Preferably, the signals 124a and 125a are generated at a plurality
of wavelengths within predetermined wavelength bands. The signal
124a from the first sensor 116 and the signal 125a from the second
sensor 118 are communicated by signal paths 124 and 125,
respectively, to a processor 123. As discussed in additional
detail, below, the processor 123 processes the signals 124a and
125a to determine a spectral characteristic of interest, such as
the attenuation and/or optical rotation of the return radiation 120
caused by the sample 114.
To determine the attenuation of the return radiation 120 caused by
the sample, the sensors 116 and 118 are positioned at
non-equivalent distances Z1 and Z2 from the sample volume 113.
Preferably, the first and second sensors 116 and 118 will be
positioned so that one of the sensors will be in the immediate
proximity of the sample volume 113 while the other sensor is
positioned at a location adjacent to the first sensor, but
displaced slightly further from the sample volume 113. It has been
found that this arrangement advantageously improves the
signal-to-noise ratio and, hence, measurement accuracy of the
spectroscopic system 110.
The attenuation of the return radiation 120 is dependent on the
distance traveled by the return radiation 120 through the sample
114. Hence, the signal 124a measured by the first sensor 116 and
the signal 125a measured by the second sensor 118 will suffer
different attenuation where the distances Z1 and Z2 from the sample
volume 113 are not equivalent. The processor 123 can be used to
process the signals 124a and 125a to determine the
wavelength-dependent attenuation of the sample 114. The attenuation
determined by the spectroscopic system 110 will reflect the effects
of light absorption and scattering by the sample 114. Where the
radiation 112 causes fluorophores in the sample 114 to
fluorescence, the attenuation of the sample 114 can be used to
determine the intrinsic fluorescence 129 of the fluorophores in the
sample volume 113. The methods by which the attenuation and
intrinsic LIF are determined are discussed in detail below.
The source 111, shown in FIG. 1, can be any radiation source, such
as a laser or lamp emitting excitation radiation 112 at a
wavelength capable of interacting with the sample 114. The sensors
116 and 118 will preferably include a detector, which can be as
simple as individual light-sensitive diodes with appropriate band
pass filters, or more complicated sensors such as an optical
spectrum analyzer. Preferably, the sensor is a suitable
spectrograph or spectrometer equipped with a suitable sensor.
Alternatively, the sensor can include a multispectral CCD
camera.
In the preferred embodiment, the source 111 is an XeCl excimer
laser emitting monochromatic ultraviolet radiation at 308 nm. Each
of the sensors 116 and 118 is a spectrograph (Model FF250, ARIES
Inc., Concorde, Mass.) associated with a 1024 element intensified
photo diode array (PDA) to image the resolved spectrum. Each PDA is
connected to an optical multichannel analyzer (OMA III, EG&G
Princeton Applied Research Corporation, Princeton, N.J.). The OMA
reads the light spectrum imaged by the PDA to produce the signals
124a and 125a. A 335 nm longpass filter (Schott WG335) is placed in
front of the aperture of each of the sensors 116 and 118 to remove
any backscattered 308 nm excitation radiation. The processor 123 is
a personal computer that is networked to each of the sensors 116
and 118 via the signal paths 124 and 125 to receive the signals
124a and 125a, respectively.
In other applications, such as measuring the optical rotation or
anisotropy of a sample, it will be desirable to position the first
and second sensors 116 and 118 at equivalent distances Z1 and Z2
from the sample volume 113. Optical rotation is observed in
optically active materials. An optically active material is
characterized by a lack of symmetry in its molecular or crystalline
structure which causes a rotation of the plane of polarization in
incident-plane-polarized radiation. The extent to which the plane
of polarization is rotated will typically vary from one optically
active material to another. In addition, the extent of rotation may
depend on the number of molecules in the path of the radiation, the
wavelength of the radiation, and the temperature of the material.
Anisotropy, on the other hand, is caused be a selective response of
the sample to radiation travelling in different directions. In
certain biological samples, for example, anisotropy can result from
microscopic inhomogeneity in the tissue structure. Use of
equivalent distances Z1 and Z2 will minimize the contribution of
the path-length-dependent attenuation to the overall attenuation
measured by the system 110. Therefore, the optical rotation or
anisotropy of the sample can be determined more easily.
The LIFA system 110 shown in FIG. 1 can be adapted to measure
optical rotation by utilizing first and second sensors 116 and 118
which are adapted to measure the polarization-dependent intensity
of the return radiation. In particular, the first sensor 116 can be
adapted to measure a first optical angle .phi..sub.1 (not shown)
yielding the maximum intensity of the first portion of the return
radiation 120. Meanwhile, the second sensor 118 can be adapted to
measure a second optical angle .phi..sub.2 (not shown) yielding the
maximum intensity of the second portion of the return radiation
120. The optical rotation of the return light 120 will be given by
the difference between the angles .phi..sub.1 and .phi..sub.2,
provided that the system has been properly calibrated. The same
sensor is used for determination of the attenuation of the sample
will find utility for monitoring the optical rotation. These
sensors can be adapted to such measurements by placing a rotatable
polarizing filter at each of the apertures 116a and 118a of the
first and second sensors 116 and 118, respectively.
An alternative embodiment of the present invention is shown in FIG.
2(a). In this embodiment, the spectroscopic system 110 of FIG. 1
has been adapted as a Laser Induced Fluorescence Attenuation
Spectroscopy ("LIFAS") system 210. In the LIFAS system 210, a
source 211 emits laser radiation 212 at an intensity and a
wavelength capable of inducing fluorescence of the sample 214. The
laser radiation 212 is transmitted through an excitation waveguide
215 to a sample volume 213 within the sample 214 where the laser
radiation 212 excites local fluorophores in the sample volume 213
to emit intrinsic fluorescence 229. The intrinsic fluorescence 229
of the fluorophores in the sample volume 213 is modulated, for
example, by the absorption and scattering of the local chromophores
(not shown) and scatterers (not shown) present in the sample 214,
respectively.
Two collection waveguides 221 and 222 having apertures 221a and
222a and numerical apertures 217 and 219 are disposed about the
excitation waveguide 215. As shown in FIG. 2(b), the collection
waveguides 221 and 222 may be positioned such that their apertures
221a and 222b are laterally displaced from the excitation waveguide
215 by the distances x1' and x2' and/or axially displaced from the
aperture 215a of the excitation waveguide 215 by the distances y1'
and y2'. Preferably, the lateral distances x1' and x2' are small or
zero. Meanwhile, the axial distances y1' and y2' are preferably
selected to be small and unequal, while one of the axial distances
is preferably zero. Also, it is desirable to position the apertures
215a, 221a and 222a of the waveguides 215, 221 and 222 in close
proximity to or in contact with the sample 214 during use of the
LIFAS system 210, as shown in FIG. 2(a).
An alternative configuration of the collection waveguides is shown
in FIG. 2(c). In this configuration, the axial distances y1' and
y2' are both zero. Meanwhile, the lateral distances x1' and x2' are
selected to be small and unequal. The apertures 240a and 242a of
the waveguides 240 and 242 are oblique such that the fields of view
244 and 246 are directed toward the sample volume 213 and to
advantageously encompass the bulk of the sample volume 213. As
shown in FIG. 2(c), the distances z1' and z2' are directed along
the respective axes of the numerical apertures 244 and 246 and
extend from the surface of the apertures 240a and 242a to the point
of intersection of the axes of the numerical apertures 244 and 246,
respectively.
The collection waveguide 221 collects a first portion 228 of the
return light 220 and transmits the first portion 228 of the return
light 220 to a first sensor 216. The first sensor 216 generates a
first signal 224a, representing the intensity I.sub.c1(.lamda.) of
the first portion 228 of the return light 220 at a plurality of
wavelengths within predetermined wavelength bands. Similarly, the
collection waveguide 222 collects a second portion 230 of the
return light 220 and transmits the second portion 230 of the return
light 220 to the second sensor 218. The second sensor 218 generates
a second signal 225a representing the intensity I.sub.c2(.lamda.)
of the second portion 230 of the return light 220 at a plurality of
wavelengths preferably within the same wavelength bands monitored
by the first sensor 216.
The processor 223 then processes the first and second signals 224a
and 225a to determine the wavelength-dependent attenuation of the
sample 214. As discussed in detail, below, once the attenuation is
known, either of the signals 224a or 225a can be processed to
minimize the effects of attenuation and determine the intrinsic LIF
229 of the fluorophores in the sample volume 213. Alternatively, if
the sensors 216 and 218 are selected to monitor the polarization of
the first portion 228 and the second portion 230 of the return
light 220, the processor 223 can be used to determine the optical
rotation of the return light 220 caused by the sample 214.
Preferably, the intensity and wavelength of the laser radiation 212
should be sufficient to create a sample volume 213 that is large
enough to have some overlap with the numerical apertures 217 and
219 of the collection waveguides 221 and 222, respectively. The
numerical apertures 217 and 219 can be selected to encompass all or
parts of the sample volume 213. Furthermore, longpass filters are
placed before the inputs of the sensors 216 and 218 to selectively
block backscattered excitation radiation.
In the preferred embodiment, the source 211 is an Xe--Cl excimer
laser emitting 308 nm ultraviolet excitation radiation 212. The
waveguides 215, 221 and 222 are advantageously comprised of optical
fibers or optical fiber bundles, which can be integrated into a
probe for ease of use and durability. The optical bundle and fibers
are made of fused silica which is transparent to ultraviolet
radiation. Specifically, a 1.4 mm diameter optical bundle is used
as the excitation waveguide 215 while the collection waveguides 221
and 222 are 0.4 mm optical fibers disposed about the excitation
bundle. In the preferred probe embodiment, the lateral range x1'
and x2' are equal to zero while the axial distance y1' is about 0.6
mm and the axial distance y2' is about zero. Furthermore, the probe
may be configured within, for example, the shaft of a hypodermic
needle, so that the in-vivo physiological or pathological
properties of biological tissue can be determined.
In the preferred embodiment, each of the sensors 216 and 218 is a
spectrograph (Model FF250, ARIES Inc., Concorde, Mass.) associated
with a 1024 element intensified photo diode array (PDA). Each PDA
is connected to an optical multichannel analyzer (OMA III, EG&G
Princeton Applied Research Corporation, Princeton, N.J.) which
measures the intensity of the light spectrum imaged by the PDA and
produces the signals 224a and 225a. The processor 223 is a personal
computer that is networked to each of the sensors 216 and 218 via
the signal paths 224 and 225 to receive the signals 224a and 225a,
respectively.
The method of determining the attenuation and intrinsic
fluorescence of a sample is better understood with reference to the
block diagram shown in FIG. .[.11.]. .Iadd.18.Iaddend., which
illustrates the main steps involved in determining the
wavelength-dependent attenuation coefficient .alpha.(.lamda.) and
the intrinsic fluorescence I.sub.T(.lamda.) of a sample in
accordance with the current invention. In the embodiment shown in
FIG. 2(a), the first and second portions 228 and 230 of the return
light 220 experience different attenuation effects due to the
unequal path lengths traversed by the return light 220 from the
sample volume 213 to the apertures 221a and 222a of the collection
waveguides 221 and 222. The first portion 228 of the return light
220 collected by the aperture 221a travels an additional
path-length of {y1'-y2'} through the tissue as compared to the
second portion 230 of the return light 220 which is collected by
the aperture 222a. Hence, the first portion 228 of the return light
220 suffers more path-length-dependent attenuation as compared to
the second portion 230 of the return light 220. Thus, the signals
224a and 225a representing the intensity I.sub.c1(.lamda.) and
I.sub.c2(.lamda.) at different wavelengths will exhibit differing
levels of modulation caused by the attenuation of the sample
214.
In addition, the signals 224a and 225a will also exhibit
wavelength-dependent modulations caused by the instrumental
effects. The wavelength-dependent modulations due to the
instrumental effects can be determined and, in turn, compensated
for by conducting a calibration of the LIFAS system 210. System
calibration can be performed using light from a standard lamp
(Quartz Halogen Lamp, Model No. 63358, Oriel Instruments,
Stratford, Conn.) having a predetermined continuous spectrum to
measure the wavelength-dependent instrumental effects of the LIFAS
system 210. Once these instrumental effects are known, the
processor 223 can be adapted to correct the measured intensities
I.sub.c1(.lamda.) and I.sub.c2(.lamda.) for modulations caused by
the wavelength-dependent instrumental effects. The corrected
intensities I.sub.c1(.lamda.).sup.c and I.sub.c2(.lamda.).sup.c
representing the intensity of the first and second portions 228 and
230 of the return light 220 at different wavelengths can then
.Iadd.be .Iaddend.used to determine the attenuation coefficient
.alpha.(.lamda.) of the sample 214 as described below.
The regions {circle around (1)} and {circle around (2)}, of FIG.
2(a) may be at an effective range, symbolized as "R," from which
the collection waveguides 221 and 222 collect the majority of the
first and second portions of return light 228 and 230,
respectively. The effective range "R" will vary with the
attenuation of the sample. However, the regions {circle around (1)}
and {circle around (2)}, are geometrically symmetric with respect
to the aperture 215a of the excitation waveguide 215 and, hence,
have identical intensity of the intrinsic fluorescence 229
represented by I.sub.T(.lamda.). Furthermore, since the lateral and
axial distances x1', x2' and y1', y2' are predetermined by the
configuration of the probe (in the preferred embodiment x1'=x2',
y1'>y2', y2'=0), the wavelength-dependent intensities
I.sub.c1(.lamda.).sup.c and I.sub.c2(.lamda.).sup.c can be
described by the following equations:
I.sub.c1(.lamda.).sup.c=I.sub.T(.lamda.)e.sup.-.alpha.(.lamda.).(R+y1')
(1)
I.sub.c2(.lamda.).sup.c=I.sub.T(.lamda.)e.sup.-.alpha.(.lamda.).(R+y-
2') (2)
The attenuation coefficient .alpha.(.lamda.) can be calculated
independent of the value of "R" from (1) and (2) as follows:
.alpha.(.lamda.)={1/{y2'-y1'}}ln{I.sub.c1(.lamda.).sup.c/I.sub.c2(.lamda.-
).sup.c} (3)
Where the difference {y2'-y1'} is a known constant, "e" is the
natural exponential function and "ln" is the natural logarithm. It
will be appreciated by those of ordinary skill in the art that the
natural exponential and logarithm can be replaced by the common
exponential and logarithm to the base 10, respectively. Similarly,
for the probe configuration in FIG. 2(c), the attenuation
coefficient can be calculated as follows:
.alpha.(.lamda.)={1/{z2'-z1'}}ln{I.sub.c1(.lamda.).sup.c/I.sub.c2(.lamda.-
).sup.c} (4)
Once the attenuation coefficient .alpha.(.lamda.) is determined,
the intrinsic fluorescence, I.sub.T(.lamda.), can be restored from
either of the signals I.sub.c1(.lamda.).sup.c or
I.sub.c2(.lamda.).sup.c (preferably I.sub.c2(.lamda.).sup.c where
y2'<<y1') by assuming an average effective range "R" from
which most of the intrinsic fluorescence is collected. For
biological tissue, the constant "R" is approximately about 0.2 mm
at 308 nm excitation radiation. Therefore, the intrinsic
fluorescence I.sub.T(.lamda.) can be obtained by substituting the
measured I.sub.c2(.lamda.).sup.c into equation (2) and solving for
I.sub.T(.lamda.) using the constant R and the known value y2':
I.sub.T(.lamda.)=I.sub.c2(.lamda.).sup.ce.sup..alpha.(.lamda.).(R+y2')
(5)
Whether the measured attenuation accounts for absorption and/or
scattering is mainly determined by the wavelength band of interest
and the nature of the sample. The optical properties of biological
tissue and the effects of tissue on LIF are greatly different for
the wavelengths below and above approximately 600 nm. Below about
600 nm, the optical attenuation of biological tissue is primarily
due to absorption and, hence, the attenuation coefficient
.alpha.(.lamda.) will represent absorptivity .[..alpha.(.lamda.).].
.Iadd.a (.lamda.).Iaddend.. Absorptivity is a property of a
substance, while absorbance is a property of a particular sample of
a substance. Therefore, absorbance will vary with the concentration
of the substance (e.g..Iadd., .Iaddend.hemoglobin) and geometry of
the tip of the probe. Thus, for samples such as biological tissue
where the attenuation of the sample is primarily due to absorption,
the absorbance A(.lamda.) and the percent transmittance %T(.lamda.)
of the sample can be calculated as follows:
A(.lamda.)=log{I.sub.c2(.lamda.).sup.c/I.sub.c1(.lamda.).sup.c} (6)
%T(.lamda.)=100{I.sub.c1(.lamda.).sup.c/I.sub.c2(.lamda.).sup.c}
(7)
In an alternative embodiment of the LIFAS system 310, shown in FIG.
3(a), the source 311 emits laser radiation 312 at a wavelength and
intensity capable of inducing fluorescence of the sample 314. The
laser radiation 312 is reflected by a dichroic mirror 326 into an
excitation-collection waveguide 321, which transmits the laser
radiation 312 to the sample volume 313 where the laser radiation
312 excites local fluorophores to emit intrinsic fluorescence 329.
Furthermore, the intensity of the laser 312 should be sufficient to
create a sample volume 313 that overlaps with the numerical
aperture 319 of the collection-only waveguide 322. The dimensions
of the sample volume 313 will also depend on the numerical aperture
of the excitation-collection waveguide 321, on the wavelength and
intensity of the excitation radiation 312 and on the optical
properties of the sample 314. The intrinsic fluorescence 329 is
modulated, for example, by the absorption and scattering of the
local chromophores (not shown) and scatterers (not shown) of the
sample 314.
The excitation-collection waveguide 321 collects a first portion
328 of the return light 320 directly from the sample volume 313.
The first portion 328 of the return light 320 is transmitted
through the dichroic mirror 326 to the first sensor 316. The first
sensor 316 generates a first signal 324a representing the intensity
I.sub.xc(.lamda.) of the first portion 328 of the return light 320
at a plurality of wavelengths within a predetermined wavelength
band.
As shown in FIG. 3(a), the collection-only waveguide 322 may be
positioned such that the aperture 322a is laterally displaced from
the excitation-collection waveguide 321 by the distance x3 and
axially displaced from the aperture 321a of the
excitation-collection waveguide 321 by the range y3. Preferably,
the lateral distance x3 is zero while the axial distance y3 is
non-zero. Also, the apertures 321a and 322a of the waveguides 321
and 322 are preferably positioned in close proximity to or in
contact with the sample 314 during use of the LIFAS system 310, as
shown in FIG. 3(a). Meanwhile, the numerical aperture 319 of the
collection-only waveguide 322 is selected to include at least a
portion of the sample volume 313.
The collection-only waveguide 322 collects a second portion 330 of
the return light 320 which is transmitted to a second sensor 318.
The second sensor 318 generates a second signal 325a representing
the intensity I.sub.co(.lamda.) of the second portion 330 of the
return light 320 at a plurality of wavelengths preferably within
the same wavelength bands as the signal 324a generated by the first
sensor 316. Furthermore, a longpass filter is placed in front of
the aperture of each of the sensors 316 and 318 to selectively
block backscattered excitation radiation.
In the embodiment shown in FIG. 3(a), the first and second portions
328 and 330 of the return light 320 experience different
attenuation effects due to the unequal pathlengths traversed by the
return light 320 from the sample volume 313 to the apertures 321a
and 322a of the waveguides 321 and 322, respectively. The second
portion 330 of the return light 220 collected by the aperture 322a
travels an additional path-length through the tissue as compared to
the first portion 328 of the return light 320 which is collected by
the aperture 321a directly from the sample volume 313. Hence, the
first portion 328 of the return light 320 suffers less
path-length-dependent attenuation as compared to the second portion
330 of the return light 320. Thus, the signals 324a and 325a
representing intensity I.sub.xc(.lamda.) and I.sub.co(.lamda.) at
different wavelengths will exhibit different levels of modulation
caused by the sample 314.
The signals 324a and 325a will also exhibit wavelength-dependent
modulations caused by the instrumental effects. The
wavelength-dependent modulations due to instrumental effects can be
determined and, in turn, compensated for by conducting a
calibration of the LIFAS system 310. System calibration is
performed using light from a standard lamp (Qaurtz Halogen Lamp,
Model No. 63358, Oriel Instruments, Stratford, Conn.) having a
predetermined continuous spectrum to measure the
wavelength-dependent instrumental effects of the LIFAS system 310.
Once these instrumental effects are known, the processor 323 can be
adapted to correct the measured intensities I.sub.xc(.lamda.) and
I.sub.co(.lamda.) for modulations caused by the
wavelength-dependent instrumental effects. The corrected
intensities I.sub.xc(.lamda.).sup.c and I.sub.co(.lamda.).sup.c
representing the intensity of the first and second portions 328 and
330 of the return light 320 at different wavelengths can then be
used by the processor 323 to determine the wavelength-dependent
attenuation .alpha.(.lamda.) of the sample 314. As discussed in
detail, below, once the attenuation is known, either of the signals
324a or 325a, preferably 324a, can be corrected for the effects of
attenuation to restore the intrinsic LIF 329 of the fluorophores in
the sample volume 313.
In the preferred embodiment, the source 311 is an Xe--Cl excimer
laser emitting ultraviolet excitation radiation at a wavelength of
308 nm. The waveguides 321 and 322 are advantageously comprised of
optical fibers or optical fiber bundles, which can be integrated
into a probe for ease of use and durability. A 1.4 mm diameter
optical bundle and a 0.4 mm optical fiber are used as the
excitation-collection and the collection-only waveguides,
respectively. Preferably, the collection-only waveguide is a
plurality of 0.4 mm optical fibers disposed about the periphery of
the excitation-collection waveguide. The optical bundle and fibers
are made of fused silica which is transparent to the 308 nm
ultraviolet radiation. Furthermore, the probe may be configured
within, for example, the shaft of a hypodermic needle, so that the
in-vivo physiological or pathological properties of biological
tissue can be determined. Each of the sensors 316 and 318 is a
spectrograph (Model FF250, ARIES Inc., Concorde, Mass.) associated
with a 1024 element intensified photo diode array (PDA) to image
the resolved spectrum. Each PDA is connected to an optical
multichannel analyzer (OMA III, EG&G Princeton Applied Research
Corporation, Princeton, N.J.) which measures the intensity of the
light spectrum imaged by the PDA and produce the signals 324a and
325a. The processor 323 is a personal computer that is networked to
each of the sensors 316 and 318 via the signal paths 324 and 325 to
receive the signals 324a and 325a, respectively.
The method of determining the attenuation coefficient
.alpha.(.lamda.) and the intrinsic fluorescence I.sub.T(.lamda.) of
a sample using the embodiment of FIG. 3(a) in accordance with the
current invention is as follows. In the embodiment shown in FIG.
3(a), "D" represents the effective range, from which the collection
waveguide 321 collects a majority of the portion 328 of the return
light 320. The portion 328 of the return light 320 is collected
directly from the sample volume 313 by the excitation-collection
waveguide 321. Therefore, the wavelength-dependent intensity
I.sub.xc(.lamda.).sup.c of the portion 328 of the return light 320
collected by the excitation-collection waveguide 321 will be
substantially similar to the wavelength-dependent intensity of the
return light 320, represented as I.sub.o(.lamda.). However, the
return light 320 with the intensity I.sub.o(.lamda.) will suffer
additional wavelength-dependent attenuation as it travels the extra
path-length to reach the aperture 322a and is collected as the
second portion 330 of the return light 320 with the intensity
I.sub.co(.lamda.).sup.c. The lateral and axial distances x3 and y3
will be predetermined by the configuration of the waveguides 321
and 322. In the preferred embodiment, the lateral distance x3=0 and
the axial distance y3>0. Thus, the wavelength-dependent
intensity I.sub.co(.lamda.).sup.c can be approximated by the
following equations:
I.sub.xc(.lamda.).sup.c.apprxeq.I.sub.o(.lamda.)=I.sub.T(.lamda.)e.sup.-.-
alpha.(.lamda.)D (8)
I.sub.co(.lamda.).sup.c.apprxeq.I.sub.o(.lamda.)e.sup.-.alpha.(.lamda.)y3
(9) .apprxeq.I.sub.xc(.lamda.).sup.ce.sup.-.alpha.(.lamda.)y3 (9)
.alpha.(.lamda.)={1/y.sup.3}ln{I .[..sub.cx.].
.Iadd..sub.xc.Iaddend.(.lamda.).sup.c/I.sub.co(.lamda.).sup.c} (10)
Similarly, for the alternative probe configuration shown in FIG.
3(b), where the lateral distance x3'>0 and the axial distance
y3'=0, the attenuation coefficient can be approximated as follows:
.alpha.(.lamda.)={1/x3'}ln{I.sub.xc(.lamda.).sup.c/I.sub.co(.lamda.).sup.-
c} (11) Once the attenuation coefficient .alpha.(.lamda.) is
determined, the intrinsic fluorescence, I.sub.T(.lamda.), can be
restored from either of the signals I.sub.xc(.lamda.).sup.c or
I.sub.co(.lamda.).sup.c, preferably I.sub.xc(.lamda.).sup.c, by
assuming an average effective range "D" from which most of the
intrinsic fluorescence is collected. For biological tissue, the
constant "D" is approximately about 0.2 mm at 308 nm excitation
radiation. By substituting the measured I.sub.xc(.lamda.).sup.c
into equation (7) and solving for I.sub.T(.lamda.):
I.sub.T(.lamda.)=I.sub.xc(.lamda.).sup.ce.sup..alpha.(.lamda.).D
(12)
As discussed in connection with the previous embodiment, the
measured attenuation may account for absorption and/or scattering
depending on the wavelength band of interest and the nature of the
sample. Below about 600 nm, the optical attenuation of biological
tissue is primarily due to absorption and, hence, the attenuation
coefficient .alpha.(.lamda.) will represent absorptivity
.[..alpha.(.lamda.) .]. .Iadd.a(.lamda.).Iaddend.. Also, the
absorbance A(.lamda.) and the percent transmittance %T(.lamda.) of
such samples can be calculated as follows:
A(.lamda.)=log{I.sub.xc(.lamda.).sup.c/I.sub.co(.lamda.).sup.c}
(13)
%T(.lamda.)={I.sub.co(.lamda.).sup.c/I.sub.xc(.lamda.).sup.c}100
(14)
In another alternative embodiment shown in FIG. 4, a LIFAS system
410 in accordance with the present invention has been adapted for
biomedical applications. In this particular embodiment, the LIFAS
system 410 has been adapted to determine the optical attenuation of
biological tissue. The laser 411 emits radiation 412 at a
wavelength capable of exciting the tissue 414 to emit fluorescence.
The radiation 412 is directed through an iris 440 at a dichroic
mirror 442 which reflects the radiation 412 onto a lens 444 which
focuses the radiation 412 onto the proximal tip 445 of an optical
fiber 446. The adjustable iris 440 is advantageously used to reduce
the energy of the radiation 412. Alternatively, the adjustable iris
440 can be replaced by any suitable attenuator.
The probe 448 includes a central optical fiber 446 that is used as
the excitation-collection fiber and peripheral optical fibers
450a-h are used as the collection-only fibers. The distal end of
the optical fibers 446 and 450a-h are incorporated into the optical
probe 448. The aperture 448a the optical fiber probe 448 is placed
in proximity to the tissue 414 so that the aperture 446a of the
excitation-collection fiber 446 and apertures 451a-h of the
collection-only fibers 450a-h are in contact with the tissue 414.
As shown in a partial perspective view in FIG. 5(a), the optical
fiber probe 448 includes a central optical fiber 446 and a
plurality of optical fibers 450a-h disposed about the periphery of
the central optical fiber 446. The apertures 451a-h of the distal
ends of the collection-only fibers 450a-h are axially displaced by
a small distance y3 with respect to the aperture 446a of the
excitation-collection fiber 446. The return light 420 collected by
apertures 451a-h of the collection-only fibers 450a-h pool into the
aperture 418a of the sensor 418. It will be appreciated by those in
the art that the optical fibers 446 and 450a-h can advantageously
be replaced by optical fiber bundles in order to obtain greater
flexibility and durability. For example, as shown in FIG. 5(b), the
central optical fiber 446 can be replaced with an optical fiber
bundle 454.
The excitation radiation 412 is transmitted through the optical
fiber 446 to the tissue 414 to induce intrinsic fluorescence of the
fluorophores of the tissue 414. The intrinsic fluorescence is
modulated, for example, by the chromophores and/or scatterers of
the tissue 414. A first portion 428 of the return light 420 is
collected by the optical fiber 446 from the tissue volume that is
directly irradiated by the excitation radiation 412 and transmitted
through the optical fiber 446 to a first lens 444 where the return
light is directed through the dichroic mirror 442 and then focused
by a second lens 449 onto the aperture 416a of a first sensor 416.
Similarly, the collection-only optical fibers 450a-h collect a
second portion 430 of the return light 420 and transmit the second
portion 430 of the return light 420 to the second sensor 418.
Preferably, longpass filters are placed in front of the apertures
416a and 418a of the sensors 416 and 418 to selectively block
backscattered excitation radiation. A first signal 424a and a
second signal 425a representing the intensity of the first and
second portions 428 and 430, respectively, of the return light 420
are generated by the first and second sensors 416 and 418 and
transmitted via signal paths 424 and 425 to the processor 423. The
processor 423 uses the first and second signals 424a and 425a
generated by the detectors 416 and 418 to determine the
wavelength-dependent attenuation of the sample 414 using equations
(8), (9) and (10) as described in the previous embodiment.
In a particularly preferred embodiment of the LIFAS probe 448 shown
in FIG. 8, an illumination source 490 emitting visible light 492 is
mounted on the probe 448 to act as a spotlight for the operator.
Because room illumination can contaminate the spectral measurements
of the system 410 by adding background light, use of the
illumination source 490 will allow the operator to see and
accurately position the probe 448 under low-light conditions. The
illumination source 490 is configured to illuminate the sample at
all times, except when the LIFAS system is monitoring return light
from the sample 414.
The biological electrical signal arising in nerves or contractile
tissue, such as muscle, is known as the "action potential" and is
caused by sudden changes in the ion conductivity of the cell
membrane. The occurrence of an action potential in contractile
tissue initiates a contraction. For example, in cardiac tissue, the
action potential propagates and spreads in a wave-like manner to
induce a local myocardial contraction wherever it travels.
An action potential propagating in the tissue local to the aperture
448a of the probe 448 can be detected by the electrode 464 which is
incorporated into the probe 448 as shown, for example, in FIG.
6(a). Voltage alternations caused by the occurrence of the action
potential are picked up by the electrode 464 in reference to the
common electrode 466 and are transmitted to the amplifier 460. The
common electrode 466 provides the electrical ground for the
amplifier 460 by maintaining contact with the tissue of interest
414 or the whole body. As discussed in connection with FIG. 7,
below, the amplified action potential 468 is transmitted to the
processor 423 to trigger the acquisition process of the LIFAS
system 410 at a pre-selected phase of the tissue contraction or of
the cardiac cycle, whichever is applicable.
In an alternative probe arrangement, a plurality of electrodes or
fibers with a conductive coating can be distributed
circumferentially about the tip of the optical fiber probe 448. For
example, as shown in FIG. 6(b), the probe 448 can be equipped with
three electrodes 470, 472 and 474 arranged in a triangular
configuration. The action potentials measured by each of the
electrodes 470, 472 and 474 are amplified through separate channels
of the amplifier 460 and transmitted to the processor 423. As
illustrated in FIG. 7, the processor 423 processes the received
signals to determine the direction of propagation of the
contraction vector 462. In particular, the processor detects the
phase lead/lag between the action potentials collected by the
electrodes 470, 472 and 474 to determine the orientation of the
contraction vector 462 with respect to the location of the
electrodes 470, 472 and 474.
For example, the contraction vector 462 shown in FIG. 7(a) is
propagating from the tissue site 476a to the site 478a and, hence,
as shown in FIG. 7(c), the action potential 474a arrives before the
action potential 472a which in turn arrives before the action
potential 470a. Moreover, the phase difference or time delay
between a pair of action potentials indicates how the contraction
vector 462 is centered between the location of the corresponding
pair of electrodes. For example, as shown in FIG. 7(b), the
contraction vector 462 propagates from the tissue site 476b to the
site 478b. As a result, the corresponding time of arrival of action
potentials 470b, 472b and 474b, as shown in FIG. 7(d), will vary.
The processor 423 processes the signals and indicates the direction
of the propagation of the contraction to the system operator. The
direction of propagation can be indicated, for example, by a
circular array 496 of light emitting diodes (LED) 498 mounted
circumferentially on the probe 448, as shown in FIG. 8. The
processor 423 transmits a signal to the LED array 496 so that, for
example, only the LED element pointing in the direction of
contraction propagation would glow.
It will be appreciated that the amplifier 460 can be replaced by
any device that can measure the electrical activity of biological
tissue such as a differential amplifier, an electrocardiogram
(ECG), an electromyogram (EMG), an electroencephalogram (EEG),
depending on the LIFAS application. Furthermore, it will be
appreciated that optical fibers with a metallic or electrically
conductive coating can be used in place of one or all of the
electrode 464, 470, 472 or 474 to measure the action potential of
the tissue. It should be noted that, conventional ECG is generally
not suitable for triggering the acquisition of LIFS or LIFAS
systems, since it does not accurately indicate the instantaneous
state of myocardial contraction at the sample volume. However,
customary ECG using limb or chest leads can be used to trigger data
acquisition of the LIFAS system 410 where the sample 414 is
non-contractile tissue.
In the LIFAS system 410 shown in FIG. 4, the light source 411 is
preferably a lamp or a laser that emits ultraviolet, visible or
infrared radiation. In the preferred embodiment, the source 411 is
an XeCl excimer laser emitting pulses of ultraviolet excitation
radiation at 308 nm. Where ultraviolet radiation is used, it is
advisable that the optical components used in the acquisition
system be made of synthetic quartz (fused silica) to ensure maximal
ultraviolet transmission and minimal instrumental fluorescence.
Alternatively, a nitrogen laser, a helium-cadmium laser, a
frequency-multiplied laser, a solid-state laser, an arc lamp or a
light-emitting diode can be used as the light source 411. The
energy of the excitation light 412 is typically between 0.001-10 m
Joules. However, it will be appreciated that the selected energy
level should be low enough to avoid tissue ablation and/or
photobleaching while still being adequate to produce detectable
LIF.
In the preferred embodiment, the sensors 416 and 418 are each
comprised of a spectrograph (Model FF250, ARIES Inc., Concorde,
Mass.) associated with a 1024 element intensified photo diode array
(PDA) detector. An optional low fluorescence, long-pass filter (not
shown) with a cutoff wavelength above 308 nm, preferably 335 nm
(Schott WG335), is placed before the entrance slit of each
spectrograph to selectively block any backscattered excitation
radiation from reaching the sensors 416 and 418. The entrance slit
of the spectrograph preferably has a width of 100 micrometers. The
spectrograph uses a 150 lines per millimeter diffraction grating to
disperse the incoming return light 420 into its spectral
components.
The spectrum formed by the spectrograph is imaged by a detector,
preferably an intensified linear photodiode array (Model 1420,
EG&G Princeton Applied Research Corporation, Princeton, N.J.)
facing the output port of the spectrograph. The photodiode array
generates a plurality of electrical signals representing the
intensity of the return light 420 at wavelengths within
predetermined wavelength bands. Alternatively, the sensors can be
constructed of any suitable materials, such as individual
light-sensitive diodes with appropriate band-pass filters for the
analysis of spectral bands of the return light or an optical
spectrum analyzer ("OSA") for analysis of a broader spectrum.
Selection of the return light monitoring device will depend on a
variety of factors, including cost, accuracy, resolution, and
whether the user is interested in monitoring a single wavelength, a
wavelength band or an entire spectrum.
Although in the preferred embodiments shown in FIGS. 4 and 5, the
optical fiber probe includes a central excitation-collection
optical fiber or optical fiber bundle with a plurality of
collection-only optical fibers disposed around its periphery, it
will be appreciated by those of ordinary skill in the art that the
probe can take many forms. Furthermore, it will be appreciated that
the central optical fiber can be used as the collection-only
waveguide while all or some of the peripheral fibers can be used
for excitation-collection or collection-only. The latter
arrangement is preferred when testing highly-attenuating samples to
achieve a better signal-to-noise ratio.
FIG. 9 shows three alternative geometrical configurations of the
collection-only optical fibers .[.950.]. .Iadd.450'.Iaddend.a-h
about the excitation-collection waveguide .[.946.].
.Iadd.446'.Iaddend.. In these embodiments, it is desirable to
couple each of the collection-only waveguides .[.950.].
.Iadd.450'.Iaddend.a-h to a separate sensor so that the intensity
of each portion of the return light collection by each fiber can be
monitored. In FIG. 9(a), the collection-only waveguides .[.950.].
.Iadd.450'.Iaddend.a-h are arranged about the excitation-collection
waveguide .[.946.]. .Iadd.446'.Iaddend.so that their apertures have
a helical configuration. Because the aperture of each successive
collection-only waveguide is shifted axially away from the aperture
.[.947.]. .Iadd.447'.Iaddend.of the excitation-collection waveguide
.[.946.]. .Iadd.446'.Iaddend., the return light collected by each
of the collection-only waveguides .[.950.]. .Iadd.450'.Iaddend.a-h
will be attenuated in varying degrees. Use of a probe arrangement
having a plurality of collection distances will be useful in
measuring the polarization and/or the attenuation of.Iadd.,
.Iaddend.especially, a sample having a higher sensitivity to
attenuation due to absorption. Furthermore, a probe incorporating a
plurality of collection distances .[.have.]. .Iadd.has
.Iaddend.application to a larger variety of samples. For example,
where the sample is highly attenuating, the collection-only
waveguides having apertures close to the excitation site can be
used to collect the return light. Where the sample is lightly
attenuating, the return light collected by the waveguides having
apertures that are further from the excitation site will be useful
in determining the attenuation.
In FIG. 9(b), the collection-only waveguides .[.950.].
.Iadd.450'.Iaddend.a-c are displaced laterally with respect to the
excitation-collection waveguide .[.946.]. .Iadd.446'.Iaddend.. Such
a probe configuration will be useful in measuring the polarization
and/or the attenuation of the sample where the sample has a higher
sensitivity to attenuation due to scattering. The return light
collected by the waveguide .[.950.]. .Iadd.450'.Iaddend.c will be
useful in measuring attenuation of a lightly attenuating sample. On
the other hand, the return light measured by the closest waveguide
.[.950.]. .Iadd.450'.Iaddend.a will be useful in measuring the
attenuation of a heavily-attenuating sample.
In FIG. 9(c), the collection-only waveguides .[.950.].
.Iadd.450'.Iaddend.a-c are both axially and laterally displaced
with respect to the excitation-collection waveguide .[.946.].
.Iadd.446'.Iaddend.. This configuration combines the advantages of
both of the aforementioned configurations shown in FIGS. 9(a) and
9(b).
In an alternative embodiment of the LIFAS system of FIG. 4, shown
in FIG. 10, a single sensor 1016 and an associated optical
multiplexer 1080 are utilized. The multiplexer 1080 is used to
switch the input of the sensor 1016 between the
excitation-collection optical path (1046, 1044, 1042, 1049, 1046')
and the collection-only optical path (1050) such that the return
light from each pathway is measured sequentially. The processor
1023 sends trigger signals to the source 1011, the sensor 1016 and
the optical multiplexer 1080 to synchronize their actions. If the
source 1011 is a pulsed laser, two or more radiation pulses are
typically required to acquire a single attenuation measurement
using the multiplexed system 1010. A single laser pulse can be used
if the laser pulse lasts long enough to sustain the emission of
return light 1020 during the minimum period of time required by the
optical multiplexer 1080 and associated sensor 1016 to acquire the
spectral measurements from both the excitation-collection and the
collection only optical paths. The preferred embodiment of the
LIFAS system 1010 is similar to that of 410, except that the system
1010 utilizes an electromagnetic dual port shutter as the optical
multiplexer 1080 associated with a single sensor 1016 in a
configuration where the portion 1028 and the portion 1030 of the
return light 1020 are measured sequentially.
Diagnosis of Hypoxia and Ischemia
The LIFAS methods and devices of the present invention can be used
advantageously for the in-vivo diagnosis of hypoxia and ischemia of
biological tissue. Hypoxia is a deficiency in the amount of oxygen
reaching the tissue, e.g., due to pneumonia, whereas, ischemia is a
localized reduction in arterial blood perfusion, e.g., due to a
narrowing of arteries by spasm or disease. Ischemia can also result
from hemorrhage of an arterial wound or during surgical procedure
that temporarily interrupts the blood flow to a body region. The
present invention describes new criteria and methods for the
discrimination between normal, ischemic and hypoxic biological
tissue, including, in particular renal and myocardial tissue, as
follows.
Utilizing LIFAS methods and devices, it has been found that
ischemic (or hypoxic) tissue exhibits a lower laser induced
fluorescence attenuation ("LIFA") than normal tissue of the same
type. Since LIFA is an absolute quantity, its magnitude can be
directly compared with predetermined standard values. Similarly, in
absorption-dominant media, the absorbance or percent transmittance
can also be utilized. It has been found that, although the LIFA of
ischemic tissue is lower than the LIFA of normal tissue over all
wavelengths, their difference is most prominent between 350 and 450
nm.
The LIFAS-derived LIFA spectra of normal and ischemic rabbit kidney
are shown in FIG. .[.12.]. .Iadd.19.Iaddend.. The LIFA spectra
shown in FIG. .[.12.]. .Iadd.19 .Iaddend.are acquired using a LIFAS
system employing 308 nm excitation radiation produced by an XeCl
excimer laser. This LIFAS system uses a 335 nm longpass filter
(Schott WG335) to cutoff backscattered excitation radiation form
the collected return light. Hence, for this particular LIFAS system
the LIFA values below 350 nm are not reliable. For biological
tissue, the LIFA values in the wavelength band about 480 nm have
the highest signal-to-noise ratio and hence measurement accuracy.
As shown in FIG. .[.12.]. .Iadd.19.Iaddend., the LIFA of ischemic
tissue is lower than the LIFA of normal tissue over the entire
spectrum and is particularly low in the region from 350 to 450
nm.
As demonstrated in FIG. .[.12.]. .Iadd.19.Iaddend., the LIFA,
absorbance or percent transmittance at a predetermined wavelength
or wavelength bands can be used for the detection of ischemia or
hypoxia. Furthermore, predictive models, spectral recognition
techniques and associated classifiers can be applied to identify
whether a given LIFA, absorbance or percent transmittance spectrum
has been acquired from normal, ischemic or hypoxic tissue. The
classifiers can be initially trained with LIFA, at a predetermined
wavelength or wavelength bands acquired from tissue with a known
state of perfusion or oxygenation.
Another parameter found to be useful in the classification of
normal, ischemic and hypoxic tissue, is the intensity of return
light from a pair of LIFAS collection pathways (i.e.
[I.sub.c1(.lamda.).sup.c, I.sub.c2(.lamda.).sup.c] or
[I.sub.xc(.lamda.).sup.c, I.sub.co(.lamda.).sup.c] for the LIFAS
embodiments described above.[...]. ).Iadd.. .Iaddend.For example,
FIG. .[.13.]. .Iadd.20 .Iaddend.shows the [I.sub.xc(.lamda.).sup.c,
I.sub.co(.lamda.).sup.c] pair at .lamda.=480 nm, symbolized as
[I.sub.xc(480).sup.c, I.sub.co(480).sup.c], measured from
hyper-oxygenated (x), normal (o) and oxygen deficient (+) rabbit
kidney. The [I.sub.xc(480).sup.c, I.sub.co(480).sup.c] from normal
and oxygen deficient tissue tend to cluster in two linearly
separable regions of the two dimensional
I.sub.xc(.lamda.).sup.c-I.sub.co(.lamda.).sup.c space. Thus, a
simple linear or nonlinear classifier function can be trained on a
set of [I.sub.xc(.lamda.).sup.c, I.sub.co(.lamda.).sup.c] pairs
measured using a LIFAS system from normal, ischemic and hypoxic
tissue. Other classifiers such as artificial neural networks (ANN)
are also being used. The trained classifier function can then be
used to classify an unknown [I.sub.xc(.lamda.).sup.c,
I.sub.co(.lamda.).sup.c] pair as normal, ischemic or hypoxic. A
"nearest neighbor" (NN) classifier has been found to perform
satisfactorily. The NN classifier checks the proximity of an
unknown [I.sub.xc(480).sup.c, I.sub.co(480).sup.c] pair to clusters
of predetermined [I.sub.xc(480).sup.c, I.sub.co(480).sup.c] pairs
measured from known normal, ischemic and hypoxic tissue. Other
classifiers such as artificial neural networks (ANN) can also be
utilized. For myocardial and renal tissue it has been found that it
is preferable to use [I.sub.xc(.lamda.).sup.c,
I.sub.co(.lamda..sup.c)] measured at 480 nm, to optimize
signal-to-noise ratio. However, [I.sub.xc(.lamda.).sup.c,
I.sub.co(.lamda.).sup.c] at other single or multiple pre-selected
wavelengths can also be used.
An additional parameter for the classification of normal, ischemic
and hypoxic tissue, is the wavelength of the peak transmittance of
the tissue, symbolized hereafter as .lamda..sub.max-T, especially
in the 450-500 nm band. An alternative to .lamda..sub.max-T is the
wavelength of the peak I.sub.co(.lamda.).sup.c in the 450-500 nm
band, symbolized hereafter as .lamda..sub.max-co. Both
.lamda..sub.max-T and .lamda..sub.max-co shift towards shorter
wavelengths as the hemoglobin in the tissue becomes deoxygenated.
For example, FIG. .[.14.]. .Iadd.21 .Iaddend.shows
I.sub.co(.lamda.).sup.c spectra that are acquired using the LIFAS
system shown in FIG. 10 employing 308 nm excitation radiation
produced by and XeCl excimer laser. It should be noted that the
.lamda..sub.max-co of normal tissue shifts to a shorter wavelength
as the tissue becomes hypoxic; whereas the .lamda..sub.max-co of
normal tissue shifts to a longer wavelength as the tissue becomes
hyperoxic. Specifically, .lamda..sub.max-co varies between about
480 and 500 nm as blood or hemoglobin oxygenation varies between
deoxygenated to oxygenated, respectively. A separation border can
be identified at about 489.5 nm to separate .lamda..sub.max-co of
normal tissue (peaks above 489.5 nm) from hypoxic/ischemic tissue
(peaks below 489.5 nm). A simple classifier can be trained to
identify tissue as hypoxic if its .lamda..sub.max-co is below
489.5, and vice versa. The degree of hypoxia is determined from the
magnitude of the shift in .lamda..sub.max-co from the normal value,
the smaller the shift the subtle the hypoxia.
The presence of renal or myocardial ischemia can be detected from
the shape of the main lobe of the common LIF (e.g.
I.sub.xc(.lamda.).sup.c) spectrum in the wavelength band 350-450
nm. The common LIF can be acquired via an excitation-collection
waveguide of a LIFAS or a conventional LIFS system. For example,
FIGS. .[.16.]. .Iadd.23.Iaddend.(a) and (b) show typical LIF
spectra acquired from normal and ischemic rabbit kidneys at an
excitation wavelength of 308 nm. As shown in FIG. .[.16.].
.Iadd.23.Iaddend.(b), the shape of the main lobe of the LIF
acquired from normal tissue is skewed to the right (a positive
skewness value). However, the shape of the main lobe of the LIF
acquired from ischemic tissue is almost symmetric (a very small
skewness value).
Skewness can be measured by a single parameter which indicates the
degree of asymmetry of a lobe or curve around its mean value. For a
symmetrical lobe the skewness is zero, however for an asymmetrical
lobe the skewness can be either positive or negative depending on
the shape. The skewness "S" is given by: .times..lamda.
.times..times..lamda..times..lamda..times..lamda. ##EQU00001##
where x is the intensity of the fluorescence at the wavelength
.lamda., x is the mean intensity of the lobe, .lamda. is the
wavelength and N is the number of wavelength measurements in the
lobe.
Therefore, tissue ischemia can be detected by monitoring the
skewness of the main lobe of a common (i.e. modulated) LIF acquired
using an excitation-collection fiber from the tissue. A zero or
negative skewness value indicates that the LIF spectra is acquired
from ischemic tissue, while a positive skewness will indicate
normally perfused tissue. The bottom of the central spectral valley
is considered as the baseline for the definition of the main lobe
as indicated in FIG. .[.16.]. .Iadd.23.Iaddend..
The intrinsic LIF of normal biological tissue will have a spectrum
with a single peak resembling that of the main tissue fluorophores,
collagen and elastin. However, in the event of ischemia or hypoxia
the intrinsic LIF develops a secondary peak or a hump at about
470-490 nm which is associated with the peak fluorescence emission
of NADH. The appearance of this hump in an intrinsic LIF spectrum
is a direct indication of oxygen deficiency resulting from hypoxia
or ischemia. Unfortunately, this NADH-related hump is not readily
detectable in common LIF spectra because of an overlapping spurious
hump created by an intensity dip associated with peak hemoglobin
absorption at about 410 nm. For example, in common and intrinsic
LIF spectrum of normal and ischemic rabbit myocardium shown in
FIGS. 15(a) and (b), respectively, the intrinsic LIF spectrum of
normal kidney does not exhibit the second hump created by the
spectral valley associated with hemoglobin absorption. To the
contrary, the intrinsic fluorescence of hypoxic myocardium exhibits
a secondary hump indicating high NADH concentration. Accordingly,
ischemia or hypoxia can be detected from a LIFAS-derived intrinsic
LIF spectrum by assessing its NADH-related secondary hump either
visually or through simple qualitative analysis. The magnitude of
the NADH hump will indicate the level of oxygen deficiency caused
by hypoxia or ischemia while its absence indicate normal
oxygenation.
Classification using Chemometric Models
Current ischemia or hypoxia detection techniques use predetermined
ratios of spectral intensities at wavelengths associated with NADH,
elastin and collagen to discriminate normal from abnormal tissue.
However, the magnitude of the intensity does not fully characterize
spectral changes associated with ischemia or hypoxia. Instead, it
is desirable to undertake spectral classification based on the
shape of wavelength bands or the whole spectrum.
The introduction of the LIFAS methods and devices of the present
invention enables the application of a new modeling approach for
the measurement of ischemia and hypoxia. The approach relies on
methods of multivariate linear regression (MLR), principle
component regression (PCR) and the method of partial least squares
(PLS). These modeling methods are well-established chemometric
techniques used in the field of analytical chemistry to estimate
the concentration of a material in a mixture from absorption
spectra of the mixture. The algorithm and theoretical basis for PLS
predictive modeling can be found in Brereton, R. G. Chemometrics:
Applications of Mathematics and Statistics to Laboratory Systems,
New York: Ellis Horwood, 1990. A basic overview of the PLS
regression can be found in Gerald and Kowalski, "Partial
Least-Squares Regression: A Tutorial" Analytical Chimica Acta 185
(1986): 1-17.
In the preferred embodiment, the MLR, PCR and the PLS models are
trained with LIFA or absorbance spectra as their input and the
concentration of both oxygenated and deoxygenated hemoglobin as
their outputs. The blood content (i.e. quantity of blood) in the
tissue is determined by combining the concentrations of both
oxygenated and deoxygenated hemoglobin. The MLR, PCR and the PLS
models are trained with LIFA spectra from normal, hypoxic or
ischemic rabbit heart and kidney.
Once a model is trained, it can be used to predict the
concentrations of oxygenated and deoxygenated hemoglobin if given a
LIFA spectrum measured from tissue with unknown oxygenation. The
presence and level of hypoxia can be determined by comparing the
latter-predicted concentrations values to those known values
previously measured from normal tissue. Hypoxic tissue has less
oxygenated and more deoxygenated hemoglobin concentrations than
normal tissue. Similarly, the presence and level of ischemia can be
determined by combining the predicted concentrations to determine
the total blood content and then comparing the total blood content
to known values of blood content measured in normal tissue.
Ischemic tissue will have a lower blood content than normally
perfused tissue. The PLS model was found to have the optimal
accuracy in detecting the presence and level of ischemia and
hypoxia.
Although LIFA or absorbance spectra are the optimal input to the
MLR, PCR or PLS models for concentration prediction, it will be
appreciated that modulated LIF, or percent transmittance spectra
can also be used due to the fact that, even though common LIF
represent fluorescence information, the LIF spectra are modulated
by wavelength-dependent tissue attenuation.
The MAM Classifier
In accordance with the invention, a new mathematical classifier has
been developed and applied to categorize spectral data for ischemia
or hypoxia detection. The recent multicriteria associative memories
(MAM) technique is modified to perform as a data classifier.
Similar to existing classifiers, the MAM requires initial training
on a set of input-output data pairs. The training process of the
MAM classifier involves the calculation of the weights matrix
M(.eta.) from the input-output data set using the following
learning rule:
M(.eta.)=.eta.RS.sup.T[.eta.SS.sup.T+(1-.eta.)I].sup.-1 (17) The
matrices "S" and "R" holds the input and output training vectors as
their columns, respectively. The superscripts .sup.T and .sup.-1
indicate matrix transpose and inversion, respectively, and "I" is
the identity matrix. The parameter .eta. is initially set to 0.98;
however, it can assume any value between 0 and 1 depending on the
noise of the system. Following the training stage, the output "r"
for an unknown input "s" can be readily calculated from the dot
product: r=M(.eta.).s (18) Finally, an appropriate transfer
function is used to assign the output "r" into one of several
predetermined classification categories.
In the present invention, the MAM classifier is initially trained
with LIFA (or absorbance) spectra acquired from normal or ischemic
tissue as the training inputs. The corresponding normal or ischemic
state can be encoded as, for example, "-1" or "1," respectively and
used as the training outputs. Therefore, the training LIFA spectra
are placed as columns of the input matrix "S" while their
corresponding state-coded values are arranged in the same order as
elements of the output row vector "R." The number of columns in "S"
and elements in "R" are equal to the number of available training
sets. A trained MAM matrix can determine whether an unknown LIFA
spectrum "s.sub.?" has been acquired from normal or ischemic tissue
by calculating the dot product r.sub.?=M(.eta.), s.sub.? and
passing the scalar result "r.sub.?" to a hard limit transfer
function. The hard limit transfer function then converts any
negative or positive values of "r.sub.?" into "0" or "1" indicating
a normal or ischemic classification, respectively.
In a similar fashion, the MAM classifier can be applied for the
detection of hypoxia and the discrimination between normal,
ischemic and hypoxic tissue. It should be apparent that common or
intrinsic LIF spectra can replace LIFA spectra as the classifier
input. In addition, this input can be an entire spectrum, a
re-sampled version of a spectrum or a set of statistical parameters
or features characterizing a spectrum. Similarly, actual ischemia
or hypoxia levels can be used as the classifier outputs instead of
the binary coded output values employed in the above demonstration.
In this case a linear transfer function may be used to categorize
the output "r" of the MAM classifier.
The MAM technique outperformed the commonly-used artificial neural
network (ANN) classifier in accurately classifying LIF/LIFA spectra
resulting from normal and ischemic tissue. The superiority of the
MAM classifier is most probably due to its insensitivity to
spectral noise that might be present in LIF/LIFA spectra measured
from biological systems. Although the foregoing MAM classifier is
currently applied to discriminate spectral data for the purpose of
ischemia or hypoxia detection, it is understood that those skilled
in the art may apply it in various ways for different
classification purposes.
Tissue Characterization
It will also be appreciated that the LIFAS devices and methods can
be applied to tissue characterization, i.e., to differentiate
between normal and diseased tissue, for tissue diagnostics and
malignancy detection. Current LIFS techniques use the intensity
spectrum of modulated LIF to identify malignant (cancerous and
pre-cancerous) tissue and classify its type. LIFAS techniques offer
a unique tissue characterizing capability not offered by
conventional LIFS, based upon measurement of the attenuation
spectrum.
A simple demonstration of LIFAS diagnostic capability is shown in
FIGS. 17(a)-(d). Although normal kidney and heart tissue are
different in nature, their common LIF spectra, shown in FIGS. 17(a)
and (b) are almost identical and, hence, are not so useful for
classification purposes. However, heart and kidney LIFA spectra,
shown in FIGS. 17(c) and d are different in terms of both shape and
peak attenuation values. Thus, LIFAS techniques offer better tissue
identification power than conventional LIFS techniques.
Other biomedical applications of the LIFAS methods and devices will
include laser removal of decorative tattoos, detection of the
in-vivo glucose level, assessment of the degree of burn trauma,
detection of atherosclerotic plaque, angioplasty, measurement of
acidity or alkalinity, pH measurement, the analysis of biochemical
fluids, and the like. For example, measurement of the in vivo skin
absorption using LIFAS methods and devices in accordance with the
present invention can aid in the selection of optimal laser
wavelengths for removing tattoos of different colors. Furthermore,
since LIFAS techniques can be used to determine absorbance from a
hypodermic sample volume, the skin color and the depth of the
tattoo dye can be more accurately characterized than in surface
reflectance techniques. Similarly, burn injury assessment can be
accomplished by using LIFAS techniques to measure the depth of burn
by probing for the presence of blood perfusion at varying locations
within the tissue. LIFAS techniques can also be used to determine
the absorbance or turbidity of a liquid in-situ without the
necessity of extracting a sample for use in a spectrometer.
It will be understood by those of ordinary skill in the art that,
although the LIFAS system and method is shown in the exemplary
method as applied to biological tissue, it is also readily
applicable to chemical and industrial material. For example, LIFAS
devices and methods can be used to measure the absorbance and/or
turbidity of materials and mixtures in medical, food, beverage,
detergent, plastic, glass, oil, paint, textile, and semiconductor
applications. Furthermore, the concentration of the pure components
of the mixture can be determined from the absorbence spectrum using
chemometric techniques such as multivariate regression (MLR),
partial least squares (PLS) or artificial neural networks described
above.
Although the foregoing discloses preferred embodiments of the
present invention, it is understood that those skilled in the art
may make various changes to the preferred embodiments without
departing from the scope of the invention. The invention is defined
only by the following claims.
* * * * *