U.S. patent application number 14/532998 was filed with the patent office on 2015-06-11 for system and method for analyzing samples labeled with 5, 10, 15, 20 tetrakis (4-carboxyphenyl) porphine (tcpp).
The applicant listed for this patent is bioAffinity Technologies, Inc.. Invention is credited to Gordon Bennett, John Cousins, Constance Dorian.
Application Number | 20150160197 14/532998 |
Document ID | / |
Family ID | 43449864 |
Filed Date | 2015-06-11 |
United States Patent
Application |
20150160197 |
Kind Code |
A1 |
Dorian; Constance ; et
al. |
June 11, 2015 |
System and Method for Analyzing Samples Labeled with 5, 10, 15, 20
Tetrakis (4-Carboxyphenyl) Porphine (TCPP)
Abstract
One embodiment of the present invention provides for a method of
determining if a sputum sample contains dysplastic or carcinomic
cells by obtaining a sputum sample containing cells. The sputum
sample is labeled with TCPP to stain cells suspected to be
dysplastic or carcinomic. The labeled sputum sample is excited with
an excitation wavelength of light of about 475 nm +/-30 nm and
emission at about 560 nm +/-30 nm is detected from cells identified
to be macrophages. An imager focuses on the plasma membrane of one
or more cells suspected to be dysplastic or carcinomic and emission
at about 655 nm +/-30 nm, if present, is detected for TCPP labeled
cells of the sputum sample after focusing on the plasma membrane of
the cells of the sputum sample. Photon flux for each pixel of a
sensor is measured to obtain a value for the imaged cell. The
measured value is scored to determine if a cell is cancerous or
dysplastic.
Inventors: |
Dorian; Constance;
(Albuquerque, NM) ; Cousins; John; (Albuquerque,
NM) ; Bennett; Gordon; (Albuquerque, NM) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
bioAffinity Technologies, Inc. |
San Antonio |
TX |
US |
|
|
Family ID: |
43449864 |
Appl. No.: |
14/532998 |
Filed: |
November 4, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12839283 |
Jul 19, 2010 |
8877509 |
|
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14532998 |
|
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61226646 |
Jul 17, 2009 |
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Current U.S.
Class: |
435/34 |
Current CPC
Class: |
G01N 33/5091 20130101;
G01N 33/582 20130101; G01N 33/57492 20130101; G01N 33/52 20130101;
G01N 33/57423 20130101; G01N 2021/6439 20130101; G01N 21/6428
20130101; G01N 2458/30 20130101; G01N 2800/12 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50; G01N 21/64 20060101 G01N021/64 |
Claims
1. A method of determining if a sputum sample contains dysplastic
or carcinomic cells the method comprising: obtaining a sputum
sample containing cells; labeling the sputum sample with TCPP to
stain cells suspected to be dysplastic or carcinomic; exciting the
labeled sputum sample with an excitation wavelength of light of
about 475 nm +/-30 nm; detecting within the sputum sample emission
at about 560 nm +/-30nm from cells identified to be macrophages;
focusing an imager on the plasma member of one or more cells
suspected to be dysplastic or carcinomic; detecting emission at
about 655 nm +/-30 nm if present for TCPP labeled cells of the
sputum sample after focusing on the plasma membrane of the cells of
the sputum sample; measuring photon flux for each pixel of a sensor
to obtain a value for the imaged cell; and scoring measured value
to determine if a cell is cancerous or dysplastic.
2. The method of claim 1 wherein the TCPP is Meso Tetra
(4-Carboxyphenyl) Porphine.
3. The method of claim 1 wherein the excitation wavelength is about
475 nm +/-5 nm.
4. The method of claim 1 wherein emission of macrophages is about
560 nm +/-5 nm.
5. The method of claim 1 wherein the imager is a fluorescent
microscope.
6. The method of claim 1 wherein the emission of TCPP labeled cells
is about 655 nm +/-5 nm.
7. The method of claim 1 wherein the sensor is a CCD camera.
8. The method of claim 1 wherein the scoring further comprises
comparing the measured value to a database of stored values for
cancerous, dysplastic and non-cancerous cells to assigning a score
based upon the results of the comparison.
9. The method of claim 1 wherein the sputum sample is from a
human.
10. A computer readable medium for enabling a computer to
characterize a sputum sample, the computer readable medium
comprising software instructions for enabling the computer to
perform predetermined operations of the following steps: exciting a
sputum sample labeled with TCPP with an excitation wavelength of
light of about 475 nm +/-30 nm; detecting within the labeled sputum
sample emission at about 560 nm +/-30nm from cells identified to be
macrophages; focusing an imager on the plasma membrane of one or
more cells suspected to be dysplastic or carcinomic; detecting
emission at about 655 nm +/-30 nm if present for TCPP labeled cells
of the sputum sample after focusing on the plasma membrane of the
cells of the sputum sample; measuring photon flux for each pixel of
a sensor to obtain a value for the imaged cell; and scoring
measured value to determine if a cell is cancerous or dysplastic.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 12/839,283, entitled "System and Method for
Analyzing Samples Labeled with 5, 10, 15, 20, Tetrakis
(4-Carboxyphenyl) Porphine (TCPP)", filed on Jul. 19, 2010, and
issued on Nov. 4, 2014, as U.S. Pat. No. 8,877,509, which claims
priority to and the benefit of the filing of U.S. Provisional
Patent Application Ser. No. 61/226,646 entitled "System and Method
for Analyzing Samples Labeled with 5, 10, 15, 20, Tetrakis
(4-Carboxyphenyl) Porphine (TCPP)", filed on Jul. 17, 2009, and the
specification and claims thereof are incorporated herein by
reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not Applicable.
INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A COMPACT
DISC
[0003] Not Applicable.
COPYRIGHTED MATERIAL
[0004] Not Applicable.
INTRODUCTION
[0005] Pathologists, who examine disease progression and analyze
tissue samples for abnormalities, including cancer, have determined
that a cellular condition called dysplasia, which refers to
abnormal formation or maturation of cells, can potentially identify
cells in a pre-cancerous condition. Unchecked, dysplasia can
progress through mild, moderate and severe stages and eventually to
cancer. About one in seven of the moderate cases of dysplasia will
progress to cancer, and as many as 83% of cases with severe
dysplasia have been reported to progress to cancer, depending on
the types of cells involved. However, removal of mild and moderate
dysplasias greatly reduces the development of cancer.
[0006] In the lung, removal of dysplastic cells not only greatly
reduces the formation of cancerous cells, but in some cases
pulmonary tissue will return to a normal morphology.
[0007] In general, the earlier cancers are detected, the better the
prognosis is for patient survival. If breast cancer is detected
early when it is still localized to a single mass, the five-year
survival rate is more than 96%. When it has spread to a distant
location, the five-year survival rate is less than 20%. For lung
cancer, when it is detected as a single mass the 5-year survival is
more than 46%. When it has spread, the five-year survival is less
than 14%. For cervical cancer, additional improvement in survival
occurs when pre-cancerous changes are found and treated before
developing into a more severe stage (Boring and Squires 1993, CA
Cancer J Clin 43:7-26 and Ferguson 1990, Hematol Oncol Clin N Am
4:1053-1168).
[0008] Lung carcinoma is presently the leading cause of cancer
mortality among men and women in the United States (Wingo et al.
1995, CA Clinical J Clin 45.8-30). In 1997, there were an estimated
160,000 deaths from lung cancer, accounting for 12% of all cancer
deaths in U.S. men and 2% in U.S. women (Boring & Squires 1993,
supra). Lung cancer is also one of the most lethal types of cancer,
as reflected in a five-year survival rate of only 14%. The poor
prognosis for lung cancer patients, relative to other types of
human cancer, is due largely to the lack of effective early
detection methods. At the time of clinical (symptomatic)
presentation, over two thirds of all patients have regional nodule
involvement or distant metastases, both of which are usually
incurable. In studies of patients with localized (Stage 0 or 1)
lung cancer, however, 5-year survival rates have ranged from 40% to
70% (Boring & Squires, 1993, supra; Ferguson, 1990, supra).
[0009] Historically, the only diagnostic tests used to detect lung
cancer before symptoms occur have been sputum cytology and chest
radiography. As a consequence, the efficacy of these tests as mass
screening tools has been extensively evaluated in studies over the
past several decades. Both tests detect presymptomatic,
earlier-stage carcinoma, particularly carcinoma of squamous
cells.
[0010] Improvements in screening methods have largely centered
around improving the utility of sputum cytology through technologic
advances in microscopy. Sputum cytology requires a visual
examination of a cell sample during which cell size, shape,
organization, and a ratio between the size of the cell's nucleus
and cytoplasm is used to determine the cell's morphology. Because
this assessment of cell morphology requires a visual inspection and
classification, the technique requires a significant amount of
expertise on behalf of the clinical observer. Various
investigations have been conducted with results suggesting that
computer-assisted, high resolution image analysis enables detection
of subvisual changes in visually normal nuclei associated with
several tissue types (Montag et al. 1991, Anal Quant Cytol Histol
13:159-167; Haroske et al. 1988, Arch Geschwulstforsch, 58:159-168;
Hutchinson et al. 1992, Anal Quant Cytol Estol 4:330-334).
Computer-assisted analysis of DNA distribution in cell samples
provided 74% correct morphological classification of nuclei without
human review of the material and without the need for visually
abnormal nuclei being present when compared with standard
cytological testing.
[0011] The morphologic assessment of cytological specimens has also
improved due to advances in the understanding of lung tumor
pathology. Much of this work has centered on the identification of
"biomarkers." Biomarkers refer to a wide range of progressive
phenotypic and genetic abnormalities of the respiratory mucosa
which may be used in determining the potential for bronchial
epithelium to fully transform into a malignant tumor. Markers have
been broadly classified as morphological changes,
immuno/histochemical markers for differentially expressed proteins,
markers for genomic instability, markers of epigenetic change
(e.g., abnormal methylation), and gene mutations (Hirsh et al.
1997, Lung Cancer 17:163-174).
[0012] The expression levels of these markers are now being
evaluated in dysplastic and neoplastic cyto/histological tissue
samples collected from high risk populations. Among those specimens
currently being targeted for exploratory marker analysis is sputum.
Interest in sputum samples for biomarker research has been
generated from the long-held belief that exfoliated cells recovered
in sputum may be the earliest possible indication of an incipient
carcinoma, since lung cancer most frequently develops in the
bronchial epithelium. Through application of sophisticated
molecular genetic techniques (e.g., PCR-based assays), studies are
providing evidence that selected biomarkers can be detected in
sputum (Mao et al. 1994, Cancer Res 54:1634-1637; Mao et al. 1994,
Proc Natl Acad Sci USA 91:9871-9875; Sidransky 1995, J Natl Cancer
Inst 87:1201-1202; Tockman et al. 1988, J Clin Oncol, 11:
1685-1693; Tockman et al. 1994, Chest, 106:385s-390s).
[0013] Commercially available cancer screening or detection
services rely on tests based on cytomorphological diagnosis by
trained clinicians who look at each sample and determine the extent
and identity of abnormal cell types. This process is not only
expensive and time-consuming, it also introduces human judgement
and therefore error into the procedure. Recently, a method has been
developed for detecting cancerous cells of the lung through use of
5, 10, 15, 20-tetrakis (carboxyphenyl)-porphine (TCPP) (U.S. Pat.
No. 5,162,231 to Cole et al). This method relies on the propensity
of cancerous cells to accumulate TCPP from their environment in a
greater amount than non-cancerous cells. Upon incubation of a cell
sample for 6-24 hours with 200 .mu.g/ml TCPP, the TCPP entered
cells and bound to the perinuclear membrane and mitochondria of
neoplastic cells. TCPP fluoresces under ultraviolet light, and
cancerous cells may thereby be diagnosed solely by the intensity of
fluorescence, without reference to morphology. The extension of the
use of this compound to identifying pre-cancerous tissue conditions
(e.g., dysplastic cells) would permit screening in high risk
populations to identify those individuals whose tissues are
progressing toward invasive cancer conditions, and thereby
facilitate catching the cancer or dysplasia at the most treatable
stage. The desirable characteristics of such a screening method
would be a procedure that is rapid, inexpensive, and requires a
minimum of technical expertise.
[0014] For the foregoing reasons, there is a need for a technique
and methodology for detecting dysplastic cells in their earliest
stages. In addition, there is a need for a technique that can
provide highly reliable diagnostic results objectively and that
does not rely on the subjective analysis of the clinician
performing the diagnosis.
SUMMARY OF THE INVENTION
[0015] One embodiment of the present invention provides for a
method of determining if a sputum sample contains dysplastic or
carcinomic cells by obtaining a sputum sample containing cells. The
sputum sample is labeled with TCPP to stain cells suspected to be
dysplastic or carcinomic. An imager focuses on the plasma membrane
of one or more cells suspected to be dysplastic or carcinomic and
emission at about 655 nm +/-30 nm, if present, is detected for TCPP
labeled cells of the sputum sample after focusing on the plasma
membrane of the cells of the sputum sample. Photon flux for each
pixel of a sensor is measured to obtain a value for the imaged
cell. The measured value is scored to determine if a cell is
cancerous or dysplastic.
[0016] Another embodiment provides for a method of determining if a
biological sample of cells contains dysplastic or carcinomic cell
by obtaining a biological sample suspected of containing dysplastic
or carcinomic cells. The biological sample is labeled with TCPP.
The sample is excited with an excitation wavelength of light of
about 475 nm +/-30 nm. An imager is focused on the plasma member of
one or more cells suspected of containing dysplastic or carcinomic
cells to obtain an image. Emission at about 655 nm +/-30 nm if
present is detected from TCPP labeled cells after focusing on the
plasma membrane of one or more cells of the biological sample
suspected of containing dysplastic or carcinomic cells. Photon flux
is measured for each pixel of the sensor to obtain a value for the
imaged cell. The measured value is scored to determine if a cell is
cancerous or dysplastic.
[0017] Yet another embodiment provides a computer readable medium
for enabling a computer to characterize a sputum sample, the
computer readable medium comprising software instructions or code
for enabling the computer to perform predetermined operations. The
predetermined operation steps include exciting a sputum sample
labeled with TCPP with an excitation wavelength of light of about
475 nm +/-30 nm; detecting within the labeled sputum sample
emission at about 560 nm +/-30nm from cells identified to be
macrophages; focusing an imager on the plasma membrane of one or
more cells suspected to be dysplastic or carcinomic; detecting
emission at about 655 nm +/-30 nm if present for TCPP labeled cells
of the sputum sample after focusing on the plasma membrane of the
cells of the sputum sample; measuring photon flux for each pixel of
a sensor to obtain a measured value for the imaged cell; and
scoring the measured value to determine if a cell is cancerous or
dysplastic.
[0018] In a preferred embodiment the TCPP is Meso Tetra
(4-Carboxyphenyl) Porphine. In another embodiment the excitation
wavelength is about 475 nm +/-5 nm. In yet another embodiment the
emission of macrophages is about 560 nm +/-5 nm. In yet another
embodiment the imager is a fluorescent microscope. In yet another
embodiment the emission of TCPP labeled cells is about 655 nm +/-5
nm. In yet another embodiment the sensor is a CCD camera. In yet
another embodiment the scoring further comprises comparing the
measured value to a database of stored values for cancerous,
dysplastic and non-cancerous cells to assigning a score based upon
the results of the comparison. In yet another embodiment the sputum
sample is from a human.
[0019] One aspect of the present invention provides for labeling
biological samples with Meso Tetra (4-Carboxyphenyl) Porphine or 5,
10, 15, 20 tetrakis (4-carboxyphenyl) porphine defined herein as
"TCPP" for the detection of cancerous and precancerous cells.
[0020] Another aspect of the present inventions provides for using
TCPP to detect cancerous cells in sputum since TCPP will bind
preferentially with cancerous and precancerous cells.
[0021] Another aspect of the present invention provides a system
and method to verify and quantify the spectral signature of TCPP
optically, and/or quantify the photon emission rates of TCPP when
used as a labeling compound.
[0022] Another aspect of the present invention provides for
analyzing TCPP labeled samples using a fluorescent system equipped
with a tuneable optical filter and Change Coupled Device (CCD).
[0023] Additional objects and advantages of the present invention
will be apparent in the following detailed description read in
conjunction with the accompanying drawing figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The file of this patent contains at least one drawing
executed in color. Copies of this patent with color drawing(s) will
be provided by the Patent and Trademark Office upon request and
payment of the necessary fee.
[0025] FIG. 1 illustrates a reconstructed 21 layer image of a cell
labeled with TCPP.
[0026] FIG. 2 illustrates fluorescence optical spectrum of TCPP
labeled plasma membrane (non-corrected units).
[0027] FIG. 3 illustrates fluorescent image of cells with area of
interested marked in red.
[0028] FIG. 4 illustrates plot of spectral signatures from FIG.
1.
[0029] FIG. 5 illustrates plot of spectral signature of
auto-fluorescence from the blue highlighted region of FIG. 6.
[0030] FIG. 6 illustrates cells imaged for auto-fluorescence and
labeled with TCPP.
[0031] FIG. 7 illustrates plot for TCPP labeled cell membrane from
the blue highlighted region of FIG. 6.
[0032] FIG. 8 illustrates 530 nm layer focused at 660 nm.
[0033] FIG. 9 illustrates 660 nm layer focused at 660 nm.
DETAILED DESCRIPTION OF THE INVENTION
[0034] As used herein "a" means one or more.
[0035] As used herein "CCD" means Charge Coupled Device.
[0036] As used herein sample "a biological sample" or "sample" or
"specimen" refers to a whole organism or a subset of its tissue,
cells or components parts (body fluids, including but not limited
to blood, mucus, lymphatic fluid, sputum, plasma, ejaculate,
mammary duct fluid, cerebrospinal fluid, urine, and fecal
stool.
[0037] According to one embodiment of the present invention, a
system and method for determining the amount of photon emission
from TCPP bound to a cell of interest thought to be cancerous
relative to the amount of photon emission from TCPP from
non-cancerous cells is provided. Since we are determining relative
amounts of fluorescence, with all the cells in the same
environment, we are not limited to the actual quantum yields of the
individual fluorophores. One embodiment of the present invention
provides for determination of photon emission from specimens with
Equation 1 (Eq. 1).
.PHI. R = .LAMBDA. TCPPpos .LAMBDA. TCPPneg Eq . 1 ##EQU00001##
Where .LAMBDA..sub.TCPP pos is the number of photons emitted per
second photon flux per unit area of cell membrane for a cancerous
cell and .LAMBDA..sub.TCPP neg is the number of photons emitted per
second per unit area for a normal cell.
[0038] According to one embodiment of the present invention, TCPP
adheres preferentially to the plasma membrane of a cell and even
more preferentially to a cancerous cell. TCPP binds to a cancerous
cell or precancerous cell preferentially as compared to
non-cancerous cell. It has been observed that cancerous cells have
an abnormally high concentration of low density lipoproteins on
their plasma membranes thereby giving rise to the quantifiably
higher fluorescence emission from TCPP at a wavelength between
about 200-900 nm, preferably between about 420-720 nm more
preferably between about 600-700 nm more preferably about 655 nm
+/-30 nm. The difference in spectral signatures from cells labeled
with TCPP versus those without TCPP label have a pronounced
spectral peak in the between 420 nm-720 nm region. In addition, a
filter for example a Liquid Crystal Tunable Filter (LCTF) within
the system facilitates demonstration of not only the location of
where TCPP is binding and/or concentrating in regards to the cell
structure, but also allows quantification of the photon emission in
the region of reference relative to the rest of the fluorescent
signature from the cell or other that is not due to TCPP.
Image Capture Device
[0039] One embodiment of the present invention provides for a
sensor, for example, a Charge Couple Device "CCD" sensor, for
example a CCD camera but not limited thereto to capture an image of
a TCPP stained specimen. In one embodiment a CCD is semiconductor
device made from an epitaxial layer of doped silicon grown on a
silicon substrate. By creating separated pixels connected to a
shift register, the image focused on a two-dimensional array of
these pixels can be stored electronically. A pixel may be described
by its size and the number of electrons it can hold. For one
application herein, the CCD sensor is used to determine the number
of photons impacting specific pixels in the array. This is
accomplished by measuring the voltage developed across the
capacitive junction of each pixel over a given time. The charge
that creates the difference in potential is directly related to the
number of photons impacting each pixel quantum mechanically. For
each photon that is absorbed by the doped silicon, a specific
number of electrons are liberated and excited into the valence
levels of the semiconductor. The quantum efficiency of a CCD sensor
is represented by a quantum efficiency curve associated with a
specific CCD sensor.
[0040] A CCD sensor allows the measurement of the number of photons
by registering the voltage developed across each pixel junction as
read through the serialized output of the shift register, basically
a sequence of binary coded hexadecimal values ordered according the
sequence in which the shift register outputs the pixel voltages. As
each of these values is directly proportional to the number of
photons impacting each pixel during a period of time, these values
can be correlated to the photon emission flux of the source of the
photons.
[0041] An extraordinary amount of information can be gathered
concerning the composition of cellular structures by isolating the
specific wavelengths of emitted photons that are being emitted by
specific structures of the cell or by probes or labeling compounds
such as TCPP that bind to a cell or portion thereof. For example,
if normal auto-fluorescence occurs in the 560 nm range, and a
certain defective structure fluoresces in the 590 nm range, the
size, shape, and other aspects of the defective structure can be
seen by filtering out all the wavelengths other than the 590 nm
wavelength and then using the selected wavelength output to create
the image on the CCD sensor. The actual number of photons per unit
area of cell structure per unit time can then be determined by
measuring the voltages developed per unit time per pixel and
correlating that value to the magnification of the optical system
and the attenuation of the individual components.
Porphyrin Fluorescence Basics
[0042] Porphyrins are planar aromatic macromolecules consisting of
four pyrole rings joined by four methane bridges. They are natural
occurring compounds that are found in plants, hemoglobin, and come
in myriads of forms. A porphyrin as used herein is a probe or
labeling compound.
[0043] When illuminated with light of the correct wavelength, most
proteins will produce fluorescent photons with wavelengths in the
about 490 nm to 600 nm range. Porphyrins, however, have quite the
varied absorption and emission spectra.
[0044] By labeling cells with porphyrins (for example TCPP),
fluorescence microscopy allows the imaging of cell structures that
are highlighted by the labeling compound (see FIG. 1). Tailoring
the labeling compound to attach to specific targets in the cell
gives the ability to highlight specific cell structures. Referring
now to FIG. 1, structures within the cell exhibit auto fluorescence
in the green wavelength while the plasma membrane fluoresces in the
red wavelength when the cell is illuminated with an excitation
wavelength of about 465 nm +/-30 nm. FIG. 4 illustrates the
spectral plot obtained from a spectral scan of the image in FIG.
1.
[0045] A chart illustrating a fluorescence optical spectrum of TCPP
labeled Plasma Membrane of a cell from a biological specimen is
shown in FIG. 2 according to one embodiment of the present
invention.
Experimental Protocol
[0046] According to one embodiment of the present invention, a
biological specimen, for example a sputum sample is processed using
a thin prep protocol onto a microscope slide. The sputum sample is
fixed in a methanol based solution which has been demonstrated to
be less corrosive to the plasma membrane of a cell from a cell
population of interest. Minimizing corrosive effects to the plasma
membrane is important as the TCPP is shown to localize on the
plasma membrane of the cell surface. The cells are processed to
separate the cells from the mucous and cell fragments. Each
prepared slide contains a monolayer of the sputum cells. After
preparing the slides, the labeling reagent TCPP is dissolved in
concentrations between 0.05 ug/ml-4.0 ug/ml in an aqueous alcohol
containing between 50% and 90% isopropanol alcohol solution. The pH
is adjusted with sodium Bicarbonate to a pH between 6.0 and 10.5.
The slide is immersed in the TCPP labeling solution, rinsed,
air-dried, and a cover-slip is placed on top. (See for example U.S.
Pat. No 7,670,799 to Garwin).
Imager
[0047] An imager such as a scope, for example, a microscope,
preferably a Fluorescent Microscope is utilized by the system
according to one embodiment of the present invention.
Excitation Source
[0048] A light source, for example, a Mercury Vapor Lamp or
preferably a laser which may be tuned to user specified wavelengths
is further utilized by the system according to one embodiment of
the present invention.
Optics
[0049] Fluorescence optics cube with a blue visible frequency notch
filter. Fluorescent light from the sample on a specimen platform,
for example, a slide then passes through a beam splitter to the
microscope objective and on to a CCD camera in a preferred
embodiment. In addition the system may also comprise a processor, a
database and computer readable instructions for obtaining a score
from an image and producing a report based upon the score.
[0050] TCPP has a pronounced molar absorption coefficient around
400 nm, called the Soret band. Although very efficient in this
region, photo-bleaching occurs. Therefore, a region of the spectrum
where the absorption by TCPP is not so efficient may be selected,
thereby eliminating most of the photo-bleaching and extending the
fluorescent lifetimes for which the samples are viable.
[0051] In one embodiment, a region in the blue spectrum 475 nm
+/-30 nm was the selected excitation wavelength. A band pass filter
centered on about 475 nm was employed. A fluorescent optical cube
also contained a dichroic beam splitter that has a fairly flat
optical transmission frequency response in the visible above 500 nm
with second pronounced transmission peak below that centered around
400 nm allowing any of the light corresponding to the Soret band
that happens to get through the excitation filter to pass through
and not be reflected to the sample.
Image Capture and Method for Obtaining Image
[0052] To gather our data an image capture system having a detector
capable of quantifying emission of photons from a TCPP labeled cell
across light spectrum from about 350 nm to about 800 nm was
employed according to one embodiment of the present invention. The
system comprises an imager as a scope for example a microscope more
preferably a fluorescent microscope. An image sensor and capture
device which may be automated for data acquisition for optimizing
emission capture of an image. The image capture device may attach
directly to an imager such as a fluorescent microscope. A filter,
for example, a Liquid Crystal Tunable Filter, (LCTF) but not
limited thereto allows the capture of images from different optical
frequencies, and the measurement of the emission at those different
frequencies. However, other filters (customized or off the shelf)
may be utilized and other filtering techniques may be utilized and
is not limited to LCTF. In addition, a light source, for example a
mercury vapor lamp or more preferably a laser which may be tuned to
a user specified wavelengths is useful for illuminating the
specimen. In one example, a mercury vapor lamp having luminous
efficacy 301 m/W luminous flux 3000 h luminous intensity 300 cd
luminance 17000 cd/cm2 and known spectral characteristics was
utilized in the system according to one embodiment of the present
invention.
[0053] Referring now to FIG. 1 is a cell labeled or stained with
TCPP according to one embodiment of the present invention. The
image is reconstructed from multiple images acquired over at
designated wavelengths over a user defined spectrum and the
resulting images are recombined. For example 21 layers of an image
with each layer acquired at a different wavelength were obtained
from the cell labeled with TCPP. The autofluorescence of the cells
is detected in the green channel 560 +/-30nm and the fluorescence
of the TCPP labeling compound on the cell is detected in the red
channel 660 +/-30 nm. One embodiment of the system and method of
the present invention provides for the isolation of specific
frequencies for imaging of the cell and measuring frequencies by
tuning the LCTF. By tuning the LCTF to different frequencies during
imaging, the system permits information to be gathered and analyzed
over a broad spectrum. Then, after capturing each image for a
specific wavelength range, the specific optical spectra of interest
may be extracted. The image with spectral enhancements to highlight
specific features from the image is displayed. Then a grey scale
image is measured with the LCTF tuned to the appropriate frequency,
see for example FIG. 3. The photon flux is measured from a
specified cell structure(s) in the image (see for example the red
circle with bulls-eye positioned over the area of interest). The
determination of a relative threshold emission value for
determining whether a cell is cancerous or not is then determined.
This also allows the separation of emissions by different cell
structures and quantifying the emissions to produce a value. In
addition one or more of the following features from the image and
or cell of interest may also be useful in scoring: ROI Number, Cube
ID, Avg Signal (counts), Avg Signal (scaled counts/s), Avg Signal,
(x10 6Photons/cm2/s), Avg Signal (OD), Std Deviation Counts, Std
Deviation Scaled Counts, Std Deviation (x10 6Photons/cm2/s), Std
Deviation (OD), Total Signal Counts, Total Signal Scaled Counts
(x10 6Photons/cm2/s), Total Signal (OD), Max Signal Counts, Max
Signal Scaled Counts, Max Signal (x10 6Photons/cm2/s), Max Signal
(OD), Area Pixels, Area (um)2, Major Axis, Minor Axis, x location,
y location, Spectrum ID, Cube Time Stamp, Cube, Visual
Fluorescence, Cell Morphology (size, shape, not limited to type,
characteristics), Spectral Signature (TCPP), Background
Fluoresence, Signal/Background Ratio, Std Deviation,
signal/Background Ratio, Fluorescence (Auto, TCPP)), Capture Image
Cube Narrow Band Width, Capture Image Cube Full Spectrum.
[0054] In one embodiment of the present invention the value
produced by the scoring is correlated to a cancerous cell or
non-cancerous cell to determine the health of a patient.
[0055] The system permits the separation of an image based upon
specific wavelengths as well as selecting specific regions in that
image in order to measure the signal from the CCD sensor, and then
export the spectral data for analysis.
DATA: Spectral Signature of TCPP
[0056] Referring now to FIG. 6, the image is from a sample of lung
sputum that was placed on a microscope slide according to the above
listed procedure. The slide was illuminated with light having an
about 475 nm wavelength from the mercury vapor source filtered
through a band-pass filter a long pass beam splitter, approximately
500 nm cutoff. The image was taken before the labeling procedure in
order to demonstrate the spectral signature of TCPP relative to the
normal auto-fluorescence of the cell structures. An area
highlighted in blue, of FIG. 6, was analyzed using color and the
graph, of FIG. 5, shows the spectral components of the image.
Referring now to FIG. 5, a plot of a spectral signature or
auto-fluorescence from the blue highlighted region in FIG. 6 is
illustrated.
[0057] The specimen of FIG. 6 was then labeled with TCPP and
re-imaged. The area highlighted in blue, of FIG. 6, was imaged with
the CCD sensor and analyzed. The plot in FIG. 7 shows the
fluorescent spectral output of TCPP (the green line) from the blue
highlighted region of FIG. 6 as imaged. The photons per second per
pixel are in arbitrary units.
[0058] The scales were set to the same value in order to
demonstrate the spectral signature of the staining compound. The
peak around 660 nm is due to the TCPP staining compound.
DATA: Location of TCPP in Cell Structures
[0059] Another feature of the present invention illustrates that
the TCPP compound localizes to the plasma membrane of a cell. By
separating the images by spectral emission it can be shown that the
emission of the TCPP labeling compound is emitted exclusively from
the plasma membrane. The microscope can be focused on the
structures that are emitting at specific wavelengths in the visible
range. Images of (either internal or external) features of the
fluorescing cell structures are obtained. Images of a cell,
portions thereof and cellular structures emitting photons at
different wavelengths are illustrated in FIG. 1.
[0060] FIG. 8 is a grayscale image taken with the LCTF tuned to
about 530 nm. The image shows internal structures that were located
below the layer that emitted the TCPP signature. FIG. 8 was taken
with the focus of the microscope set on cell structures that were
seen with the LCTF set to 660 nm. In order to bring this image into
focus the field of focus had to be physically lowered. The image
that resulted (not shown) from lowering the field of focus
demonstrated more definition and was in better focus and the plasma
membrane is better defined than FIG. 8. This is due to the fact
that some of the light being emitted from the lower cell structures
is occluded by the plasma membrane.
[0061] The image in FIG. 9 was obtained with the LCTF tuned to 660
nm. The field of focus was raised relative to the focal field for
the 530 nm image. As the excitation light was coming from above the
slide, combined with the fact that only the exposed surface of the
cells were subjected to the staining compound during the staining
process, the images support the premise that TCPP adheres only to
surface features and does not migrate into internal cell
structures. When this is taken into consideration with FIG. 9, it
demonstrates that the objects emitting a 530 nm signature were
located physically below those emitting an about 660 nm signature.
The plasma membrane in FIG. 9 is focused.
Data: Measurement of the fluorescing flux of a cancer cell labeled
with TCPP
[0062] Determination of the photon flux from the fluorescing cell
is based on the saturation level and quantum efficiency of the CCD
sensor. The basis for the values calculated is as follows:
[0063] According to one embodiment of the present invention, the
photons emitted by the fluorescing structures pass through 12
mediums before impacting the CCD. These consist of the slide cover,
the optics in the objective, the optics cube, the beam splitter for
the microscope, the LCTF, and the air gaps between each. We have
taken into consideration the transmission coefficients for each of
the mediums at the relevant wavelengths, along with the wavelength
dependant attenuation of the LCTF and quantum efficiency of the CCD
in order to arrive at a value for the number of photons emitted per
second arriving at each pixel of the CCD. Of course there is a
bandwidth consideration due to the bandwidth of the LCTF. Each of
the bandwidths in question have a specification of 20 nm full width
at half max (FWHM).
[0064] The equation below gives the basic form of the expression
used.
(PCCCD)/[(0.99)(MO)(OC)(LCTF)(QECCD)]=photon flux from cell per
pixel
Where PCCCD is the photon count from the CCD, MO is the attenuation
attributed to the microscope optics, OC is the attenuation of the
optics in the fluorescent optics cube, LCTF is the attenuation due
to the liquid crystal tunable filter, and QECCD is the quantum
efficiency of the CCD chip. The 0.99 term is to account for the
absorption of photons in the slide cover and the scattering and
other losses due to the air gaps and Fresnel reflections.
[0065] All the data was collected using a 20.times. objective with
a numerical aperture of 0.7. This allows the data to be correlated
to the actual size of the emission area of the cell structure. The
data analysis allows the measurement of the photon count over
specified areas of the image. In this particular case, the image
capture device for example a CCD which may consists of a
1392.times.1040 pixel array. Determining the actual dimensions of
the measured area is simply a matter of geometry.
[0066] Once an image is captured the relevant grayscale layer is
isolated and a region of interest is specified. The charge on each
element of the CCD sensor is acquired. Based upon the charge, a
value for the number of photons absorbed by that element at that
wavelength is determined. Using this value, the actual number of
photons emitted by the fluorescing source can be estimated with the
above relationship. The value is scored against controls and a
score is assigned. The assigned score determines whether the cell
screened is cancerous or non-cancerous.
[0067] The information for the specified region in terms of the
number of pixels in the specified region, the total number of
counts in the specified region, the number of counts from the pixel
with the highest value and the standard deviation for the frequency
distribution is calculated.
[0068] Although the invention has been described in detail with
particular reference to these preferred embodiments, other
embodiments can achieve the same results. Variations and
modifications of the present invention will be obvious to those
skilled in the art and it is intended to cover in the appended
claims all such modifications and equivalents. For example,
wavelengths are provided as about a specific wavelength and about
specified ranges of wavelengths. It should be understood that some
embodiments permit +/-30 nm flux. Also specific examples are
provided that relate to sputum samples but biological samples may
be of any type and obtained by different means as is identified in
U.S. Pat. No. 6,316,215 to Adair. The entire disclosures of all
references, applications, patents, and publications cited above are
hereby incorporated by reference.
[0069] The present invention has been described in terms of
preferred embodiments, however, it will be appreciated that various
modifications and improvements may be made to the described
embodiments without departing from the scope of the invention. The
entire disclosures of all references, applications, patents, and
publications cited above and/or in the attachments, and of the
corresponding application(s), are hereby incorporated by
reference.
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