Note: Descriptions are shown in the official language in which they were submitted.
CA 02842464 2017-01-27
PATENT APPLICATION
SYSTEM AND METHOD FOR ANALYZING SAMPLES LABELED WITH 5, 10, 15, 20 TETRAKIS
(4-CARBOXYPHENYL) PORPHINE (TCPP)
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0001] Not Applicable.
INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC
[0002] Not Applicable.
COPYRIGHTED MATERIAL
[0003] Not Applicable.
INTRODUCTION
[0004] 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. 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.
[0005] 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).
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[0006] 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).
[0007] 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.
[0008] 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.
[0009] 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, immunoThistochemical 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).
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[0010] 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 lnst
87:1201-1202; Tockman
et al. 1988, J Clin Oncol, 11: 1685-1693; Tockman et al. 1994, Chest, 106:385s-
390s).
[0011] 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
(carboxyphenyI)-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
pg/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.
[0012] 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
[0013] 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
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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.
[0014] 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.
[0015] 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.
[0016] 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 +1- 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.
[0017] 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.
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[0018] 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.
[0019] 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.
[0020] 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).
[0021] 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
[0022] 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.
[0023] Fig. 1 illustrates a reconstructed 21 layer image of a cell labeled
with TCPP.
[0024] Fig. 2 illustrates fluorescence optical spectrum of TCPP labeled
plasma membrane
(non-corrected units).
[0025] Fig. 3 illustrates fluorescent image of cells with area of
interested marked in red.
[0026] Fig. 4 illustrates plot of spectral signatures from figure 1.
[0027] Fig. 5 illustrates plot of spectral signature of auto-fluorescence
from the blue
highlighted region of figure 6.
[0028] Fig. 6 illustrates cells imaged for auto-fluorescence and labeled
with TCPP.
[0029] Fig. 7 illustrates plot for TCPP labeled cell membrane from the
blue highlighted region
of figure 6.
[0030] Fig. 8 illustrates 530 nm layer focused at 660nm.
[0031] Fig. 9 illustrates 660 nm layer focused at 660 nm.
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DETAILED DESCRIPTION OF THE INVENTION
[0032] As used herein "a" means one or more.
[0033] As used herein "CCD" means Charge Coupled Device.
[0034] 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.
[0035] 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).
ATCPP pos
OR= _________________________ Eq. 1
ATCPP neg
Where ATCPP pos is the number of photons emitted per second photon flux per
unit area of cell
membrane for a cancerous cell and ATCPP neg is the number of photons emitted
per second per unit
area for a normal cell.
[0036] 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 200-900nm, preferably between about 420-
720nm more
preferably between about 600-700nm more preferably about 655nm +/- 30nm. The
difference in
spectral signatures from cells labeled with TCPP versus those without TCPP
label have a pronounced
spectral peak in the between 420nm ¨ 720nm 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
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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
[0037] 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.
[0038] 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.
[0039] 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
560nm range, and a certain
defective structure fluoresces in the 590nm range, the size, shape, and other
aspects of the defective
structure can be seen by filtering out all the wavelengths other than the
590nm 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.
Porphvrin Fluorescence Basics
[0040] 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,
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hemoglobin, and come in myriads of forms. A porphyrin as used herein is a
probe or labeling
compound.
[0041] When illuminated with light of the correct wavelength, most
proteins will produce
fluorescent photons with wavelengths in the about 490nm to 600nnn range.
Porphyrins, however,
have quite the varied absorption and emission spectra.
[0042] By labeling cells with porphyrins (for example TCPP), fluorescence
microscopy allows
the imaging of cell structures that are highlighted by the labeling compound
(see figure 1). Tailoring
the labeling compound to attach to specific targets in the cell gives the
ability to highlight specific cell
structures. Referring now to figure 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 465nm +/- 30nm. Figure 4 illustrates
the spectral plot obtained
from a spectral scan of the image in figure 1.
[0043] A chart illustrating a fluorescence optical spectrum of TCPP
labeled Plasma
Membrane of a cell from a biological specimen is shown in figure 2 according
to one embodiment of
the present invention.
Experimental Protocol
[0044] 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 0.05ug/m1¨
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
[0045] 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
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[0046] 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_
[0047] 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.
[0048] TCPP has a pronounced molar absorption coefficient around 400nm,
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.
[0049] In one embodiment, a region in the blue spectrum 475nm +1- 30nm was
the selected
excitation wavelength. A band pass filter centered on about 475nm 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 500nm with second pronounced transmission peak
below that centered
around 400nm 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
[0050] 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
350nm to about
800nm 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 30Im/W luminous flux 3000h luminous intensity 300cd
luminance
17000cd/cm2 and known spectral characteristics was utilized in the system
according to one
embodiment of the present invention.
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[0051] Referring now to figure 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 +/- 30nm. 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
figure 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, (x10A6Photons/cm2/s), Avg Signal (OD), Std Deviation Counts, Std
Deviation Scaled Counts,
Std Deviation (x10A6Photons/cm2/s), Std Deviation (OD), Total Signal Counts,
Total Signal Scaled
Counts (x10A6Photons/cm2/s), Total Signal (OD), Max Signal Counts, Max Signal
Scaled Counts,
Max Signal (x10A6Photons/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.
[0052] 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.
[0053] 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
[0054] Referring now to figure 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 475nm wavelength from the mercury vapor source filtered
through a band-pass filter
a long pass beam splitter, approximately 500nm cutoff. The image was taken
before the labeling
CA 02842464 2017-01-27
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 figure 6,
was analyzed using color
and the graph, of figure 5, shows the spectral components of the image.
Referring now to figure 5, a
plot of a spectral signature or auto-fluorescence from the blue highlighted
region in figure 6 is
illustrated.
[0055] The specimen of figure 6 was then labeled with TCPP and re-imaged.
The area
highlighted in blue, of figure 6, was imaged with the CCD sensor and analyzed.
The plot in figure 7
shows the fluorescent spectral output of TCPP (the green line) from the blue
highlighted region of
figure 6 as imaged. The photons per second per pixel are in arbitrary units.
[0056] The scales were set to the same value in order to demonstrate the
spectral signature
of the staining compound. The peak around 660nm is due to the TCPP staining
compound.
DATA: Location of TCPP in Cell Structures
[0057] 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 figure 1.
[0058] Figure 8 is a grayscale image taken with the LCTF tuned to about
530nm. The image
shows internal structures that were located below the layer that emitted the
TCPP signature. Figure 8
was taken with the focus of the microscope set on cell structures that were
seen with the LCTF set to
660nm. 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 figure 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.
[0059] The image in figure 9 was obtained with the LCTF tuned to 660nm.
The field of focus
was raised relative to the focal field for the 530nm 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 figure 9, it demonstrates that the objects emitting a
530nm signature were
located physically below those emitting an about 660nm signature. The plasma
membrane in figure 9
is focused.
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Data: Measurement of the fluorescing flux of a cancer cell labeled with TCPP
[0060] 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:
[0061] 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).
[0062] The equation below gives the basic form of the expression used.
(PCCCD)/[(0.99)(M0)(0C)(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.
[0063] All the data was collected using a 20X 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 X 1040
pixel array. Determining the actual dimensions of the measured area is simply
a matter of geometry.
[0064] 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.
[0065] 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.
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CA 02842464 2017-01-27
[0066] 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 +1- 30nm 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.
[0067] 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.
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