U.S. patent number 5,983,120 [Application Number 08/860,363] was granted by the patent office on 1999-11-09 for method and apparatus for reflected imaging analysis.
This patent grant is currently assigned to Cytometrics, Inc.. Invention is credited to Warren Groner, Richard G. Nadeau.
United States Patent |
5,983,120 |
Groner , et al. |
November 9, 1999 |
**Please see images for:
( Certificate of Correction ) ** |
Method and apparatus for reflected imaging analysis
Abstract
Reflected imaging is used to perform non-invasive, in vivo
analysis of a subject's vascular system. A raw reflected image is
normalized with respect to the background to form a corrected
reflected image. An analysis image is segmented from the corrected
reflected image to include a scene of interest for analysis. The
method and apparatus can be used to determine such characteristics
as the hemoglobin concentration per unit volume of blood, the
number of white blood cells per unit volume of blood, a mean cell
volume, the number of platelets per unit volume of blood, and the
hematocrit. Cross-polarizers can be used to improve visualization
of the reflected image.
Inventors: |
Groner; Warren (Great Neck,
NY), Nadeau; Richard G. (North East, MD) |
Assignee: |
Cytometrics, Inc.
(Philadelphia, PA)
|
Family
ID: |
27555521 |
Appl.
No.: |
08/860,363 |
Filed: |
June 5, 1997 |
PCT
Filed: |
October 21, 1996 |
PCT No.: |
PCT/US96/16905 |
371
Date: |
June 05, 1997 |
102(e)
Date: |
June 05, 1997 |
PCT
Pub. No.: |
WO97/15229 |
PCT
Pub. Date: |
May 01, 1997 |
Current U.S.
Class: |
600/310; 356/364;
356/39; 600/476; 382/134 |
Current CPC
Class: |
G01N
15/06 (20130101); A61B 5/14535 (20130101); A61B
5/1455 (20130101); A61B 5/0261 (20130101); A61B
5/411 (20130101); G01N 2015/1497 (20130101); G01N
2015/0084 (20130101); G01N 15/1475 (20130101); G01N
2015/008 (20130101); G01N 15/05 (20130101) |
Current International
Class: |
A61B
5/00 (20060101); A61B 5/026 (20060101); G01N
15/14 (20060101); G01N 15/00 (20060101); G01N
15/04 (20060101); G01N 15/05 (20060101); A61B
005/00 () |
Field of
Search: |
;600/310,322,473,476,479
;356/39-42,364,369,445 ;382/128,130,133,134,274 |
References Cited
[Referenced By]
U.S. Patent Documents
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WO 93/07801 |
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WO |
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WO 93/13706 |
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Jul 1993 |
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WO |
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WO 97/24066 |
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Jul 1997 |
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WO |
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|
Primary Examiner: Winakur; Eric F.
Attorney, Agent or Firm: Sterne, Kessler, Goldstein &
Fox, P.L.L.C.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to: Appl. No. 60/005,836, filed
Oct. 23, 1995; Appl. No. 60/016,040, filed Apr. 23, 1996; Appl. No.
60/016,036, filed Apr. 23, 1996; Appl. No. 60/016,037, filed Apr.
23, 1996; Appl. No.60/016,039, filed Apr. 23, 1996; and Appl. No.
60/020,685, filed Jun. 27, 1996; the disclosure of all of the
foregoing applications is incorporated herein by reference as
though set forth in full hereinafter.
Claims
What is claimed is:
1. An apparatus for analysis of blood by use of reflected spectral
imaging, comprising:
a light source for illuminating blood, wherein a light path is
formed between said light source and the illuminated blood;
a first polarizer for polarizing light from said light source;
image capturing means for capturing a reflected image reflected
from the illuminated blood at a depth less than a multiple
scattering length, the reflected image traveling along a reflected
light path between the illuminated blood and said image capturing
means; and
a second polarizer disposed in the reflected light path between the
illuminated blood and said image capturing means, wherein a plane
of polarization of said second polarizer is 90.degree. relative to
a plane of polarization of said first polarizer.
2. The apparatus according to claim 1, wherein said light source
comprises said first polarizer.
3. The apparatus according to claim 2, wherein said light source
comprises a laser diode.
4. The apparatus according to claim 1, wherein said image capturing
means comprises a charge coupled device (CCD) camera.
5. The apparatus according to claim 4, wherein said image capturing
means further comprises a photodetector.
6. The apparatus according to claim 1, wherein said image capturing
means comprises a photodetector.
7. The apparatus according to claim 1, further comprising:
a spectral selection means disposed in the reflected light path
between said second polarizer and said image capturing means.
8. The apparatus according to claim 1, wherein said light source is
a pulsed light.
9. The apparatus according to claim 1, further comprising:
image separating means disposed in the reflected light path between
said second polarizer and said image capturing means for separating
the reflected image into a first portion and a second portion.
10. The apparatus according to claim 9, wherein said image
separating means comprises a dichroic mirror, wherein the first
portion of the reflected image is transmitted through said dichroic
mirror to said image capturing means, and the second portion of the
reflected image is reflected by said dichroic mirror.
11. The apparatus of claim 10, further comprising:
a second image capturing means for capturing the second portion of
the reflected image.
12. The apparatus of claim 11, wherein said second image capturing
means comprises a charge coupled device (CCD) camera.
13. The apparatus of claim 11, further comprising:
a spectral selection means disposed in the reflected light path
between said dichroic mirror and said second image capturing
means.
14. The apparatus of claim 1, further comprising:
an image separating means disposed in the reflected light path
between said second polarizer and said image capturing means for
separating the reflected image into a plurality of portions,
wherein a first portion of the reflected image is captured by said
image capturing means.
15. The apparatus according to claim 14, further comprising:
a second image capturing means; and
wherein said plurality of portions is two, and a second portion of
the reflected image is captured by said second image capturing
means.
16. The apparatus according to claim 15, further comprising:
a first spectral selection means disposed in the reflected light
path between said image separating means and said image capturing
means; and
a second spectral selection means disposed in the reflected light
path between said image separating means and said second image
capturing means.
17. The apparatus according to claim 16, wherein said first
spectral selection means is centered at a wavelength of 550 nm and
said second spectral selection means is centered at a wavelength of
650 nm.
18. The apparatus according to claim 1, further comprising:
image correcting and analyzing means coupled to said image
capturing means for correcting and analyzing the reflected
image.
19. The apparatus according to claim 18, wherein said image
correcting and analyzing means comprises a computer.
20. An apparatus for analysis of blood by use of reflected spectral
imaging, comprising:
a light source for illuminating blood;
a beam splitter for forming a light path between said light source
and the illuminated blood and a reflected light path for a
reflected image reflected from the illuminated blood at a depth
less than a multiple scattering length;
a first polarizer for polarizing light from said light source;
a camera for capturing the reflected image; and
a second polarizer disposed in the reflected light path between the
illuminated blood and said camera, wherein a plane of polarization
of said second polarizer is 90.degree. relative to a plane of
polarization of said first polarizer.
21. The apparatus according to claim 20, wherein said light source
comprises said first polarizer.
22. The apparatus according to claim 21, wherein said light source
comprises a laser diode.
23. The apparatus according to claim 20, wherein said light source
is a pulsed light.
24. The apparatus according to claim 20, further comprising:
a heat rejection filter disposed in the light path between said
light source and the illuminated blood.
25. The apparatus according to claim 20, further comprising:
an objective lens disposed between said beam splitter and the
illuminated blood for magnifying the reflected image, wherein said
camera is in a magnified image plane of said objective lens.
26. The apparatus of claim 20, further comprising:
a dichroic mirror disposed in the light path between said second
polarizer and said camera for separating the reflected image,
wherein a first portion of the reflected image is transmitted
through said dichroic mirror to said camera, and a second portion
of the reflected image is reflected by said dichroic mirror;
and
an image capturing means for capturing the second portion of the
reflected image.
27. The apparatus according to claim 26, wherein said image
capturing means comprises a second camera.
28. The apparatus according to claim 27, further comprising:
a first spectral selection filter disposed in the light path
between said dichroic mirror and said camera; and
a second spectral selection filter disposed in the light path
between said dichroic mirror and said second camera.
29. The apparatus according to claim 28, wherein said first
spectral selection filter is centered at a wavelength of 550nm and
said second spectral selection filter is centered at a wavelength
of 650 nm.
30. The apparatus according to claim 26, wherein said image
capturing means comprises a photodetector.
31. The apparatus according to claim 20, further comprising:
a computer coupled to said camera for analyzing the reflected
image.
32. A method for analysis of blood, comprising:
(1) imaging blood to produce a raw reflected image reflected from a
depth less than a multiple scattering length, comprising
(a) illuminating the blood with light polarized by a first
polarizer and
(b) capturing a reflected image reflected from the blood, wherein
the reflected image is passed through a second polarizer having a
lane of polarization 90.degree. relative to a plane of polarization
of the first polarizer to produce the raw reflected image;
(2) applying a correction to the raw reflected image to form a
corrected reflected image;
(3) segmenting a scene from the corrected reflected image to form
an analysis image; and
(4) analyzing the analysis image for a characteristic of the
blood.
33. The method of claim 32, wherein step (2) comprises:
(a) applying a first wavelength filter to the raw reflected image
to form a first filtered image;
(b) applying a second wavelength filter to the raw reflected image
to form a second filtered image;
(c) taking the negative logarithm of the quotient obtained by
dividing the first filtered image by the second filtered image to
form the corrected reflected image.
34. The method of claim 33, wherein the first wavelength filter is
centered at a first wavelength located at an isobestic point.
35. The method of claim 33, wherein the first wavelength filter is
centered at a first wavelength of 550 nm and the second wavelength
filter is centered at a second wavelength of 650 nm.
36. The method of claim 32, wherein step (2) comprises applying a
velocity correction so that a moving portion of the raw reflected
image is extracted from a stationary portion of the raw reflected
image to form the corrected reflected image.
37. The method of claim 32, wherein step (3) comprises:
(a) applying an optical intensity criterion to the corrected
reflected image.
38. The method of claim 37, wherein step (3) further comprises:
(b) applying a size criterion to the corrected reflected image.
39. The method of claim 38, wherein the size criterion is used to
segment large vessels into the analysis image, wherein large
vessels are of sufficient size so that a plurality of red blood
cells flow side-by-side through them.
40. The method of claim 38, wherein the size criterion is used to
segment small vessels into the analysis image, wherein small
vessels are of a size so that red blood cells flow substantially
single file through them.
41. The method of claim 38, wherein step (3) further comprises:
(c) applying a shape criterion to the corrected reflected
image.
42. The method of claim 32, wherein step (3) comprises using
spatial frequency to segment the scene from the corrected reflected
image.
43. The method of claim 32, wherein step (4) comprises:
(a) determining a mean reflected light intensity of the analysis
image; and
(b) converting the mean reflected light intensity of the analysis
image into a hemoglobin concentration per unit volume of blood.
44. The method of claim 32, wherein step (4) comprises:
(a) counting white blood cells in the analysis image to determine a
number of white blood cells per unit volume of blood.
45. The method of claim 32, wherein step (4) comprises:
(a) determining a mean cell volume from the analysis image.
46. The method of claim 32, wherein step (4) comprises:
(a) counting platelets in the analysis image to determine a number
of platelets per unit volume of blood.
47. The method of claim 32, wherein step (4) comprises:
(a) measuring a volume of cells per unit volume of blood in the
analysis image to determine hematocrit.
48. The method of claim 32, wherein step (4) comprises:
(a) measuring a volume of cells per unit volume of blood in the
analysis image to determine hematocrit (Hct);
(b) determining a mean reflected light intensity of the analysis
image;
(c) converting the mean reflected light intensity of the analysis
image into a hemoglobin concentration per unit volume of blood
(Hb); and
(d) determining a mean cell volume (MCV) from the analysis
image.
49. The method of claim 48, further comprising:
(5) determining a red blood cell count (RBC) from the equation
50. The method of claim 48, further comprising:
(5) determining a mean cell hemoglobin concentration (MCHC) from
the analysis image.
51. The method of claim 50, further comprising:
(5) determining a mean cell hemoglobin from the equation
52. The method of claim 32, wherein the analysis image is segmented
from the corrected reflected image so that the analysis image
includes capillary plasma.
53. The method of claim 52, wherein step (4) comprises:
(a) determining a mean reflected light intensity of the analysis
image; and
(b) converting the mean reflected light intensity of the analysis
image into a bilirubin concentration per unit volume of blood.
54. The method of claim 53, wherein step (2) comprises:
(a) applying a first wavelength filter to the raw reflected image
to form a first filtered image;
(b) applying a second wavelength filter to the raw reflected image
to form a second filtered image;
(c) taking the negative logarithm of the quotient obtained by
dividing the first filtered image by the second filtered image to
form the corrected reflected image.
55. The method of claim 54, wherein the first wavelength filter is
centered at a first wavelength of 450 nm and the second wavelength
filter is centered at a second wavelength of 600 nm.
56. The method of claim 52, wherein step (4) comprises:
(a) detecting optical contrast produced by a marker introduced into
the blood.
57. The method of claim 56, wherein the marker is used to detect
compounds in the capillary plasma.
58. The method of claim 56, wherein the marker is used to detect
compounds that attach to cells in the blood.
59. The method of claim 52, wherein step (4) comprises:
(a) detecting natural constituents of plasma.
60. The method of claim 52, wherein step (4) comprises:
(a) detecting non-natural components of plasma.
61. A method for non-invasive, in vivo analysis of blood in large
vessels, comprising:
(1) illuminating a portion of a subject's vascular system with
light polarized by a first polarizer;
(2) capturing a reflected image reflected from the illuminated
portion, wherein the reflected image is passed through a second
polarizer having a plane of polarization 90.degree. relative to a
plane of polarization of the first polarizer to produce a raw
reflected image;
(3) applying a correction to the raw reflected image to form a
corrected reflected image;
(4) segmenting a scene from the corrected reflected image that
includes large vessels to form an analysis image, wherein large
vessels are of sufficient size so that a plurality of red blood
cells flow side-by-side through them; and
(5) analyzing the analysis image for a characteristic of blood in
large vessels.
62. The method of claim 61, wherein step (4) comprises applying at
least one of:
(a) an optical intensity criterion to the corrected reflected image
to form an intensity-corrected reflected image;
(b) a size criterion to the intensity-corrected reflected image to
form an intensity-and-size-corrected reflected image; and
(c) a shape criterion to the intensity-and-size-corrected reflected
image to form the analysis image.
63. The method of claim 62, wherein step (5) comprises:
(a) determining a mean reflected light intensity of the analysis
image; and
(b) converting the mean reflected light intensity of the analysis
image into a hemoglobin concentration per unit volume of blood
(Hb).
64. The method of claim 63, wherein step (3) comprises applying a
bi-chromatic correction using a first wavelength centered at 550 nm
and a second wavelength centered at 650 nm.
65. The method of claim 62, wherein step (5) comprises:
(a) measuring a volume of cells per unit volume of blood in the
analysis image to determine hematocrit (Hct).
66. The method of claim 61, wherein step (2) is performed using
stop action.
67. The method of claim 66, wherein step (5) comprises:
(a) counting white blood cells in the analysis image to determine a
number of white blood cells per unit volume of blood.
68. The method of claim 67, wherein step (1) is performed using
light in the range from 400 nm to 600 nm.
69. The method of claim 66, wherein step (5) comprises:
(a) counting granulocytes in the analysis image; and
(b) counting agranulocytes in the analysis image.
70. The method of claim 61, wherein step (5) comprises:
(a) detecting optical contrast produced by a marker introduced into
the subject's vascular system.
71. The method of claim 70, wherein the marker is used to detect
compounds that attach to cells in the subject's vascular
system.
72. The method of claim 61, wherein step (5) comprises:
(a) detecting a hemoglobin complex.
73. A method for non-invasive, in vivo analysis of blood in small
vessels, comprising:
(1) illuminating a portion of a subject's vascular system with
light polarized by a first polarizer;
(2) capturing a reflected image reflected from the illuminated
portion, wherein the reflected image passes through a second
polarizer having a plane of polarization 90.degree. relative to a
plane of polarization of the first polarizer to produce a raw
reflected image;
(3) applying a correction to the raw reflected image to form a
corrected reflected image;
(4) segmenting a scene from the corrected reflected image that
includes small vessels to form an analysis image, wherein small
vessels are of a size so that red blood cells flow substantially
single file through them; and
(5) analyzing the analysis image for a characteristic of blood in
small vessels.
74. The method of claim 73, wherein step (2) is performed using
stop action.
75. The method of claim 74, wherein step (5) comprises:
(a) counting platelets in the analysis image to determine a number
of platelets per unit volume of blood.
76. The method of claim 73, wherein the analysis image is segmented
from the corrected reflected image so that the analysis image
includes capillary plasma.
77. The method of claim 76, wherein step (5) comprises:
(a) determining a mean reflected light intensity of the analysis
image; and
(b) converting the mean reflected light intensity of the analysis
image into a bilirubin concentration per unit volume of blood.
78. The method of claim 77, wherein step (3) comprises applying a
bi-chromatic correction using a first wavelength centered at 450 nm
and a second wavelength centered at 600 nm.
79. The method of claim 76, wherein step (5) comprises:
(a) detecting optical contrast produced by a marker.
80. The method of claim 79, wherein the marker is used to detect
compounds in the capillary plasma.
81. The method of claim 79, wherein the marker is used to detect
compounds that attach to cells.
82. The method of claim 73, wherein step (5) comprises:
(a) determining a mean cell volume from the analysis image.
83. The method of claim 73, wherein step (5) comprises:
(a) determining a mean cell hemoglobin concentration (MCHC) from
the analysis image.
84. The method of claim 83, wherein step (3) comprises:
(a) applying a bi-chromatic correction using a first wavelength
centered at 550 nm and a second wavelength centered at 650 nm.
85. The method of claim 73, wherein step (3) comprises:
(a) applying a velocity correction to the raw reflected image to
from the corrected reflected image.
86. The method of claim 73, wherein step (4) comprises applying at
least one of:
(a) an optical intensity criterion to the corrected reflected image
to form an intensity-corrected reflected image;
(b) a size criterion to the intensity-corrected reflected image to
form an intensity-and-size-corrected reflected image; and
(c) a shape criterion to the intensity-and-size-corrected reflected
image to form the analysis image.
87. The method of claim 73, wherein step (5) comprises:
(a) detecting natural constituents of plasma.
88. The method of claim 73, wherein step (5) comprises:
(a) detecting non-natural components of plasma.
89. Apparatus for optically penetrating an object and detecting
subsurface optical characteristics of an object, comprising:
a light source for illuminating an object at a wavelength such that
the multiple scattering depth is small compared to the penetration
depth of the illuminating light;
a first polarizer for polarizing light from said light source;
imaging means for detecting an image reflected from beneath the
surface of the illuminated object; and
a second polarizer disposed in a reflected light path between the
object and said imaging means through which the scattered light
passes, wherein a plane of polarization of said second polarizer is
substantially orthogonal relative to a plane of polarization of
said first polarizer.
90. The apparatus according to claim 89, further comprising:
an objective lens disposed between said first polarizer and the
object, wherein said imaging means is in a magnified image plane of
said objective lens.
91. The apparatus according to claim 89, further comprising:
reflected light separating means disposed in the reflected light
path between said second polarizer and said imaging means for
separating the reflected light reflected from the illuminated
object, wherein a first portion of the reflected light is
transmitted through said reflected light separating means to said
imaging means, and a second portion of the reflected light is
reflected by said reflected light separating means; and
a second imaging means for imaging the second portion of the
reflected light.
92. The apparatus according to claim 91, further comprising:
a first spectral selection means disposed in the reflected light
path between said reflected light separating means and said imaging
means; and
a second spectral selection means disposed in the reflected light
path between said reflected light separating means and said second
imaging means.
93. The apparatus according to claim 89, wherein said imaging means
comprises a camera.
94. The apparatus according to claim 93, wherein said imaging means
further comprises a photodetector.
95. The apparatus according to claim 89, wherein said imaging means
comprises a photodetector.
96. The apparatus according to claim 89, further comprising:
a dichroic separator disposed in the reflected light path between
said second polarizer and said imaging means.
97. The apparatus according to claim 89, wherein said light source
comprises said first polarizer.
98. The apparatus according to claim 97 wherein said light source
comprises a laser diode.
99. The apparatus according to claim 89, wherein said light source
is monochromatic.
100. The apparatus according to claim 99, wherein said light source
comprises a light emitting diode (LED).
101. The apparatus according to claim 99, wherein said light source
is polarized.
102. The apparatus according to claim 101, wherein said light
source comprises a laser.
103. Apparatus for quantitatively measuring absorption properties
of an imaged object, comprising:
a light source for illuminating an object to be imaged;
a first polarizer for polarizing light from said light source;
imaging means for detecting an image reflected from the illuminated
object;
a second polarizer disposed in a reflected light path between the
object and said imaging means, wherein a plane of polarization of
said second polarizer is substantially orthogonal relative to a
plane of polarization of said first polarizer; and
measuring means coupled to said imaging means for quantitatively
measuring differences in absorption properties between imaged
structures using said reflected light image.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is related to reflected light analysis. More
particularly, the present invention is related to the use of
reflected spectral imaging to perform non-invasive analysis of a
subject's vascular system. The present invention is also related to
the use of cross-polarizers in reflected spectral imaging
analysis.
2. Related Art
Widely accepted medical school doctrine teaches that the complete
blood count including the white blood cell differential (CBC+Diff)
is one of the best tests to assess a patient's overall health. With
it, a physician can detect or diagnose anemia, infection, blood
loss, acute and chronic diseases, allergies, and other conditions.
CBC+Diff analyses provide comprehensive information on constituents
in blood, including the number of red cells, the hematocrit, the
hemoglobin concentration, and indices that portray the size, shape,
and oxygen-carrying characteristics of the entire red blood cell
(RBC) population. The CBC+Diff also includes the number and types
of white blood cells and the number of platelets. The CBC+Diff is
one of the most frequently requested diagnostic tests with about
two billion done in the United States per year.
A conventional CBC+Diff test is done in an "invasive" manner in
which a sample of venous blood is drawn from a patient through a
needle, and submitted to a laboratory for analysis. For example, a
phlebotomist (an individual specially trained in drawing blood)
collects a sample of venous blood into a tube containing an
anticoagulant to prevent the blood from clotting. The sample is
then sent to a hematology laboratory to be processed, typically on
automated, multiparameter analytical instruments, such as those
manufactured by Coulter Diagnostics of Miami, Fla. The CBC+Diff
test results are returned to the requesting physician, typically on
the next day.
In medical diagnosis it is often necessary to measure other types
of blood components, such as non-cellular constituents present in
the plasma component of blood. Such constituents can include, for
example, blood gases and bilirubin. Bilirubin is a reddish to
yellow pigment produced in the metabolic breakdown of hemoglobin
and other proteins. Bilirubin is removed from the blood by the
liver and is excreted from the body. However, the livers of newborn
children, especially premature babies, cannot process bilirubin
effectively.
The birth process often results in extensive bruising, resulting in
blood escaping into the tissues where it is broken down
metabolically. For this and other medical causes, bilirubin may
accumulate in the blood stream. If bilirubin levels rise high
enough, it begins to be deposited in other body tissues causing
jaundice. Its first appearance is in the eye. At still higher
levels, deposition begins in deeper tissues, including the brain,
and can result in permanent brain damage.
The most common method for bilirubin analysis is through an in
vitro process. In such an in vitro process, a blood sample is
invasively drawn from the patient. The formed elements (red blood
cells and other cells) are separated by centrifugation and the
remaining fluid is reacted chemically and analyzed
spectrophotometrically.
Invasive techniques, such as for conventional CBC+Diff tests and
bilirubin analysis, pose particular problems for newborns because
their circulatory system is not yet fully developed. Blood is
typically drawn using a "heel stick" procedure wherein one or more
punctures are made in the heel of the newborn, and blood is
repeatedly squeezed out into a collecting tube. This procedure is
traumatic even for an infant in good health. More importantly, this
procedure poses the risk of having to do a blood transfusion
because of the low total blood volume of the infant. The total
blood volume of the newborn infant is 60-70 cc/kg body weight.
Thus, the total blood volume of low birth weight infants (under
2500 grams) cared for in newborn intensive care units ranges from
45-175 cc. Because of their low blood volume and delay in
production of red cells after birth, blood sampling from preterm
infants and other sick infants frequently necessitates transfusions
for these infants. Blood bank use for transfusion of infants in
neonatal intensive care units is second only to the usage for
cardiothoracic surgery in blood banking requirements. In addition
to newborns, invasive techniques are also particularly stressful
for, and/or difficult to carry out on, children, elderly patients,
burn patients, and patients in special care units.
A hierarchical relationship exists between the laboratory findings
and those obtained at the physical examination. The demarcation
between the physical findings of the patient and the laboratory
findings are, in general, the result of technical limitations. For
instance, in the diagnosis of anemia (defined as low hemoglobin
concentration), it is frequently necessary to quantify the
hemoglobin concentration or the hematocrit in order to verify the
observation of pallor. Pallor is the lack of the pink color of skin
which frequently signals the absence or reduced concentration of
the heavily red pigmented hemoglobin. However, there are some
instances in which pallor may result from other causes, such as
constriction of peripheral vessels, or being hidden by skin
pigmentation. Because certain parts of the integument are less
affected by these factors, clinicians have found that the pallor
associated with anemia can more accurately be detected in the
mucous membrane of the mouth, the conjunctivae, the lips, and the
nail beds. A device which is able to rapidly and non-invasively
quantitatively determine the hemoglobin concentration directly from
an examination of one or more of the foregoing areas would
eliminate the need to draw a venous blood sample to ascertain
anemia. Such a device would also eliminate the delay in waiting for
the laboratory results in the evaluation of the patient. Such a
device also has the advantage of added patient comfort.
Soft tissue, such as mucosal membranes or unpigmented skin, do not
absorb light in the visible and near-infrared, i.e., they do not
absorb light in the spectral region where hemoglobin absorbs light.
This allows the vascularization to be differentiated by spectral
absorption from surrounding soft tissue background. However, the
surface of soft tissue strongly reflects light and the soft tissue
itself effectively scatters light after penetration of only 100
microns. Therefore, in vivo visualization of the circulation is
difficult because of poor resolution, and generally impractical
because of the complexities involved in compensating for multiple
scattering and for specular reflection from the surface. Studies on
the visualization of cells in the microcirculation consequently
have been almost exclusively invasive, using a thin section (less
than the distance for multiple scattering) of tissue containing the
microcirculation, such as the mesentery, that can be observed by a
microscope using light transmitted through the tissue section.
Other studies have experimented with producing images of tissues
from within the multiple scattering region by time gating (see,
Yodh, A. and B. Chance, Physics Today, March, 1995, 34-40).
However, the resolution of such images is limited because of the
scattering of light, and the computations for the scattering factor
are complex.
Spectrophotometry involves analysis based on the absorption or
attenuation of electromagnetic radiation by matter at one or more
wavelengths of light. The instruments used in this analysis are
referred to as spectrophotometers. A simple spectrophotometer
includes: source of radiation, such as, e.g., a light bulb; a means
of spectral selection such as a monochromator containing a prism or
grating or colored filter; and one or more detectors, such as,
e.g., photocells, which measure the amount of light transmitted
and/or reflected by the sample in the selected spectral region.
In opaque samples, such as solids or highly absorbing solutions,
the radiation reflected from the surface of the sample may be
measured and compared with the radiation reflected from a
non-absorbing or white sample. If this reflectance intensity is
plotted as a function of wavelength, it gives a reflectance
spectrum. Reflectance spectra are commonly used in matching colors
of dyed fabrics or painted surfaces. However, because of its
limited range and inaccuracy, reflection spectrophotometry has been
used primarily in qualitative rather than quantitative analysis. On
the other hand, transmission spectrophotometry is conventionally
used for quantitative analysis because Beer's law (inversely
relating the logarithm of measured intensity linearly to
concentration) can be used.
Reflective spectrophotometry is conventionally avoided for
quantitative analysis because specularly reflected light from a
surface limits the available contrast (black to white or signal to
noise ratio), and, consequently, the measurement range and
linearity. Because of surface effects, measurements are usually
made at an angle to the surface. However, only for the special case
of a Lambertian surface will the reflected intensity be independent
of the angle of viewing. Light reflected from a Lambertian surface
appears equally bright in all directions (cosine law). However,
good Lambertian surfaces are difficult to obtain. Conventional
reflection spectrophotometry presents an even more complicated
relationship between reflected light intensity and concentration
than exists for transmission spectrophotometry which follows Beer's
law. Under the Kubelka-Munk theory applicable in reflection
spectrophotometry, the intensity of reflected light can be related
indirectly to concentration through the ratio of absorption to
scattering.
Some imaging studies have been done in the reflected light of the
microcirculation of the nail beds on patients with Raynauds,
diabetes, and sickle cell disease. These studies were done to
obtain experimental data regarding capillary density, capillary
shape, and blood flow velocity, and were limited to gross physical
measurements on capillaries. No spectral measurements, or
individual cellular measurements, were made, and Doppler techniques
were used to assess velocity. The non-invasive procedure employed
in these studies could be applied to most patients, and in a
comfortable manner.
One non-invasive device for in vivo analysis is disclosed in U.S.
Pat. No. 4,998,533 to Winkelman. The Winkelman device uses image
analysis and reflectance spectrophotometry to measure individual
cell parameters such as cell size. Measurements are taken only
within small vessels, such as capillaries where individual cells
can be visualized. Because the Winkelman device takes measurements
only in capillaries, measurements made by the Winkelman device will
not accurately reflect measurements for larger vessels. This
inaccuracy results from the constantly changing relationship of
volume of cells to volume of blood in small capillaries resulting
from the non-Newtonian viscosity characteristic of blood.
Consequently, the Winkelman device is not capable of measuring the
central or true hematocrit, or the total hemoglobin concentration,
which depend upon the ratio of the volume of red blood cells to
that of the whole blood in a large vessel such as a vein.
The Winkelman device measures the number of white blood cells
relative to the number of red blood cells by counting individual
cells as they flow through a micro-capillary. The Winkelman device
depends upon accumulating a statistically reliable number of white
blood cells in order to estimate the concentration. However, blood
flowing through a micro-capillary will contain approximately 1000
red cells for every white cell, making this an impractical method.
The Winkelman device does not provide any means by which platelets
can be visualized and counted. Further, the Winkelman device does
not provide any means by which the capillary plasma can be
visualized, or the constituents of the capillary plasma quantified.
The Winkelman device also does not provide a means by which
abnormal constituents of blood, such as tumor cells, can be
detected.
Thus, there is a need in the art for a device that provides for
complete non-invasive in vivo analysis of the vascular system.
There is a need for a device that provides for high resolution
visualization of: blood cell components (red blood cells, white
blood cells, and platelets); blood rheology; the vessels in which
blood travels; and vascularization throughout the vascular system.
There is a further need for a non-invasive device that allows
quantitative determinations to be made for blood cells, normal and
abnormal contents of blood cells, as well as for normal and
abnormal constituents of blood plasma.
SUMMARY OF THE INVENTION
The present invention is directed to a method and apparatus for
analysis of blood by use of reflected spectral imaging analysis. In
one embodiment, the apparatus can include a light source that
illuminates blood to form a light path between the light source and
the illuminated blood. A first polarizer is used to polarize light
from the light source. Image capturing means is used to capture the
reflected image reflected from the illuminated object at a depth
less than a multiple scattering length. The reflected image travels
along a reflected light path between the illuminated blood and the
image capturing means. A second polarizer is placed in the
reflected light path between the illuminated blood and the image
capturing means. The second polarizer has a plane of polarization
that is 90.degree. relative to the plane of polarization of the
first polarizer. In one aspect of the present invention, the light
source comprises the first polarizer, so that the second polarizer
has a plane of polarization 90.degree. relative to the plane of
polarization of the polarized light produced by the light
source.
In a further aspect of the present invention, the apparatus
includes image separating means that are placed in the reflected
light path between the second polarizer and the image capturing
means. The image separating means separates the reflected image
into multiple image portions. Additional image capturing means can
be used to capture the multiple portions of the reflected image.
Spectral selection means can be placed in the reflected light path
between the image separating means and the image capturing
means.
In a further aspect of the present invention, a method for analysis
of blood is provided. The method includes the following steps: (1)
imaging blood to produce a raw reflected image reflected from a
depth less than a multiple scattering length; (2) applying a
correction to the raw reflected image to form a corrected reflected
image; (3) segmenting a scene from the corrected reflected image to
form an analysis image; and (4) analyzing the analysis image for a
characteristic of the blood.
In a further aspect of the method of the present invention, the
step of applying a correction to the raw reflected image can be
carried out using the following steps: (a) applying a first
wavelength filter to the raw reflected image to form a first
filtered image; (b) applying a second wavelength filter to the raw
reflected image to form a second filtered image; and (c) taking the
negative logarithm of the quotient obtained by dividing the first
filtered image by the second filtered image to form the corrected
reflected image. Alternatively, the correction may be performed by
taking the difference of the logarithm of the first and second
filtered images.
In another aspect of the method of the present invention, a step of
segmenting a scene from the corrected reflected image to form an
analysis image includes applying one or more criterion to the
corrected reflected image. These criteria can include an optical
intensity criterion, a size criterion, a shape criterion, or other
spatial filtering techniques.
The method of the present invention can be used to determine
various characteristics of blood. Such characteristics can include
the hemoglobin concentration per unit volume of blood, the number
of white blood cells per unit volume of blood, a mean cell volume,
a mean cell hemoglobin concentration, the number of platelets per
unit volume of blood, and the hematocrit.
In still a further aspect of the method of the present invention,
the blood can be illuminated with light polarized by a first
polarizer. The reflected image can be passed through a second
polarizer or analyzer having a plane of polarization 90.degree.
relative to a plane of polarization of the first polarizer to
produce the raw reflected image.
In further aspects of the present invention, the method is used to
perform in vivo analysis of blood in large vessels, and in vivo
analysis of blood in small vessels to determine blood parameters
such as concentrations and blood cell counts. The method of the
present invention can also be used to conduct non-invasive in vivo
analysis of non-cellular characteristics of capillary plasma.
In yet a further embodiment of the present invention, an apparatus
is provided for detecting optical characteristics of an object. The
apparatus includes a light source for illuminating the object, and
detecting means for detecting reflected light that is reflected
from the illuminated object. A first polarizer is used to polarize
light from the light source. A second polarizer is placed in a
reflected light path between the object and the detecting means.
The plane of polarization of the second polarizer is 90.degree.
relative to a plane of polarization of the first polarizer. In
further aspects of the present invention, the light source is
monochromatic, polarized, or monochromatic and polarized.
FEATURES AND ADVANTAGES
It is a feature of the present invention that it provides for
non-invasive in vivo analysis of the vascular system. It is a
further feature of the present invention that quantitative analyses
of formed blood cell components (red blood cells, white blood
cells, and platelets) can be done. It is also a further feature of
the present invention that quantitative analyses of non-formed
blood components, such as capillary plasma, can also be done.
It is yet a further feature of the present invention that per unit
volume or concentration measurements, such as hemoglobin,
hematocrit, and blood cell counts, can be made through the use of
reflected spectral images of the vascular system.
It is yet a further feature of the present invention that blood
cells, blood vessels, and capillary plasma can be visualized and
segmented into an analysis image.
A still further feature of the present invention is that it can be
used to determine characteristics such as the hemoglobin
concentration per unit volume of blood, the number of white blood
cells per unit volume of blood, the mean cell volume, the mean cell
hemoglobin concentration, the number of platelets per unit volume
of blood, and the hematocrit through the use of reflected spectral
imaging.
An advantage of the present invention is that it provides a means
for the rapid, non-invasive measurement of clinically significant
parameters of the CBC+Diff test. It advantageously provides
immediate results. As such, it can be used for point-of-care
testing and diagnosis.
A further advantage of the present invention is that it eliminates
the invasive technique of drawing blood. This eliminates the pain
and difficulty of drawing blood from newborns, children, elderly
patients, burn patients, and patients in special care units. The
present invention is also advantageous in that it obviates the risk
of exposure to AIDS, hepatitis, and other blood-borne diseases.
A still further advantage of the present invention is that it
provides for overall cost savings by eliminating sample
transportation, handling, and disposal costs associated with
conventional invasive techniques.
A still further advantage of the present invention is that it
provides for substantially improved range and accuracy for
reflection spectrophotometry. The present invention is also
advantageous in that it permits use of a simple relationship
between concentration and intensity.
A still further advantage of the present invention is that it
provides for improved visualization of reflected images of any
object, and for quantitative and qualitative analyses of these
reflected images.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention is described with reference to the
accompanying drawings. In the drawings, like reference numbers
indicate identical or functionally similar elements. Additionally,
the left-most digit(s) of a reference number identifies the drawing
in which the reference number first appears.
FIG. 1 shows a block diagram of one embodiment of a method of the
present invention;
FIG. 2 shows a block diagram of an embodiment of the present
invention for imaging a subject's vascular system;
FIG. 3 shows a block diagram illustrating step 220 shown in FIG.
2;
FIG. 4 shows a block diagram illustrating step 230 shown in FIG.
2;
FIG. 5 shows a chart entitled Approach to Hematologic
Disorders;
FIG. 6A illustrates a feasibility model;
FIG. 6B illustrates a block diagram of the feasibility model shown
in FIG. 6A;
FIG. 7 shows a more detailed illustration of the hydro-dynamic
focused flow-cell shown in FIG. 6A;
FIG. 8A shows a chart summarizing the agreement between laboratory
results on a Coulter device and the feasibility model;
FIG. 8B shows a graph illustrating a Comparative Determination of
Hemoglobin with Various Levels of Anemia (hemoglobin gm/dL as a
function of % of blood volume lost against total blood volume);
FIG. 8C shows a graph illustrating a Comparative Determination of
Hemoglobin on 23 "Healthy" Human Subjects;
FIG. 9 shows images of red cells and platelets using the
feasibility model;
FIG. 10 shows an area of interest around a background (region A),
to be used as a reference, and an area of interest inside a flowing
stream (region B);
FIG. 11 shows a chart of optical density versus concentration of
bilirubin;
FIG. 12 illustrates a chopper stabilized reflectance
spectrophotometer;
FIG. 13 shows a reflectance spectral scan for leukopheresed
plasma;
FIG. 14 shows a leukemic blood sample image obtained with the
feasibility model;
FIG. 15A shows a block diagram illustrating one embodiment of an in
vivo apparatus;
FIG. 15B shows further detail of the in vivo apparatus shown in
FIG. 15A;
FIG. 16 shows a block diagram of a computer system suitable for use
in the present invention;
FIGS. 17A and 17B show embodiments of the present invention
suitable for use with a subject;
FIG. 18A shows an ink-jet cross visualized in conventional
reflected light;
FIG. 18B shows an ink-jet cross visualized in reflected light using
the cross polarization technique of the present invention;
FIG. 19 shows a plot illustrating reflected spectrophotometry of
red aniline dye; and
FIG. 20 shows a block diagram of one embodiment of a reflection
colorimeter apparatus of the present invention.
DETAILED DESCRIPTION OF THE EMBODIMENTS
1. Overview
The present invention is directed to a method and apparatus for
analysis, particularly non-invasive, in vivo analysis of a
subject's vascular system. The method is carried out by imaging a
portion of the subject's vascular system. The tissue covering the
imaged portion must be traversed by light without multiple
scattering to obtain a reflected image. In order to form an image,
two criteria must be met. First, there must be image contrast
resulting from a difference in the optical properties, such as
absorption, index of refraction, or scattering characteristics,
between the subject to be imaged and its surround or background.
Second, the light that is collected from the subject must reach an
image capturing means without substantial scattering, i.e., the
reflected image must be captured from a depth that is less than the
multiple scattering length. As used herein, "image" refers to any
image that satisfies the foregoing two criteria. As used herein,
"reflected image" refers to the image of a subject in reflected
light. The resolution required for capturing the image is dictated
by the spatial homogeneity of the imaged portion. For example, a
reflected image of individual cells requires high resolution. A
reflected image of large vessels can be done with low resolution. A
reflected image suitable for making a determination based on pallor
requires very low resolution.
The tissue covering the imaged portion is thus preferably
transparent to light, and relatively thin, such as the mucosal
membrane on the inside of the lip of a human subject. As used
herein, "light" refers generally to electromagnetic radiation of
any wavelength, including the infrared, visible, and ultraviolet
portions of the spectrum. A particularly preferred portion of the
spectrum is that portion where there is relative transparency of
tissue, such as in the visible and near-infrared wavelengths. It is
to be understood that for the present invention, light can be
coherent light or incoherent light, and illumination may be steady
or in pulses of light.
The reflected image is corrected to form a corrected reflected
image. The correction to the reflected image is done, for example,
to isolate particular wavelengths of interest, or to extract a
moving portion of the image from a stationary portion of the image.
A scene is segmented from the corrected reflected image to form an
analysis image. The analysis image is then analyzed for the desired
characteristic of the subject's vascular system.
The method of the present invention can be used for analysis in
large vessels, small vessels, and in capillary plasma. As used
herein, "large vessel" refers to a vessel in the vascular system of
sufficient size so that a plurality of red blood cells flow
side-by-side through it. "Small vessel" refers to a vessel in the
vascular system of a size so that red blood cells flow
substantially "single file" through it. As explained in more detail
below, the present invention uses reflectance, not transmission,
for the images that are analyzed. That is, the image is made by
"looking at" the vascular system, rather than by "looking through"
the vascular system. Per unit volume or concentration measurements
can be made directly from the images.
By using the method of the present invention to provide a reflected
spectral image of large vessels, the hemoglobin (Hb), hematocrit
(Hct), and white blood cell count (WBC) parameters can be directly
determined. By using the method of the present invention to provide
a reflected spectral image of small vessels, mean cell volume
(MCV), mean cell hemoglobin concentration (MCHC), and platelet
count (Plt) can be directly determined.
To implement the method of the present invention, a light source is
used to illuminate the portion of the subject's vascular system to
be imaged. The reflected light is captured by an image capturing
means. By image capturing means is meant a device capable of
capturing an image as defined herein. Suitable image capturing
means include, but are not limited to, a camera, a film medium, a
photocell, a photodiode, or a charge coupled device camera. An
image correcting and analyzing means, such as a computer, is
coupled to the image capturing means for carrying out image
correction, scene segmentation, and blood characteristic
analysis.
The image correction can be a poly-chromatic correction using a
primary wavelength and one or more secondary wavelengths. For
example, to implement a bi-chromatic correction, the reflected
image is separated into two portions. This can be implemented using
an image separating means, such as a dichroic mirror. In this
manner, one image capturing means is used to capture the portion of
the image transmitted through the dichroic mirror, and one image
capturing means is used to capture the portion of the image
reflected by the dichroic mirror. Image correcting and analyzing
means coupled to both image capturing means carries out a
correction of one image portion relative to the other image portion
to provide the corrected image.
Cross-polarizers are preferably used in implementing the present
invention. One polarizer is placed in the light path between the
light source and the illuminated portion of the subject's vascular
system. A second polarizer or "analyzer" is placed in the reflected
light path between the illuminated portion and the image capturing
means. The second polarizer has a plane of polarization 90.degree.
relative to the plane of polarization of the first polarizer. The
cross-polarizer configuration improves the collection of light that
has interacted with the illuminated portion of the subject's
vascular system and tissue by eliminating light that has simply
been reflected and has not fully interacted with the illuminated
portion. Therefore, light with no information regarding the
illuminated subject is eliminated. In this manner, the image
contrast for the reflected image is vastly increased, thereby
improving visualization in the illuminated portion.
The cross-polarization technique of the present invention can be
used in any application that requires optically measuring or
visually observing reflecting characteristics of an object. The
cross-polarizers of the present invention can be used to increase
the working range, and permit the use of a simple relationship
between intensity and concentration for analytical instruments that
detect differences in reflected intensity. The cross-polarization
technique of the present invention is applicable in such fields as
dye lot control, fabric color control, strip testing using paper,
film, or latex, borescopic and orthoscopic applications.
2. Methods of the Present Invention
In order to see into an object using reflected light, there must be
light coming back from the object which has interacted with the
object below the surface. In using a reflected image, two
properties must be considered: (1) the absorption of light within
the object; and (2) the scattering of light within the object. A
reflected image is a three dimensional image that has an area and a
depth of penetration. The depth of penetration or path length for
the light is controlled by three parameters: (1) the wavelength of
light; (2) the size of the particles with which the light
interacts; and (3) the index of refraction. If the wavelength of
light, the particle size, and the index of refraction are constant,
then the depth of penetration is constant. Therefore, a measurement
made per unit area in such a reflected image is proportional to a
measurement per unit volume because the depth of penetration is
constant. An area measurement is a volume measurement with a
constant third dimension (depth).
With reference now to FIG. 1, a block diagram 100 of one embodiment
of a method of the present invention for reflected spectral imaging
analysis is shown. Block diagram 100 illustrates a process used to
convert a raw reflected image 110 into a result 140. By raw
reflected image is meant the reflected image prior to application
of a correction function 115.
Correction function 115 is applied to raw reflected image 110 to
produce a corrected reflected image 120. Correction function 115
normalizes raw reflected image 110 with respect to the image
background. In one embodiment, correction function 115 is
implemented by way of a bi-chromatic correction. For a bi-chromatic
correction, two wavelengths, .lambda..sub.1 and .lambda..sub.2, are
selected. By subtracting the .lambda..sub.2 image from the
.lambda..sub.1 image, all parameters that affect both
.lambda..sub.1 and .lambda..sub.2 in the same manner cancel out,
and are thus eliminated, in the resulting (.lambda..sub.1
-.lambda..sub.2) image. The resulting (.lambda..sub.1
-.lambda..sub.2) image incorporates the effect of only those
parameters that affect .lambda..sub.1 and .lambda..sub.2
differently.
In another embodiment, correction function 115 is implemented by
way of a velocity or speed correction. For a velocity correction,
corrected reflected image 120 is formed by taking the difference
between raw reflected image 110 at a time t.sub.0 and at a time
t.sub.1. For this purpose, means must be provided to pulse the
light, and/or shutter an image capturing means such as a camera, so
that two different images in time are obtained. A velocity
correction allows a moving portion of raw reflected image 110 to be
extracted from a stationary portion of raw reflected image 110. In
this manner, corrected reflected image 120 is formed to contain
either the moving portion or the stationary portion of raw
reflected image 110.
A segmentation function 125 is applied to corrected reflected image
120 to form an analysis image 130. Segmentation function 125
segments or separates a scene of interest from corrected reflected
image 120 to form analysis image 130. An analysis function 135 is
applied to analysis image 130 to produce result 140. The scene of
interest segmented by segmentation function 125 can depend upon the
type of analysis performed by analysis function 135. In this
manner, corrected reflected image 120 may contain many scenes of
interests that are segmented differently by various segmentation
functions.
With reference now to FIG. 2, a block diagram 200 of a method of
the present invention for reflected spectral imaging of a subject's
vascular system is shown. In a step 210, a portion of a subject's
vascular system is imaged to produce raw reflected image 110 (see
FIG. 1). The tissue covering the imaged portion must be traversed
by light to obtain a reflected image without multiple scattering.
The reflected image is essentially from a single scattering of the
reflected light. The tissue covering the imaged portion should be
transparent to light. Particularly suitable tissues are the mucosal
membranes found in a variety of places in a human subject, such as
the nose, mouth, conjunctivae, rectum, and vagina. Alternatively,
for a premature baby, the skin itself is suitably transparent to
light. The inside of the lip of a human subject provides a suitable
area for imaging a portion of a human subject's vascular system. In
this manner, light from a light source penetrates the mucosal
membrane to produce a raw reflected image of the microvascular
system. The microvascular system contains both large vessels and
small vessels. As such, it is representative of vessels throughout
the vascular system. The raw reflected image of the microvascular
system from the inside of the lip is from a depth of approximately
50 .mu., to 500 .mu.. Alternatively, the microvascular system could
be imaged through skin tissue, such as on the finger or the toe of
a human subject.
In a step 220, a correction is applied to raw reflected image 110
to form corrected reflected image 120. For example, correction
function 115 can be applied to raw reflected image 110 to normalize
it with respect to the background. A poly-chromatic correction,
such as a bi-chromatic correction, can be used to eliminate the
effects of light intensity, depth, and angle of light from the
corrected reflected image. A poly-chromatic correction can
eliminate the effect of pigmentation of the tissue through which
the light travels to illuminate the imaged portion of the vascular
system. The tissue pigmentation will affect some wavelengths of
light in the same manner, so that the tissue pigmentation effect is
canceled out through use of a poly-chromatic correction. A velocity
correction could be applied to extract moving cells from a
stationary background. The velocity correction could be used alone,
or in conjunction with, a poly-chromatic correction.
In a step 230, a scene is segmented from corrected reflected image
120 to form analysis image 130. The analysis image is formed so
that it contains the subject matter needed for analyzing a
characteristic of the subject's vascular system. For example, the
characteristic to be analyzed may be one for which large vessels
should be analyzed, such as hemoglobin concentration per unit
volume of blood, or the number of white blood cells per unit volume
of blood. For these characteristics, analysis image 130 is formed
so that it contains large vessels. As another example, the
characteristic to be analyzed may be one for which small vessels
should be analyzed, such as the number of platelets per unit volume
of blood, or the concentration per unit volume of blood of
components in capillary plasma, such as bilirubin. For these
characteristics, analysis image 130 is formed so that it contains
small vessels. In a step 240, analysis image 130 is analyzed using
analysis function 135 for a characteristic of the subject's
vascular system.
In an alternate embodiment, the scene can be segmented from the raw
reflected image, and the scene corrected to form the analysis
image. The method of the present invention can also be carried out
without applying a correction function, so that the analysis image
is formed from the raw reflected image.
FIG. 3 shows a block diagram illustrating one embodiment of step
220 shown in FIG. 2 for applying a correction to raw reflected
image 110 to form corrected reflected image 120. In a step 302, a
first wavelength filter (.lambda..sub.1 filter) is applied to raw
reflected image 110 to form a first filtered image. In a step 304,
a second wavelength filter (.lambda..sub.2 filter) is applied to
raw reflected image 110 to form a second filtered image. In a
similar manner, additional wavelength filters (.lambda..sub.3,
.lambda..sub.4, .lambda..sub.5, etc.) can be used to form
additional filtered images.
In a step 306, the second filtered image is subtracted from the
first filtered image to form corrected reflected image 120. By
using the bi-chromatic correction of steps 302-306, those
parameters that affect both .lambda..sub.1 and .lambda..sub.2 in
the same manner have been eliminated from or canceled out of raw
reflected image 110. The eliminated parameters include variations
of light intensity, depth of penetration, angle of light, and
pigmentation of tissue covering the imaged portion of the subject's
vascular system. The corrected reflected image, (.lambda..sub.1
-.lambda..sub.2) image, includes those items that are
differentially affected by the two wavelengths. For raw reflected
image 110, the light intensity, the depth of penetration of the
light, and the angle of the light are all parameters that affect
.lambda..sub.1 and .lambda..sub.2 in the same manner. Therefore, a
bi-chromatic correction eliminates from raw reflected image 110 the
effects of light intensity variation, depth of penetration of the
light, and angle of the light to produce corrected reflected image
120.
Step 306 is preferably carried out in a manner that follows Beer's
Law so that the logarithm of the reflected intensity of corrected
image 120 is inversely proportional to concentration of a
component, such as hemoglobin, within the corrected reflected
image. Under Beer's law, the negative logarithm of measured
reflected light intensity is linearly related to concentration. In
one embodiment, step 306 is carried out so that the logarithm of
the second filtered image is subtracted from the logarithm of the
first filtered image to form corrected reflected image 120. In this
manner, the logarithm of the reflected intensity of the corrected
reflected image is proportional to concentration within the
corrected reflected image. In an alternate embodiment, step 306 is
carried out so that corrected reflected image 120 is formed by
taking the negative logarithm of the quotient obtained by dividing
the first filtered image by the second filtered image. In this
manner as well, the logarithm of the reflected intensity of the
corrected reflected image is proportional to concentration within
the corrected reflected image.
By properly selecting .lambda..sub.1 and .lambda..sub.2, it is
possible to normalize corrected reflected image 120 so that all
that remains within the image is something that is proportional to
hemoglobin concentration. To do so, one wavelength, such as
.lambda..sub.1, should be an absorbing wavelength for hemoglobin.
Blood in the vascular system of a human subject is made up of
arterial blood and venous blood. Arterial blood is that blood
having hemoglobin rich in oxygen (oxy-hemoglobin) to be carried
from the lungs to other parts of the body. Venous blood is that
blood having hemoglobin poor in oxygen (deoxy-hemoglobin) to be
carried from other parts of the body to the lungs to be replenished
with oxygen. Arterial and venous blood differ in color. This color
difference can be used to determine the degree of oxygen (O.sub.2)
saturation. The color signature of oxy-hemoglobin and
deoxy-hemoglobin can be used to detect these hemoglobin complexes.
Other hemoglobin complexes, such as carboxy-hemoglobin (carbon
monoxide poisoning) or glycosolated hemoglobin (glucose hemoglobin
complex monitored in diabetics) have spectral signatures allowing
their measurement.
There are only certain wavelengths which are absorbed equally by
both arterial blood and by venous blood. A wavelength which is
absorbed equally by both arterial and venous blood is called an
isobestic point. One such isobestic point for hemoglobin is located
at 546 nm. In a preferred embodiment, .lambda..sub.1 is selected so
that it is located near the center of an absorption band for
hemoglobin, and so that it is located near or at an isobestic
point. A suitable .lambda..sub.1 is 550 nm. In this manner, the
hemoglobin concentration can be determined from reflected spectral
imaging of a large vessel, irrespective of whether the large vessel
is an artery carrying arterial blood or a vein carrying venous
blood.
The other wavelength referred to as a "blank", such as
.lambda..sub.2, should be a non-absorbing wavelength for
hemoglobin. .lambda..sub.2 should be selected so that it is
sufficiently close to .lambda..sub.1 so that parameters such as
light intensity, depth of penetration, angle of light, and tissue
pigmentation have the same effect on both .lambda..sub.2 and
.lambda..sub.1. .lambda..sub.2 should be selected so that it is
sufficiently far from .lambda..sub.1 so that sufficient signal is
obtained for the (.lambda..sub.1 -.lambda..sub.2) image. The
spectral spread between .lambda..sub.1 and .lambda..sub.2 should be
selected to provide sufficient signal without introducing the
effect of the other parameters listed above. It would be readily
apparent to one of skill in the relevant arts how to select an
appropriate spectral spread. An appropriate spectral spread for
hemoglobin measurement is a first wavelength of 550 nm (absorbing
wavelength) and a second wavelength of 650 nm (non-absorbing
wavelength). With such a spectral spread, the difference in
intensity of reflected light is a function of the concentration of
hemoglobin.
FIG. 4 shows a block diagram illustrating one embodiment of step
230 shown in FIG. 2 for segmenting a scene from corrected reflected
image 120 to form analysis image 130. In a step 402, an optical
intensity criterion is applied to corrected reflected image 120 to
form an intensity-corrected reflected image. For example, the
optical intensity criterion can operate to delete all portions of
the corrected reflected image that have an optical intensity below
a certain threshold. Alternatively, the optical intensity criterion
can operate to delete all portions of the corrected reflected image
that have an optical intensity above a certain threshold. As yet
another alternative, the optical intensity criterion can operate to
retain only those portions of the corrected reflected image that
have an optical intensity within a predetermined range.
In a step 404, a size criterion is applied to the
intensity-corrected reflected image to form an intensity-and-size
corrected reflected image. For example, the size criterion can
operate to delete all portions of the intensity-corrected reflected
image that are below a size threshold. Alternatively, the size
criterion can operate to delete all portions of the
intensity-corrected reflected image that are above a size
threshold. As yet another alternative, the size criterion can
operate to retain only those portions of the intensity-corrected
reflected image that have a size within a predetermined range.
In a step 406, a shape criterion is applied to the
intensity-and-size corrected reflected image to form analysis image
130. For example, the shape criterion can operate to retain only
those portions of the intensity-and-size corrected reflected image
that have a shape defined by a predetermined distance from an axis.
Alternatively, the shape criterion can operate to retain only those
portions of the intensity-and-size corrected reflected image that
have a shape characteristic as defined by a smoothly shaped
boundary. For example, the curvature of the boundary can be
integrated to determine how the points of curvature are changing.
Using a smoothly shaped boundary as a shape characteristic, a
perfect circle would have a shape characteristic one (1). If the
shape of the item in the image was not a perfect circle, its shape
characteristic would be a value less than one. The smaller the
value of the shape characteristic, the less smooth is the boundary
of the item in the image. As an example, an item in the image
shaped as an ellipse would have a shape characteristic
approximately equal to 0.8. As another example, an item in the
image having an elongated and thin shape would have a shape
characteristic approximately equal to 0.1. The shape criterion can
operate to retain only those portions of the intensity-and-size
corrected reflected image that have a shape characteristic within a
predetermined range. Alternatively, the shape criterion can operate
to delete those portions of the intensity-and-size corrected
reflected image that have a shape characteristic within a
predetermined range.
Steps 402-406 represent one embodiment for segmenting a scene from
corrected reflected image 120 to form analysis image 130. As such,
steps 402-406 represent one embodiment of segmentation function
125. It is to be understood that the present invention is not
limited to this embodiment. For example, a size criterion could be
applied directly to corrected reflected image 120. A shape
criterion could also be applied directly to corrected reflected
image 120. As yet another example, the optical intensity criterion,
size criterion, and shape criterion could be applied sequentially
in a different order. Other suitable criteria could also be used to
segment a scene from corrected reflected image 120, and the present
invention is not limited to the use of optical intensity, size, and
shape criteria. For example, motion can be used as a criterion to
segment a scene from corrected reflected image 120. Motion can be
used to discriminate between moving portions of the image, such as
blood cells, from non-moving or slower-moving portions of the
image, such as tissue. Additionally, other image contrast
enhancement and scene segmentation techniques known to one of skill
in the relevant arts could be used, such as for example, spatial
frequency, optical flow, variance operators, and intensity
histograms.
The method illustrated in FIGS. 1-4 can be used to carry out
non-invasive in vivo analysis of blood parameters for the purpose
of diagnosis or monitoring. FIG. 5, adapted from Wintrobe's
Clinical Hematology, Ninth Edition, illustrates a graphical
relationship between the three elements of the diagnosis of
disorders of the blood: (1) history; (2) physical findings; and (3)
laboratory findings. It is implicit in FIG. 5 that a hierarchical
relationship exists between the laboratory findings and the
physical findings obtained at a physical examination (P.E.). By
rapidly and non-invasively determining the hemoglobin concentration
and the mean cell volume along with the physical examination, the
need to draw a venous blood sample is eliminated, along with the
delay in waiting for the laboratory results in the evaluation of a
patient.
In a similar manner, a rapid and non-invasive determination of the
white blood cell count could aid in the diagnosis of infection
and/or inflammation. The patient with fever could be examined to
determine whether or not the concentration of white blood cells was
elevated or decreased from normal.
Blood is made up of plasma and formed elements, and it flows
throughout the vascular system through small vessels and large
vessels as defined above. The formed elements of blood include red
blood cells, white blood cells, and platelets. As used herein,
"blood cells" refers to the formed elements of blood, and includes
red blood cells, white blood cells, and platelets. The
concentration of cells per unit volume of blood is a constant for
large vessels and is a reliable predictor of the concentration in
even larger vessels throughout the vascular system, such as vessels
large enough for insertion of a needle for drawing blood. In
contrast, a per unit volume (concentration) measurement made in a
small vessel where red blood cells flow substantially single file
is not a reliable predictor of the concentration measurement in
larger vessels from which blood may drawn by insertion of a needle.
The relationship between the concentration of cells and blood
volume is constantly changing in a small vessel, and, as such,
cannot be used as a reliable predictor of cell concentration in
larger vessels. This effect is so variable between different
individuals, and between different sites within one individual,
that even averaging does not provide a reliable predictor for
larger vessels.
A complete blood count (CBC) without white blood cell differential
measures eight parameters: (1) hemoglobin (Hb); (2) hematocrit
(Hct); (3) red blood cell count (RBC); (4) mean cell volume (MCV);
(5) mean cell hemoglobin (MCH); (6) mean cell hemoglobin
concentration (MCHC); (7) white blood cell count (WBC); and (8)
platelet count (Plt). The first six parameters are referred to
herein as RBC parameters. Concentration measurements (measurements
per unit volume of blood) are necessary for producing values for
Hb, Hct, RBC, WBC, and Plt. Hb is the hemoglobin concentration per
unit volume of blood. Hct is the volume of cells per unit volume of
blood. Hct can be expressed as a percentage
RBC is the number of red blood cells per unit volume of blood. WBC
is the number of white blood cells per unit volume of blood. Plt is
the number of platelets per unit volume of blood.
Red cell indices (MCV, MCH, and MCHC) are cellular parameters that
depict the volume, hemoglobin content, and hemoglobin
concentration, respectively, of the average red cell. The red cell
indices may be determined by making measurements on individual
cells, and averaging the individual cell measurements. Red cells do
not change volume or lose hemoglobin as they move through the
vascular system. Therefore, red cell indices are constant
throughout the circulation, and can be reliably measured in small
vessels. The three red cell indices are related by the equation
Thus, only two red cell indices are independent variables.
To determine values for the six RBC parameters listed above, the
following two criteria must be met. First, three of the parameters
must be independently measured or determined. That is, three of the
parameters must be measured or determined without reference to any
of the other of the six parameters. Second, at least one of the
three independently measured or determined parameters must be a
concentration parameter (per unit volume of blood). Therefore,
values for the six key parameters can be determined by making three
independent measurements, at least one of which is a concentration
measurement which cannot be made in a small vessel.
In one embodiment of the present invention, Hb and Hct are directly
measured by reflected spectral imaging of large vessels, and MCV is
directly measured by reflected spectral imaging of small vessels.
In this manner, three parameters are independently measured, and
two of the parameters (Hb and Hct) are concentration parameters
measured per unit volume of blood. In such an embodiment, the six
RBC parameters listed above can be determined in the following
manner.
______________________________________ Hb Directly measured Hct
Directly measured RBC Hct .div. MCV MCV Directly measured MCH MCV
.times. (Hb .div. Hct) MCHC Hb .div. Hct
______________________________________
In an alternate embodiment of the present invention, Hb is directly
measured by reflected spectral imaging of large vessels, and MCV
and MCHC are directly measured by reflected spectral imaging of
small vessels. In this manner, three parameters are independently
measured, and one of the parameters (Hb) is a concentration
parameter measured per unit volume of blood. In such an alternate
embodiment, the six RBC parameters listed above can be determined
in the following manner.
______________________________________ Hb Directly measured Hct Hb
.div. MCHC RBC Hb .div. (MCV .times. MCHC) MCV Directly measured
MCH MCV .times. MCHC MCHC Directly measured
______________________________________
Concentration measurements are measurements per unit volume. As
discussed above, a measurement made per unit area is proportional
to a measurement made per unit volume (volume measurement with
constant depth) when the depth of penetration is constant. The
depth of penetration is a function of wavelength, the size of the
particles with which it interacts, and refractive index. For blood,
the particle size and index of refraction are essentially constant.
Consequently, the depth of penetration will be constant for a
particular wavelength.
Hemoglobin is the main component of red blood cells. Hemoglobin is
a protein that serves as a vehicle for the transportation of oxygen
and carbon dioxide throughout the vascular system. Hemoglobin
absorbs light at particular absorbing wavelengths, such as 550 nm,
and does not absorb light at other non-absorbing wavelengths, such
as 650 nm. Under Beer's law, the logarithm of the measured
transmitted light intensity is linearly and inversely related to
concentration. As explained more fully below in Section 4, the
apparatus of the present invention is configured so that reflected
light intensity follows Beer's law. Assuming Beer's law applies,
the concentration of hemoglobin in a particular sample of blood is
linearly related to the negative logarithm of light reflected by
the hemoglobin. The more 550 nm light absorbed by a blood sample,
the lower the reflected light intensity at 550 nm, and the higher
the concentration of hemoglobin in that blood sample. The
concentration of hemoglobin can be computed by taking the negative
logarithm of the measured reflected light intensity at an absorbing
wavelength such as 550 nm. Therefore, if the reflected light
intensity from a particular sample of blood is measured, the
concentration in the blood of such components as hemoglobin can be
directly determined.
a. Quantitative Blood Concentration Measurements
The method illustrated in FIGS. 1-4 can be used to carry out
non-invasive in vivo quantitative blood concentration measurements
of Hb and Hct. For example, the microvascular system beneath the
mucosal membranes on the inside of the lip of a human subject can
be imaged to produce the raw reflected image. To measure Hb, the
raw reflected image is corrected using a bi-chromatic correction so
that the logarithm of the reflected light intensity of the
corrected reflected image is inversely proportional to the
concentration of hemoglobin. Suitable wavelengths for such a
bi-chromatic correction are .lambda..sub.1 =550 nm and
.lambda..sub.2 =650 nm. The analysis image is segmented from the
corrected reflected image so that the analysis image includes large
vessels. The mean reflected light intensity in an area in the
center of a large vessel is measured. As discussed above, the area
measurements correspond to volume or concentration measurements.
Consequently, the hemoglobin concentration per unit volume can be
obtained by measuring the reflected light intensity in an area near
the center of a large vessel.
The method of the present invention can also be used to determine
the hematocrit (Hct). The difference between hemoglobin (which is
the grams of hemoglobin per volume of blood) and hematocrit (which
is the volume of blood cells per volume of blood) is determined by
the concentration of hemoglobin within the cells which determines
the index of refraction of the cells. Hence, measurements in which
the image contrast between the circulation and the background is
achieved principally by the scattering properties of the
circulation will be related to the hematocrit and those obtained
principally by the absorbing properties will be related primarily
to the hemoglobin. For example, the microvascular system beneath
the mucosal membrane on the inside of the lip of a human subject
can be imaged to produce a raw reflected image whose contrast is
determined by a difference in the scattering properties of the
blood cells. To determine the volume of cells, the raw reflected
image is corrected using a bi-chromatic correction such that the
intensity is inversely proportional to the cell concentration.
Suitable wavelengths for such a bi-chromatic correction are 900 nm
(a wavelength that makes red blood cells appear dark due to their
scattering properties) and 700 nm for which the contrast is minimal
between the cells and their background.
b. Blood Cell Counts
Human blood is made up of formed elements and plasma. There are
three basic types of formed blood cell components: red blood cells
(erythrocytes); white blood cells (leukocytes); and platelets. As
noted above, red blood cells contain hemoglobin that carries oxygen
from the lungs to the tissues of the body. White blood cells are of
approximately the same size as red blood cells, but do not contain
hemoglobin. A normal healthy individual will have approximately
5,000,000 red blood cells per cubic millimeter of blood, and
approximately 7,500 white blood cells per cubic millimeter of
blood. Therefore, a normal healthy individual will have
approximately one white blood cell for every 670 red blood cells
circulating in the vascular system.
The method of the present invention can be used to determine the
number of white blood cells per unit volume of blood. As discussed
in more detail below in section e. regarding Blood Flow
Characteristics, white blood cells are pushed to the perimeter of
the blood flow, and travel in the margin where they can be seen in
contrast and counted. For example, the microvascular system beneath
the mucosal membranes on the inside of the lip of a human subject
can be imaged to produce a raw reflected image whose contrast is
determined by a difference in the optical properties of the white
blood cells. This is best done where there is a spectral difference
between the white blood cells and the bulk circulation (red blood
cells). This occurs typically in the blue and green portions of the
visual spectrum where the hemoglobin absorbs light and the white
blood cells do not. Thus, for this purpose, a broad spectral region
may be used (for example, from 400 to 600 nm) and bi-chromatic
correction is not necessary.
The analysis image is segmented from the raw reflected image so
that the analysis image includes large vessels. The number of white
blood cells per unit area of blood can be counted in the analysis
image. For the reasons discussed above, this will be proportional
to the number of white blood cells per unit volume of blood.
Platelets are the smallest of the formed blood cell components,
being typically less than 1 .mu. in diameter. Platelets are less
abundant than red cells, but more abundant than white cells. A
normal healthy individual will have approximately one platelet for
every 17 red blood cells circulating in the vascular system for a
total of about two trillion. Platelets help in blood clotting,
since they are able to stick together under certain circumstances
and help plug any holes that may develop in the walls of the blood
vessels. It is evident that platelets are essential during an
occurrence of injuries or other mishaps. Red blood cells are
colored due to the presence of hemoglobin which absorbs particular
wavelengths of light. White cells and platelets have no visual
color, i.e., they contain no light-absorbing constituents in the
visible range.
The platelet concentration measurement should be checked regularly
as one measure of expected speed of clotting in cases of an injury
to the blood vessels. Shortage of platelets (e.g.,
thrombocytopenia) can result in leaks in the blood vessel walls,
which may be harmful or even fatal. Prior to this invention, in
order to obtain platelet concentration measurement, it was
necessary to extract the blood from a patient and analyze its
content with regard to platelets and other blood cells, using
invasive methods known in the art. It would be more advantageous to
obtain an accurate platelet concentration measurement using
non-invasive techniques. The current invention enables an accurate
measurement of patient's blood, without any risks associated with
drawing blood (e.g., AIDS, hepatitis, etc.).
The method of the present invention can be used to determine the
number of platelets per unit volume of blood. For example, the
microvascular system beneath the mucosal membranes on the inside of
the lip of a human subject can be imaged to produce the raw
reflected image. To count the number of platelets, the raw
reflected image can be corrected using a velocity correction. To
carry out the velocity correction, the corrected reflected image is
formed by taking the difference between the raw reflected image of
a particular field or scene at a time t.sub.0 and the raw reflected
image of the same field or scene at a time t.sub.1. A corrected
reflected image formed by use of such a velocity correction allows
moving cells to be extracted from a stationary background.
The analysis image is segmented from the corrected reflected image
so that the analysis image includes small vessels. The number of
platelets per unit area can be counted in the analysis image. The
counting of platelets can be done in white light, and it is not
necessary to do a color or chromatic correction. The number of
platelets per unit volume can be estimated by counting the number
of platelets in an area in a small vessel, and relating them to the
number of red blood cells in the same area. The ratio of platelets
to red blood cells in healthy individuals is fairly constant at 1
to 17. In illness, this ration can vary widely in both directions,
from approximately 1 to 5 to approximately 1 to 100. Therefore, the
relative number of platelets to red blood cells can be used as a
diagnostic tool.
In summary, the method of the present invention can be used to
determine various characteristics of the vascular system through
the use of known relationships between parameters.
c. Blood Cell Indices
The method of the present invention can be used to determine the
mean cell volume (MCV). For example, the microvascular system
beneath the mucosal membranes on the inside of the lip of a human
subject can be imaged to produce the raw reflected image. Images of
individual blood cells may be captured by using "stop action",
i.e., stopping the action with pulsed illumination and/or
shuttering. To determine the mean cell volume, the raw reflected
image may be corrected using a velocity correction. To carry out
the velocity correction, the corrected reflected image is formed by
taking the difference between the raw reflected image of a
particular field or scene at a time to and the raw reflected image
of the same field or scene at a time t,. A corrected reflected
image formed by use of such a velocity correction allows moving
cells to be extracted from a stationary background.
The analysis image is segmented from the corrected reflected image
so that the analysis image includes small vessels. The area of the
cells in the analysis image can be determined on a pixel by pixel
basis. By averaging the area of a number of cells, a relationship
between average area and average or mean cell volume can be
empirically established. The relationship between volume and area
of an object of consistent shape, such as a human red blood cell,
is determined by the equation
where K is an empirically determined shape factor. One of skill in
the relevant arts can readily empirically determine shape factor K
as is currently done for conventional in vitro apparatus.
Consequently, the mean cell volume can be estimated from the area
of cells in a small vessel.
The method of the present invention can be used to determine the
mean cell hemoglobin concentration (MCHC). The determination of
MCHC is carried out in a manner similar to that for determining Hb
in a large vessel, except that the determination is made for Hb
using individual cells in a small vessel. For example, the
microvascular system beneath the mucosal membranes on the inside of
the lip of a human subject can be imaged to produce the raw
reflected image. The raw reflected image is corrected using a
bi-chromatic correction so that the logarithm of the reflected
light intensity of the corrected reflected image is proportional to
the concentration of hemoglobin. Suitable wavelengths for such a
bi-chromatic correction are .lambda..sub.1 =550 nm and
.lambda..sub.2 =650 nm. The analysis image is segmented from the
corrected reflected image so that the analysis image includes
individual cells in small vessels. The mean reflected light
intensity for a cell is measured from which the mean cell
hemoglobin concentration can be determined.
Mean cell hemoglobin concentration (MCHC) can alternatively be
determined from the equation
Hct and Hb can be directly determined from the analysis image in
the manner discussed above.
Mean cell hemoglobin (MCH) can be determined from the equation
MCV and MCHC can be directly determined from the analysis image in
the manner discussed above.
The method of the present invention can be used to differentiate
between various types of white blood cells. There are five (5)
types of mature white blood cells. These five types can be grouped
into two categories: granulocytes that contain small granules in
the cytoplasm; and agranulocytes that do not contain granules in
the cytoplasm. Granulocytes can be differentiated from
agranulocytes in the analysis image as a result of their scattering
properties. Therefore, the method described above for determining
the number of white blood cells per unit volume can be used to
determine the number of granulocytes per unit volume and the number
of agranulocytes per unit volume. Such information is clinically
relevant. An elevated granulocyte count is indicative of a
bacterial infection, and an elevated agranulocyte count is
indicative of a viral infection.
d. Plasma Constituents and Components
Plasma is the fluid part of blood that occupies the space in the
vessels outside of the formed blood cell components. Plasma
contains a variety of constituents, one of which is bilirubin.
Bilirubin is a degradation product of hemoglobin that is in
solution in blood plasma. Plasma contains a variety of other
compounds that are in solution, as well as compounds that are
attached to formed blood cell components.
The method of the present invention can be used to determine the
concentration of constituents in capillary plasma. For example, the
microvascular system beneath the mucosal membranes on the inside of
the lip of a human subject can be imaged to produce the raw
reflected image. To determine the concentration of a constituent in
capillary plasma, the raw reflected image is corrected using a
bi-chromatic correction so that the logarithm of the reflected
light intensity of the corrected reflected image is inversely
proportional to the concentration of the constituent. For example,
to determine the concentration of bilirubin in capillary plasma, a
bi-chromatic correction is applied with .lambda..sub.1 =450 nm (an
absorbing wavelength for bilirubin) and .lambda..sub.2 =600 nm (a
non-absorbing wavelength for bilirubin to provide
normalization).
The analysis image is segmented from the corrected reflected image
so that the analysis image includes capillary plasma, such may be
found in a small vessel. The mean reflected light intensity in an
area in the capillary plasma is measured. Reflected light intensity
measured in the analysis image can be converted to bilirubin
concentration in the analysis image. As discussed above, the
reflected images are independent of depth so that the density per
unit area measurements correspond to concentration. Consequently,
the bilirubin concentration per unit volume can be obtained by
measuring the reflected light intensity in an area of capillary
plasma in a small vessel.
The method of the present invention can be used to measure natural
constituents of plasma, such as bilirubin, as explained above. The
present invention can also be used to measure non-natural
components of plasma, such as drugs. For example, the concentration
of drugs in plasma can be measured by using a bi-chromatic
correction with .lambda..sub.1 equal to an absorbing wavelength for
the drug and .lambda..sub.2 equal to a non-absorbing wavelength for
the drug in a manner similar to that for measuring the
concentration of bilirubin. Alternatively, non-natural components
can be detected by using an optical characteristic, e.g., native
fluorescence, that appears in the analysis image.
The present invention can also be used to measure cellular and
noncellular constituents of blood (natural and non-natural) through
the use of a marker, label, or tag. For example, the marker, label,
or tag can be introduced into the subject's vascular system in a
well known manner, for example, orally or by injection. The marker
can be selected so that it attaches to components dissolved in the
capillary plasma to form labeled plasma components. Alternatively,
the marker can be selected so that it attaches to a component that
is itself attached to a formed element of the blood, such as a
cell, thereby forming labeled cells. For example, a marker with
identifying optical characteristics, such as a fluorescent protein,
can be introduced into the vascular system so that it attaches to a
certain type of cell, such as a circulating tumor cell. This
technique can also be used to measure sub-classes of normal white
blood cells, like lymphocyte sub-sets. The identifying optical
characteristic will appear in the analysis image, such as with a
fluorescent "flash". In this manner, the fluorescence or other
optical contrast produced by the marker can be detected, indicating
the presence of components or cells to which the marker attaches.
Such determinations are useful for evaluating drug delivery. Other
optical characteristics, such as near infrared or ultraviolet
absorption, can also be used to detect the presence of components
in capillary plasma. Tumor or other type of abnormal cells could
also be detected through the use of an identifying spectral
fingerprint.
e. Blood Flow Characteristics
In the particular case of the white blood cells, examination of the
blood flow in vivo could also potentially provide useful
information currently unavailable even with the most sophisticated
laboratory techniques. The white blood cells or leukocytes are
mechanically pushed to the perimeter of the blood flow. It has been
shown that the process by which the white blood cells emigrate from
the blood to an afflicted area is a two-step process. Because of
their "sticky" interaction with the wall of the blood vessel, in
the first step the white cells travel in the margin of the flow,
and at a slower velocity than the red blood cells. In the second
step, the white blood cells leave the circulation by migrating
through the vessel wall. This distinction in the location and
velocity can be used to distinguish white blood cells in the
analysis image. The determination of the relative velocity of white
blood cells would aid the clinician greatly in assessing the stage
and severity of an infection or inflammation: lowered velocity with
moderate concentration indicating an early phase, with normal
velocity and elevated concentration indicating a later phase of
infection or inflammation.
The method of the present invention can also be used to determine
the speed or velocity of white blood cells. For example, the
microvascular system beneath the mucosal membranes on the inside of
the lip of a human subject can be imaged to produce the raw
reflected image. To determine the speed of white blood cells, the
raw reflected image can be corrected using a velocity correction.
To carry out the velocity correction, the corrected reflected image
is formed by taking the difference between the raw reflected image
of a particular field or scene at a time t.sub.0 and the raw
reflected image of the same field or scene at a time t.sub.1 where
the difference in time between t.sub.0 and t.sub.1 is known. A
corrected reflected image formed by use of such a velocity
correction allows moving cells to be extracted from a stationary
background.
The analysis image is segmented from the corrected reflected image
so that the analysis image includes large vessels. The speed of
white blood cells can be determined by tracking their movement per
unit time. The speed of white blood cells can be used as an
indicator of the presence of infection/inflammation which may be
more specific than the erythrocyte sedimentation rate (ESR).
3. Feasibility Model
A feasibility model, apparatus 600 (see FIGS. 6A and 6B), was
developed by the inventors to verify that the method of the present
invention provided results that were accurate, reliable,
reproducible, and statistically significant with respect to
measurements made using conventional invasive techniques for
measuring blood parameters. Apparatus 600 includes a visual image
receiver 612 for capturing the raw reflected image. A lens unit
from a Zeiss Axiovert Model 135 microscope was used for visual
image receiver 612. Light from a focusable light source 614 is
focused through visual image receiver 612 to reflect off a
hydro-dynamic focused flow cell 630 (explained in more detail below
with respect to FIG. 7) and back through visual image receiver 612
in a co-axial manner. An exemplary light source 614 is a pulsed
xenon arc light, such as is available from EG&G, Cambridge,
Mass.
The reflected light carrying the raw reflected image traverses a
path from flow cell 630, through visual image receiver 612, up to a
high resolution video camera 618. Camera 618 is preferably an
electronically shuttered, high resolution (1024.times.512 pixels)
camera. An exemplary video camera is a Hamamatsu C2400-77 high
resolution (768.times.497 pixels) charge coupled device (CCD)
camera. Alternatively, camera 618 can be a high framing rate (300
Hz), high resolution digital video camera that can capture
approximately 1,000,000 pixels of size 9 .mu.m.times.9 .mu.m,
available from EG&G, Cambridge, Mass. Reflected light is
collected coaxially and the reflected image is focused on the face
of camera 618. Pulsed light source (strobe) 614 can be driven by a
syncpulse separator 640 (FIG. 6B) to provide synchronization
between camera 618 framing rate and light source 614 pulse rate.
Camera 618 is in the magnified image plane of visual image receiver
612.
An eye piece 615 is inserted in the reflected light path between
visual image receiver 612 and camera 618 for viewing of the
reflected image. An image filter 616 is also inserted in the
reflected light path between visual image receiver 612 and camera
618. Image filter 616 functions as a spectral selection filter to
filter the reflected image by wavelength.
Camera 618 is coupled to a computer 620, such as a Compaq-P5, 75
MHZ computer, operating Image Pro Plus image analysis software,
available from Media Cybernetics, Silver Spring, Md. The filtered
image captured by camera 618 is transferred from camera 618 to
computer 620 for analysis. Computer 620 includes a "frame grabber
board", such as an Occulus F64 Image Capture Board 646, available
from Coreco, Montreal, Canada to capture the signal with the image
data from camera 618. Preferably, a 10-bit frame grabber board is
used to provide sufficient digital resolution. Computer 620 is
coupled to one or more output devices 622 for display processing
and/or storage. Output device 622 can be a hard disk drive or other
type of storage device such as DAT tape drive 654, a central
processing unit (CPU), a printer such as laser jet printer 652, a
computer screen or monitor such as video image monitor 648, a
computer monitor such as control monitor 650, etc.
The output of camera 618 can also be sent to a recorder 644, such
as a super VHS recorder. A time stamp 642 can be added to the
output of camera 618 before it is sent to computer 620 and recorder
644.
Advances in video camera technology have greatly increased the
number of pixels capable of operating at high framing rates.
Captured images of blood flow containing as many as 1 million
pixels separated by a few milliseconds can be used to visualize red
blood cells, in vivo, and to analyze blood flow in sites separated
from the external environment by thin layers of transparent or
translucent material. The movement of individual red blood cells
between frames of high speed sequential images can be used to
enhance signal to noise ratio and to suppress the stationary
background.
The fundamental accuracy of apparatus 600 to dynamically capture
particle sizes ranging from 0.94 to 10 .mu.m with a correlation
coefficient >0.99 using commercially available polystyrene beads
has been established.
Apparatus 600 was used to obtain reflected images of flowing blood
to provide a feasibility model for in vivo reflected images of the
vascular system. Blood from a blood supply 624 is pumped by a
peristaltic pump 628A through flow cell 630 in the direction shown
by arrow A to a waste reservoir 632. At the same time, sheath fluid
from a sheath fluid supply 626 is pumped by a peristaltic pump 628B
through flow cell 630 in the direction shown by arrow A to waste
reservoir 632. The sheath fluid is an isotonic medium that is used
to simulate the walls of the blood vessels and other tissues
surrounding blood. As shown in more detail in FIG. 7, the
cross-section of sample flow path 702 is smallest in the region
above visual image receiver 612. By controlling the ratio by which
the cross-section of sample flow path 702 narrows, laminar flow can
be maintained. The cross-section of sample flow path 702 can be
adjusted so that individual cells can be seen in the reflected
image.
The model used is an adaptation of the hydrodynamic focused flow
cells developed for use in flow cytometers. In this flow cell, a
narrow "sample stream" containing the blood is encased in an inert
sheath fluid, and the combined fluidic system is caused to flow
between two glass plates separated by approximately 250 .mu.m.
Altering the flow of the sheath fluid has the effect of reducing
the sample stream size from greater than 100 .mu.m to approximately
10 .mu.m in diameter, thus very closely representing typical
capillaries of the microvascular system.
With this flow cell, important parameters of the experiment can be
independently varied as follows:
The blood cell velocity, which is controlled by the volume flow of
the sheath fluid, can be varied from 100 to 1,000 .mu.m per
second.
Sample stream diameter, controlled by the relative sheath to sample
flow volume, can be varied over a range from 10 .mu.m to 100
.mu.m.
Pulsing can be introduced both in diameter and in velocity by
varying the flow rates of the sample and sheath stream.
The effect of a thin layer of tissue (Mucous Membrane) can be
simulated by adding a scattering element either to the stationary
glass window or in the sheath fluid.
To improve visualization of the reflected image of the blood
flowing through flow cell 630, two polarizers were used in
apparatus 600 (see FIG. 6A) A first polarizer 613A was placed in
the light path between light source 614 and visual image receiver
612. A second polarizer 613B was placed in the reflected light path
between the flow cell 630 containing the imaged blood flow, and
camera 618. The two polarizers are "crossed" in that the planes of
polarization are 90.degree. relative to each other. For example,
the plane of polarization of polarizer 613B is 90.degree. relative
to the plane of polarization of polarizer 613A. Likewise, the plane
of polarization of polarizer 613A is 90.degree. relative to the
plane of polarization of polarizer 613B. Through the use of
polarizer 613A and 613B, the reflected image that is captured by
camera 618 is enhanced, making the blood cell components more
sharply contrasted with respect to the background, thereby
providing an improved visualization of the blood flow. The improved
visualization through the use of cross-polarizers will be explained
in more detail in Section 4 below regarding an in vivo apparatus. A
diffuse reflector 635 was also used with apparatus 600 to reflect
light and simulate reflection from a tissue background.
With apparatus 600, the basic feasibility of using images in
reflected light to quantify four RBC parameters (RBC, MCV, Hb, and
MCHC) has been demonstrated. The following table summarizes the
correlation between values obtained using the feasibility model
(apparatus 600) and those obtained using a Coulter Stk.S (N=number
of samples; r=correlation coefficient).
______________________________________ Parameter N r
______________________________________ Hb 70 0.9 MCHC 20 0.55 MCV
45 0.70 RBC 38 0.63 ______________________________________
Using apparatus 600, the velocity and dimensions of the blood flow
can be experimentally manipulated in flow cell 630 to simulate in
vivo conditions. Apparatus 600 can also be used to measure blood
flow, for example, in a patient's finger located above visual image
receiver 612 in place of flow cell 630.
Apparatus 600 was used by the inventors in an agreement and
correlation study to confirm that the present invention could be
clinically used to provide accurate non-invasive in vivo results.
Hemoglobin concentration was measured using apparatus 600 and
seventy patient blood samples obtained from a hospital for blood
supply 624. Blood from the same seventy blood samples was analyzed
using a conventional Coulter Stk.S analyzer, manufactured by
Coulter Diagnostics of Miami, Fla. For each of the seventy samples
run on apparatus 600 and the Coulter device, a determination was
made whether the hemoglobin concentration indicated an anemic
condition or a normal condition. As shown in FIG. 8A, the
determination made using the feasibility model agreed with the
determination made using the Coulter device for 67 of the 70
samples, for a 96% agreement. As compared to the Coulter device,
apparatus 600 provided 1 false high (anemia when Coulter indicated
normal) and 2 false lows (normal when Coulter indicated
anemia).
A number of experiments were performed using apparatus 600 to
measure various characteristics of the vascular system. For
example, apparatus 600 was used to generate images of blood flowing
through flow cell 630. Light in the range between 400 nm and 1000
nm was emitted from light source 614. Images were captured at 450
nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm,
and 900 nm by selection of appropriate image filters 616. One
example of an image obtained with apparatus 600 is shown in FIG. 9.
Platelets are shown as white dots, in comparison with red blood
cells which are of a darker shade.
Apparatus 600 was also used to confirm that measurements could be
made in the capillary plasma, or space between the cells in a small
vessel such as a small capillary. By eliminating or ignoring the
portions of the images relating to blood cells, etc., measurements
relating to non-formed constituents may be made. Using a
hydrodynamically focused flow cell, four different concentrations
of bilirubin were added to a 1/4 whole blood dilution. The final
bilirubin levels ranged from 6.2 g/dl to 24.8 g/dl. A xenon
flashlamp was used to illuminate the field and images were recorded
with two different optical filters, one at a principle absorption
wavelength for bilirubin (450 nm) and one outside the absorption
band of bilirubin (600 nm).
After the images were taken, the analysis involved defining an area
or scene of interest around a background (region A) to be used as a
reference (see FIG. 10). An area of interest inside the stream
flowing through flow cell 630 (region B) was also defined. Region B
was segmented to eliminate the images of the red blood cells. This
was done by using an intensity threshold that separates the red
blood cells out of region B. The optical density was then
determined for the remaining area of region B to perform the
analysis.
FIG. 11 shows a plot of optical density and concentration of
bilirubin (mg/dl). FIG. 11 shows a marked increase in the optical
density at the absorbing wavelength (450 nm) relative to the
non-absorbing wavelength (600 nm).
Experiments were conducted with a chopper stabilized reflectance
spectrophotometer (see FIG. 12) to determine the reflection
characteristics of white blood cells or leukocytes. The resulting
reflectance spectral scan is shown in FIG. 13 (reflectance % as a
function of wavelength in nm). FIG. 13 shows that the reflected
light for white blood cells is relatively high in the spectral
region around 550 nm, where the reflected light of red blood cells
is attenuated by the absorption of hemoglobin. A spectral selection
filter centered at 550 nm (image filter 616) was used in apparatus
600 to obtain an image of leukemic blood in a 100 .mu.m glass
capillary (see FIG. 14). The leukemic blood shown in FIG. 14 has a
higher number of white blood cells (44,000/.mu.l) than does normal
or healthy (7500/.mu.l) blood. White blood cells are visible in
FIG. 14 as bright spots against the darker background of the red
blood cells.
4. In Vivo Apparatus
FIG. 15A shows a block diagram illustrating one embodiment of an in
vivo apparatus 1500 for non-invasive in vivo analysis of a
subject's vascular system. Apparatus 1500 includes a light source
1502 for illuminating tissue of a subject (shown generally at
1504). Although one light source is shown, it is to be understood
that the present invention is not limited to the use of one light
source, and more than one light source can be used. In an
embodiment where more than one light source is used, each light
source can be monochromatic or polychromatic. Light source 1502 can
be a light capable of being pulsed, a non-pulsed light source
providing continuous light, or one capable of either type of
operation. Light source 1502, can include, for example, a pulsed
xenon arc light, a mercury arc light, a halogen light, a tungsten
light, a laser, a laser diode, or a light emitting diode (LED).
Light source 1502 can be a source for coherent light, or a source
for incoherent light.
A first polarizer 1510 is placed between light source 1502 and
subject 1504. First polarizer 1510 polarizes light from light
source 1502. A second polarizer 1520 is placed between subject 1504
and an image separating means 1540. Polarizers 1510 and 1520
preferably have planes of polarization oriented 90.degree. relative
to each other.
In one embodiment of the present invention, light source 1502
comprises first polarizer 1510 so that a separate first polarizer
1510 is not required. In such an embodiment, light source 1502 is a
source of polarized light, for example, a laser or a laser diode,
and second polarizer 1520 has a plane of polarization oriented
90.degree. relative to the plane of polarization of polarized light
source 1502.
The reflected spectral image from subject 1504 is reflected from a
depth less than a multiple scattering length. Image separating
means 1540 separates the reflected spectral image from subject 1504
into two or more image portions. Each image portion is captured by
an image capturing means, such as image capturing means 1560, 1570,
and 1565. Each image capturing means is coupled to an image
correcting and analyzing means 1580 for carrying out image
correction and analysis to produce result 140.
FIG. 15B shows further detail of in vivo apparatus 1500. A beam
splitter 1518 is used to form a light path 1506 between light
source 1502 and the illuminated tissue and a reflected light path
1507. The reflected image that is reflected from a depth less than
the multiple scattering length in the illuminated tissue travels
along reflected light path 1507 to image capturing means 1560 for
capturing the reflected image. Suitable image capturing means 1560
include those devices capable of capturing a high resolution image
as defined above. The image capturing means captures all or part of
an image for purpose of analysis. Suitable image capturing means
include, but are not limited to, a camera, a film medium, a
photocell, a photodiode, a photodetector, or a charge coupled
device camera. For example, video cameras and charge coupled device
(CCD) cameras having a 1024.times.512 pixel resolution and 300 Hz
framing rate can be used. A particularly preferred image capturing
means is a Hamamatsu C2400-77 high resolution CCD camera.
The resolution required for the image capturing means can depend
upon the type of measurement and analysis being performed by the in
vivo apparatus. For example, the image resolution required for
determining the hemoglobin concentration (Hb) is lower than the
image resolution required for making cellular measurements, such as
MCV or cell counts. For example, hemoglobin concentration
measurements can be carried out using photocells, such as one red
photocell and one green photocell, as the image capturing
means.
First polarizer 1520 is placed in light path 1506 between light
source 1502 and the illuminated tissue. First polarizer 1510
polarizes light from light source 1502. Polarizer 1510 has a plane
of polarization, shown generally at 1512. Second polarizer 1520 is
placed in reflected light path 1507 between the illuminated tissue
and image capturing means 1560. Polarizer 1520 has a plane of
polarization, shown generally at 1522. As shown in FIG. 15B, planes
of polarization 1512 and 1522 are oriented 90.degree. relative to
each other. Polarizers, such as polarizers 1510 and 1520, having
planes of polarization oriented 90.degree. relative to each other
are referred to herein as "cross-polarizers".
The efficiency of a polarizer is a function of the percentage of
the input light that is transmitted through the polarizer. For each
unit of unpolarized (randomly polarized) light input to a
polarizer, a perfectly efficient polarizer would transmit out 50%
of the inputted light. When randomly polarized light is input to
two perfect polarizers (regardless of efficiency) configured as
cross-polarizers, all light is extinguished, i.e., no light is
transmitted through the second polarizer. The more light that is
extinguished by cross-polarizers (i.e., the less randomly polarized
light that is transmitted through the cross-polarizers), the
greater the extinction of the cross-polarizers. Cross-polarizers
having an extinction coefficient of at least 10.sup.-3 (for each
unit of randomly polarized light input into the cross-polarizers,
1/1000 is transmitted through the cross-polarizers) are suitable
for use with the present invention. Suitable cross-polarizers are
available as sheet polarizers from Polaroid Corp.,
Massachusetts.
As stated above, virtually all of the light is eliminated when high
extinction polarizers are crossed. Therefore, the expected result
using crossed-polarizers as described for the apparatus of the
present invention, would be to extinguish all of the illuminated
image. The unexpected result of how the use of cross-polarizers in
the apparatus of the present invention increases visualization of
reflected images, and increases the ability to perform quantitative
analysis using reflected images, will now be explained. Reflected
light has three components. First is the "mirror or specular
reflection" component that preserves the image of the source in a
reflection. The second component is a "rough surface scattering"
component. The rough surface scattering component is scattered
light that is scattered by a rough surface, and does not preserve
the image of the source. However, both the mirror reflection
component and the rough surface scattering component retain
polarization. The third and final component is a "small particle
scattering" component, commonly known as a "Rayleigh scattering"
component. The Rayleigh scattering component is light that is
scattered by particles that are small compared to the wavelength of
the illuminated light. Rayleigh scattering de-polarizes light.
Therefore, the Rayleigh scattering component is the only component
of reflected light that is de-polarized so that the original
polarization is lost.
The only component of reflected light that passes through
"cross-polarizers", such as polarizers 1510 and 1520 oriented at
90.degree. relative to each other, is the Rayleigh scattering
component. The mirror reflection component and the rough surface
scattering component retain polarization. Therefore, if the mirror
reflection component and the rough surface scattering component of
light polarized in a first direction (such as by polarizer 1510)
are passed through a polarizer oriented at 90.degree. relative to
the first direction (such as polarizer 1520), these two components
of the reflected light are extinguished. In contrast, the Rayleigh
scattering component of light polarized in a first direction is
de-polarized. Therefore, the Rayleigh scattering component is not
extinguished when it passes through a polarizer oriented at
90.degree. relative to the first direction of polarization. Also,
the Rayleigh scattering component has lost the image of the source,
and effectively provides a uniform source of reflected light from
within the object.
With reference to FIG. 15B, light from light source 1502 is
polarized in a first direction 1512 by polarizer 1510. The light
thus polarized reflects from object 1504. The Rayleigh scattering
component of the reflected light is depolarized. However, the
mirror reflection component and the rough surface scattering
component retain the polarization from polarizer 1510. When the
reflected light passes through polarizer 1520 oriented in a second
direction 1522 90.degree. relative to first direction 1512, the
mirror reflection component and the rough surface component are
extinguished. Therefore, the only component of reflected light that
passes through polarizer 1520 is the Rayleigh scattering component
that has been de-polarized in direction 1522, and which exhibits an
intensity which varies as the cosine of the angle with the incident
light.
When cross-polarizers are used, such as polarizers 1510 and 1520
oriented at 90.degree. relative to each other, only the
de-polarized reflected light, i.e., the Rayleigh scattering
component is observed. The mirror reflection component and the
rough surface scattering component are eliminated. Contrary to the
expectation noted above that no light would be transmitted with
cross-polarizers, the light depolarized by Rayleigh scattering
provides a virtual backlighting effect that significantly increases
the contrast and visualization of reflected images, and the ability
to perform quantitative analyses using reflected images. The
increased visualization and quantitative ability are due to two
consequences resulting from the use of cross-polarizers as
described above. First, the mirror reflection component and the
rough surface scattering component, which are both a source of
noise for quantitative measurement, are eliminated. Second, the
Rayleigh scattering component is "Lambertian" in that it behaves in
the same manner as light reflected from a Lambertian surface.
Therefore, the Rayleigh scattering component is independent of the
angle of viewing, and allows concentration to be computed more
simply from measured reflected light intensity (analogous to Beer's
Law).
Under Lambert's law, the luminous intensity in a given direction
radiated or reflected by a perfectly reflecting plane surface
varies as the cosine of the angle between that direction and the
normal to the surface. A Lambertian surface is an ideal, perfectly
reflecting surface for which the brightness of reflected radiation
is independent of direction. Light reflected from a Lambertian
surface appears equally bright from all angles, like freshly fallen
snow. Most surfaces are not Lambertian, and the intensity of
reflected light depends upon the angle of viewing because of
surface effects.
The increased visualization that is achieved through the use of the
cross-polarization technique of the present invention can be seen
by comparing FIGS. 18A and 18B. FIG. 18A illustrates a black
ink-jet cross visualized using conventional optics without
cross-polarizers. Visualization of the ink-jet cross is difficult
because of the relatively poor contrast between the ink-jet cross
and the background. FIG. 18B illustrates the same black ink-jet
cross used in FIG. 18A visualized using the cross-polarization
technique of the present invention, i.e., polarizers such as 1510
and 1520 oriented at 90.degree. relative to each other. FIG. 18B
dramatically demonstrates the improvement in visualization and
contrast obtained by using cross-polarizers. This image is the same
as the one that would be observed if the image was from transmitted
light. The reflection seen with conventional reflected light in
FIG. 18A has been removed in FIG. 18B through the use of
cross-polarizers.
As shown in FIG. 15B, an objective lens 1517 is placed co-axially
in light path 1506 and reflected light path 1507. Objective lens
1517 magnifies the reflected image. Image capturing means 1560 is
located in a magnified image plane of objective lens 1517.
Preferably, objective lens 1517 is selected with the lowest
magnification level required to visualize the illuminated tissue.
The magnification required is a function of the size of the object
in the illuminated tissue to be visualized, along with the size of
the pixels used for the image. Low magnification provides a high
depth of field, but more crudeness to the image. High magnification
provides a low depth of field, but is more susceptible to blurring
caused by motion. Blood vessels in the microvascular system are
typically 10-40 .mu. in diameter. Ten to twenty (10-20) pixels per
blood vessel diameter provide a suitable image with a 10X lens.
Lower magnification could be used with pixels of smaller size.
Lenses 1516 can be used on either side of polarizer 1510 for
focusing the light in light path 1506. A heat rejection filter 1508
is preferably placed in front of light source 1502 to reject heat.
Filter 1508 is a blocking filter to block out infra-red
wavelengths. Filter 1508 is preferably configured to block
wavelengths greater than or equal to 1000 nm.
Image separating means 1540 is placed in reflected light path 1507
between second polarizer 1520 and image capturing means 1560 for
separating the reflected image into a first portion 1532 and a
second portion 1534. It is to be understood that image separating
means 1540 can separate the reflected image into a plurality of
portions, and is not limited to two portions. First portion 1532 of
the reflected image is captured by image capturing means 1560.
Second portion 1534 is captured by second image capturing means
1570. Second image capturing means 1570 can be the same as or
different from image capturing means 1560. Second image capturing
means 1570 is disposed in the magnified image plane of objective
lens 1517. Additional image capturing means can be used to capture
further image portions separated by image separating means 1540. In
an alternative embodiment, a single image capturing means can be
used to capture first portion 1532 and second portion 1534 of the
reflected image.
One particularly preferred image separating means is a dichroic
mirror or other type of dichroic separator that transmits all light
less than a particular wavelength, and reflects all light greater
than the particular wavelength. Alternatively, an image separating
means can be used that reflects all light less than a particular
wavelength, and transmits all light greater than the particular
wavelength. Other suitable image separating means can also be
used.
A spectral selection means 1552 can be placed in reflected light
path 1507 between second polarizer 1520 and image capturing means
1560. Spectral selection means 1552 can be, for example, a
monochromator, a spectral filter, prism, or grating. Similarly, a
spectral selection means 1554 can be placed in reflected light path
1507 between second polarizer 1520 and second image capturing means
1570. Spectral selection means 1554 can also be, for example, a
monochromator, a spectral filter, prism, or grating. The center
values for spectral selection means 1552 and 1554 can be chosen
based upon the type of analysis to be conducted. For example, if
hemoglobin concentration is to be determined, then one of spectral
selection means 1552 or 1554 is preferably centered at 550 nm and
the other of spectral selection means 1552 or 1554 is preferably
centered at 650 nm. As another example, if bilirubin concentration
is to be determined, then one of spectral selection means 1552 or
1554 is preferably centered at 450 nm and the other of spectral
selection means 1552 or 1554 is preferably centered at 600 nm.
In a particularly preferred embodiment, light source 1502 is
configured as a plurality of LED's, each LED emitting a different
wavelength of light. For example, three LED's can be used to
provide a source of green, blue, and red light. Use of light source
1502 configured to emit a particular wavelength of light, such as
an LED, can eliminate the need for separate spectral selection
means 1552 and 1554. A single image capturing means 1560 can be
used to capture the reflected image from each of the three LED's.
For example, a single color camera sensitive to multiple
wavelengths (green, blue, and red) can be used to capture the
reflected image from each of the three (green, blue, and red)
LED's.
Image capturing means 1560 is coupled to image correcting and
analyzing means 1580. Image correcting and analyzing means 1580 can
be a computer or other type of processing system (explained in more
detail below with respect to FIG. 16). A signal 1562 representing
the reflected image captured by image capturing means 1560 is sent
by image capturing means 1560 and received by image correcting and
analyzing means 1580. Similarly, image capturing means 1570 is
coupled to image correcting and analyzing means 1580. A signal 1572
representing the reflected image captured by image capturing means
1570 is sent by image capturing means 1570 and received by image
correcting and analyzing means 1580. Image correcting and analyzing
means 1580 carries out the processing and analysis of the reflected
images received. Particularly, image correcting and analyzing means
1580 can be used to carry out steps 220-240 shown in FIG. 2. Image
correcting and analyzing means 1580 can be configured to carry out
these steps through hardware, software, or a combination of
hardware and software.
An exemplary image correcting and analyzing means 1580 for use in
the present invention is shown as a computer system 1600 in FIG.
16. Computer system 1600 includes one or more processors, such as
processor 1604. Processor 1604 is connected to a communication bus
1606. Various software embodiments are described in terms of this
exemplary computer system. After reading this description, it will
become apparent to a person skilled in the relevant art how to
implement the invention using other computer systems and/or
computer architectures.
Computer system 1600 also includes a main memory 1608, preferably
random access memory (RAM), and can also include a secondary memory
1610. Secondary memory 1610 can include, for example, a hard disk
drive 1612 and/or a removable storage drive 1614, representing a
floppy disk drive, a magnetic tape drive, an optical disk drive,
etc. Removable storage drive 1614 reads from and/or writes to a
removable storage unit 1618 in a well known manner. Removable
storage unit 1618 represents a floppy disk, magnetic tape, optical
disk, etc. which is read by and written to by removable storage
drive 1614. As will be appreciated, removable storage unit 1618
includes a computer usable storage medium having stored therein
computer software and/or data.
In alternative embodiments, secondary memory 1610 may include other
similar means for allowing computer programs or other instructions
to be loaded into computer system 1600. Such means can include, for
example, a removable storage unit 1622 and an interface 1620.
Examples of such can include a program cartridge and cartridge
interface (such as that found in video game devices), a removable
memory chip (such as an EPROM, or PROM) and associated socket, and
other removable storage units 1622 and interfaces 1620 which allow
software and data to be transferred from removable storage unit
1622 to computer system 1600.
Computer system 1600 can also include a communications interface
1624. Communications interface 1624 allows software and data to be
transferred between computer system 1600 and external devices, such
as image capturing means 1560 and 1570. Examples of communications
interface 1624 can include a modem, a network interface (such as an
Ethernet card), a communications port, a PCMCIA slot and card, etc.
Software and data transferred via communications interface 1624 are
in the form of signals which can be electronic, electromagnetic,
optical or other signals capable of being received by
communications interface 1624. For example, signals 1562 and 1572
are provided to communications interface via a channel 1628.
Channel 1628 carries signals 1562 and 1572 and can be implemented
using wire or cable, fiber optics, a phone line, a cellular phone
link, an RF link and other communications channels.
In this document, the terms "computer program medium" and "computer
usable medium" are used to generally refer to media such as
removable storage device 1618, a hard disk installed in hard disk
drive 1612, and signals provided via channel 1628. These computer
program products are means for providing software to computer
system 1600.
Computer programs (also called computer control logic) are stored
in main memory 1608 and/or secondary memory 1610. Computer programs
can also be received via communications interface 1624. Such
computer programs, when executed, enable computer system 1600 to
perform the features of the present invention as discussed herein.
In particular, the computer programs, when executed, enable
processor 1604 to perform the features of the present invention.
Accordingly, such computer programs represent controllers of
computer system 1600.
In an embodiment where the invention is implemented using software,
the software may be stored in a computer program product and loaded
into computer system 1600 using removable storage drive 1614, hard
drive 1612 or communications interface 1624. The control logic
(software), when executed by the processor 1604, causes processor
1604 to perform the functions of the invention as described
herein.
In another embodiment, the invention is implemented primarily in
hardware using, for example, hardware components such as
application specific integrated circuits (ASICs). Implementation of
the hardware state machine so as to perform the functions described
herein will be apparent to persons skilled in the relevant
art(s).
In yet another embodiment, the invention is implemented using a
combination of both hardware and software.
FIGS. 17A and 17B show embodiments of the present invention
suitable for use with a subject for performing non-invasive in vivo
analysis. FIG. 17A shows a console unit 1702 that contains a probe
1704, a printer 1706, and a processing and storage unit 1708. Probe
1704 is used to image the portion of the subject's vascular system,
such as the inside of the lower lip. An index matching medium, such
as ethyl cellulose available under the trade name "K Y" Jelly from
Johnson & Johnson, or a sugar syrup, is preferably applied to
probe 1704 to provide a good optical contact or optical seal
between probe 1704 and the inside of the lower lip.
Probe 1704 is preferably equipped with the elements shown in FIG.
15 from light source 1502 through one or more image capturing
means. To ensure optimal performance of the apparatus of the
present invention, there should not be anything in the light path
between polarizer 1510 and polarizer 1520 that de-polarizes the
light. For example, the presence of dust in the light path between
polarizer 1510 and polarizer 1520 will degrade the performance of
the apparatus. Further, the components of probe 1704 are preferably
made of non-depolarizing material so that the materials will not
de-polarize the light. A particularly preferred material for the
components of probe 1704 in the light path is a non-depolarizing
plastic material available from Kodak with the trade name KODACEL.
Other suitable materials for components in the light path are glass
or quartz. A preferred material for the imaging end of probe 1704
is glass. A signal (such as signal 1562 or 1572 shown in FIG. 15)
is transmitted from probe 1704 to processing and storage unit 1708
for processing and storage.
FIG. 17B shows a mobile unit 1722. Mobile unit 1722 includes a
probe 1724 and a belt unit 1726. Probe 1724 can be configured in a
similar manner to probe 1704 shown in FIG. 17A. Belt unit 1726
includes a data storage and transmission unit 1728. Data storage
and transmission unit 1728 receives signals from probe 1724. These
signals can be stored by data storage and transmission unit 1728
for processing at a later time. Alternatively, these signals can be
transmitted by data storage and transmission unit 1728 to a central
processing station (not shown) for processing and storage. The
central processing station can be configured to provide permanent
storage for the processed data, as well as to print and display the
results in a well known manner. Belt unit 1726 also includes a
location 1729 for batteries or other suitable power supply.
The in vivo apparatus of the present invention can be used to carry
out the methods of the present invention discussed above.
Particularly, the in vivo apparatus can be used to determine
hemoglobin and bilirubin concentrations per unit volume of blood.
The in vivo apparatus can also be used to determine the hematocrit
and the mean cell volume. The in vivo apparatus can also be used to
determine the number of white blood cells and the number of
platelets per unit volume of blood. For determining the number of
cells, such as white blood cells or platelets, the light source is
configured as a pulsed light source or flash to "stop action" in
the analysis image so the count can be made. The stop action
achieved with the pulsed light source avoids the blurring
associated with movement in the analysis image. The pulsed light
source is preferably synchronized with the frame rate of the image
capturing means. Stop action can also be achieved by controlling
shuttering on the image capturing means. A stop action image is
preferred any time a count of cells is to be made in the analysis
image. A stop action image can also be used to determine other
non-cell-count parameters, such as Hb or Hct. However, such other
parameters such as Hb and Hct can be determined with a non-stop
action image as well.
An experiment was conducted to compare laboratory measurements
using a conventional laboratory apparatus with measurements using
an in vivo apparatus at different levels of anemia in a piglet. The
piglet was bled down while a saline drip kept the piglet's fluid
volume constant. Hemoglobin was measured at various points during
the bleed down by extracting blood samples and using a conventional
Coulter laboratory apparatus and the in vivo apparatus. FIG. 8B
shows a graph illustrating a Comparative Determination of
Hemoglobin with Various Levels of Anemia induced by bleed down
(hemoglobin gm/dL as a function of % of blood volume lost against
total blood-volume) for a Coulter Stk.S device and the in vivo
apparatus FIG. 8B shows a high correlation (r=0.91) between the
Coulter Stk. S results and the HEMOSCAN (in vivo apparatus)
results.
An experiment using an in vivo apparatus was carried out on 23
"healthy" human subjects. The procedure involved collecting
reflected spectral images using a probe on the lip. The probe was
placed inside the surface of a subject's lip (transmucosal
membrane) with an optical contact or optical seal obtained by a
small amount of "KY" jelly. FIG. 8C shows a graph illustrating the
Comparative Determination of Hemoglobin on 23 "Healthy" Human
Subjects using a Coulter Stk.S device and a HEMOSCAN in vivo
apparatus. The results showed a predictive agreement of 83% with a
correlation of r=0.68.
5. In Vitro and Other Analytical Applications
The cross-polarization technique of the present invention can be
used to improve visualization of reflected images, and to improve
the ability to perform quantitative analyses using reflected images
in many applications other than non-invasive in vivo analysis of
the vascular system. The cross-polarization technique of the
present invention could readily be used for in vitro analysis of
blood characteristics. The in vivo measurements discussed above
could also be performed in vitro by imaging blood in, for example,
a tube or flow cell. The cross-polarization technique of the
present invention can be used to make in vitro measurements of
quantitative blood concentration (Hb, Hct), blood counts (WBC, RBC,
Plt), blood cell characteristics (MCV, MCHC, and granulocytes), and
plasma constituents (bilirubin, labeled plasma components, and
labeled cells).
The cross-polarization technique of the present invention can also
be used to perform quantitative analytical measurement of dyes,
inks, and chemical reactants that have been coated, for example, on
an opaque surface. Such quantitative analyses are done in "strip
testing" or strip readers, such as may be used in blood tests,
pregnancy tests or glucose tests. The present invention can be used
to make in vitro measurements of blood constituents on paper
strips. The cross-polarization technique can also be used in
applications requiring color matching between two or more color
samples. The cross-polarization technique of the present invention
can also be used in borescopic applications, or in an endoscope and
orthoscope for clinical applications.
FIG. 19 shows a graph of absorbance units as a function of dye
concentration for four samples of red aniline dye (concentrations
of 1, 3X, 10X, and 20X; see Table 1 below). Line 1902 represents
data obtained for the four samples using a conventional reflected
spectrophotometry instrument. Such conventional instruments
typically have a small working range, generally from 0.0 to 0.5
Absorbance Unit, and, in the best case, from 0.0 to 1 Absorbance
Unit. One Absorbance Unit represents a factor of 10 change in light
intensity, either transmitted or reflected. The conventional
apparatus becomes flat and non-responsive above 0.5 Absorbance
Unit, and could not distinguish between the last two points
(concentrations of 10X and 20X). Line 1902 is flat in this region.
In contrast, line 1904 represents data obtained for the same four
samples using a reflection colorimeter apparatus 2020 as shown in
FIG. 20 that includes cross-polarizers. The working range using the
reflection colorimeter apparatus of the present invention has been
extended to more than two Absorbance Units (factor of 100). The
limitation in measuring the last concentration (20X) using the
reflection colorimeter apparatus of the present invention is the
number of bits (8 bits) that were used in making the computations.
An eight bit resolution (2.sup.8 =256) corresponds to approximately
2.41 Absorbance Units. To increase the number of Absorbance Units,
additional bits are required. For example, ten bits (2.sup.10
=1024) corresponds approximately to three Absorbance Units (factor
of 1000). To measure the 20X concentration, 15 bits would be
required. The results of FIG. 19 indicate that the
cross-polarization technique of the present invention can be used
for color control of presses, fabric and dye lot control, strip
testing, such as with paper, film, or latex, as well as in other
areas requiring color differentiation.
TABLE 1 ______________________________________ Concentration
X-Polarized Conventional ______________________________________ 20X
2.28 0.47 10X 2.23 0.49 3X 0.87 0.27 1 0.20 0.08 0 0.00 0.00
______________________________________
One embodiment of reflection colorimeter apparatus 2020 is shown in
FIG. 20. Apparatus 2020 includes a light source 2022 and a
condenser lens 2024. A first polarizer 2026, having a plane of
polarization shown generally at 2027, is used to polarize light
from light source 2022. First polarizer 2026 is disposed in a light
path between light source 2022 and an object or reflecting
substrate 2042 to be illuminated. In one embodiment, light source
2022 comprises first polarizer 2026 so that a separate first
polarizer 2026 is not required. In such an embodiment, light source
2022 is a source of polarized light, such as a laser or a laser
diode. A beam splitter 2028 reflects the light polarized by first
polarizer 2026 through an objective lens 2030 onto object 2042.
A second polarizer 2034, having a plane of polarization shown
generally at 2035, is disposed in a reflected light path between
object 2042 and a detecting means 2036. Plane of polarization 2035
is 90.degree. relative to plane of polarization 2027. A spectral
selection means 2032, for example, a filter, for wavelength
selection is disposed in the reflected light path.
In operation, illuminating light 2038 from light source 2022 passes
through condenser lens 2024 and is polarized by first polarizer
2026. Polarized light 2040 reflects off beam splitter 2028 and is
focused through objective lens 2030 onto object 2042. Reflected
light 2044 from object 2042 passes through lens 2030, beam splitter
2028, spectral selection means 2032, and second polarizer 2034.
Cross-polarized reflected light 2046 is detected by detecting means
2036.
Detecting means 2036 can be any device suitable for detecting
reflected light 2046. Suitable detecting means include a
photodetector, a photocell, or other device capable of detecting
the reflected light intensity of reflected light 2046. Suitable
detecting means 2036 also includes a camera. Apparatus 2020 can
also be used to perform analysis of blood using blood in a tube or
flow cell.
6. Conclusion
While various embodiments of the present invention have been
described above, it should be understood that they have been
presented by way of example only, and not limitation. For example,
the cross-polarization technique of the present invention can be
applied wherever it is desired to view circulation through tissue.
The cross-polarization technique of the present invention can also
be used to image stained tissue in situ. The cross-polarization
technique of the present invention can be used in any analytical,
in vivo, or in vitro application that requires optically measuring
or visually observing reflecting characteristics of an object.
Thus, the breadth and scope of the present invention should not be
limited by any of the above-described exemplary embodiments, but
should be defined only in accordance with the following claims and
their equivalents.
* * * * *