U.S. patent application number 11/925028 was filed with the patent office on 2008-09-04 for image processing method for a microscope system.
This patent application is currently assigned to Ikonisys, Inc.. Invention is credited to Youngmin Kim, Triantafyllos Tafas, Xiuzhong Wang, Yanning Zhu.
Application Number | 20080212172 11/925028 |
Document ID | / |
Family ID | 39033581 |
Filed Date | 2008-09-04 |
United States Patent
Application |
20080212172 |
Kind Code |
A1 |
Zhu; Yanning ; et
al. |
September 4, 2008 |
Image Processing Method for a Microscope System
Abstract
An embodiment is disclosed for performing the image processing
for analyzing the results of a fluorescence in situ hybridization
(FISH) microscopic automated sample analysis to determine specific
chromosomal characteristics.
Inventors: |
Zhu; Yanning; (Hamden,
CT) ; Tafas; Triantafyllos; (Rocky Hill, CT) ;
Kim; Youngmin; (Wallingford, CT) ; Wang;
Xiuzhong; (Hamden, CT) |
Correspondence
Address: |
KELLEY DRYE & WARREN LLP
400 ALTLANTIC STREET , 13TH FLOOR
STAMFORD
CT
06901
US
|
Assignee: |
Ikonisys, Inc.
New Haven
CT
|
Family ID: |
39033581 |
Appl. No.: |
11/925028 |
Filed: |
October 26, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11833204 |
Aug 2, 2007 |
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11925028 |
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60821536 |
Aug 4, 2006 |
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Current U.S.
Class: |
359/383 ;
359/385 |
Current CPC
Class: |
G01N 21/6458
20130101 |
Class at
Publication: |
359/383 ;
359/385 |
International
Class: |
G02B 21/00 20060101
G02B021/00; G02B 21/06 20060101 G02B021/06 |
Claims
1. A computer-usable medium having computer readable instructions
stored thereon for execution by a processor to perform a method
comprising: adjusting a microscope to bring a specimen into focus
at a focal plane within the depth of the said specimen; irradiating
the said specimen with fluorescence exciting illumination;
adjusting the exposure parameters of an electronic imaging device;
capturing the image of the said specimen with said electronic
imaging device; enumerating objects-of-interest; identifying
nuclei; segmenting said nuclei; counting and characterizing the
color of bright fluorescent light signals occurring within said
nuclei; and interpreting and reporting the results.
Description
[0001] This application is continuation application of U.S. patent
application Ser. No. 11/833,204, filed on Aug. 2, 2007, which
claims priority from U.S. Provisional Application Ser. No.
60/821,536 filed Aug. 4, 2006. All references cited in this
specification, and their references, are incorporated by reference
herein where appropriate for teachings of additional or alternative
details, features, and/or technical background.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention generally relates to image processing
methods employed for performing automated microscopic analysis for
fluorescence in situ hybridization (FISH) detection of genetic
characteristics.
[0004] 2. Description of the Related Art
[0005] Conventional optical microscopy generally employs a
microscope slide to which the normal human complement of
chromosomes consists of the sex chromosomes (designated X and Y)
and 22 autosomes (numbered 1-22). It has been estimated that a
minimum of 1 in 10 human conceptions has a chromosome abnormality.
As a general rule, an abnormal number of sex chromosomes is not
lethal, although infertility can result. In contrast, an abnormal
number of autosomes typically results in early death. Of the three
autosomal trisomies found in live-born babies (trisomy 21, 18 and
13), only individuals with trisomy 21 (more commonly known as Down
syndrome), survive past infancy.
[0006] Although Down syndrome is easily diagnosed after birth,
prenatal diagnosis is problematic. To date, karyotyping of fetal
cells remains the established method for the diagnosis of Down
syndrome and other genetic abnormalities associated with an
aberration in chromosomal number and/or arrangement. Such genetic
abnormalities include, for example, chromosomal additions,
deletions, amplifications, translocations and rearrangements. The
assessment of such abnormalities is made with respect to the
chromosomes of a healthy individual, i.e., an individual having the
above-described normal complement and arrangement of human
chromosomes.
[0007] Genetic abnormalities include the above-noted trisomies,
such as Down syndrome, as well as monosomies and disomies. Genetic
abnormalities also include additions and/or deletions of whole
chromosomes and/or chromosome segments. Alterations such as these
have been reported to be present in many malignant tumors. Thus,
aberrations in chromosome number and/or distribution (e.g.,
rearrangements, translocations) represent a major cause of mental
retardation and malformation syndromes (du Manoir et al., et al.,
Human Genetics 90(6): 590-610 (1993)) and possibly, oncogenesis.
See also, e.g., (Harrison's Principles of Internal Medicine, 12th
edition, ed. Wilson et al., McGraw Hill, N.Y., N.Y., pp. 24-46
(1991)), for a partial list of human genetic diseases that have
been mapped to specific chromosomes, and in particular, for a list
of X chromosome linked disorders. In view of the growing number of
genetic disorders associated with chromosomal aberrations, various
attempts have been reported in connection with developing simple,
accurate, automated assays for genetic abnormality assessment.
[0008] In general, karyotyping is used to diagnose genetic
abnormalities that are based upon additions, deletions,
amplifications, translocations and rearrangements of an
individual's nucleic acid. The "karyotype" refers to the number and
structure of the chromosomes of an individual. Typically, the
individual's karyotype is obtained by, for example, culturing the
individual's peripheral blood lymphocytes until active cell
proliferation occurs, preparing single, proliferating (e.g.
metaphase, and possibly interphase) cells for chromosome
visualization, fixing the cells to a solid support and subjecting
the fixed cells to in situ hybridization to specifically visualize
discrete portions of the individual's chromosomes.
[0009] The sample contains at least one target nucleic acid, the
distribution of which is indicative of the genetic abnormality. By
"distribution", it is meant the presence, absence, relative amount
and/or relative location in one or more nucleic acids (e.g.,
chromosomes) known to include the target nucleic acid. In a
particularly preferred embodiment, the target nucleic acid is
indicative of a trisomy 21 and thus, the method is useful for
diagnosing Down syndrome. In a particularly preferred embodiment,
the sample intended for Down syndrome analysis is derived from
maternal peripheral blood. More particularly, lymphocytes are
isolated from peripheral blood according to standard procedures,
the cells are attached to a solid support (e.g., by centrifuging
onto glass slides), and fixed thereto according to standard
procedures (see, e.g., the Examples) to permit detection of the
target nucleic acid.
[0010] Nucleic acid hybridization techniques are based upon the
ability of a single stranded oligonucleotide probe to base-pair,
i.e., hybridize, with a complementary nucleic acid strand.
Fluorescence in situ hybridization ("FISH") techniques, in which
the nucleic acid probes are labeled with a fluorophore (i.e., a
fluorescent tag or label that fluoresces when excited with light of
a particular wavelength), represents a powerful tool for the
analysis of numerical, as well as structural aberrations
chromosomal aberrations. The method involves contacting a fixed
cell with an antibody labeled with a first fluorophore for
phenotyping the cell via histochemical staining, followed by
contacting the fixed cell with a DNA probe labeled with a second
fluorophore for genotyping the cell. The first and second
fluorophores fluoresce at different wavelengths from one another,
thereby allowing the phenotypic and genetic analysis on the
identical fixed sample.
[0011] Fluorescence in situ hybridization refers to a nucleic acid
hybridization technique which employs a fluorophore-labeled probe
to specifically hybridize to and thereby, facilitate visualization
of, a target nucleic acid. Such methods are well known to those of
ordinary skill in the art and are disclosed, for example, in U.S.
Pat. No. 5,225,326; U.S. patent application Ser. No. 07/668,751;
PCT WO 94/02646, the entire contents of which are incorporated
herein by reference. In general, in situ hybridization is useful
for determining the distribution of a nucleic acid in a nucleic
acid-containing sample such as is contained in, for example,
tissues at the single cell level. Such techniques have been used
for karyotyping applications, as well as for detecting the
presence, absence and/or arrangement of specific genes contained in
a cell. However, for karyotyping, the cells in the sample typically
are allowed to proliferate until metaphase (or interphase) to
obtain a "metaphase-spread" prior to attaching the cells to a solid
support for performance of the in situ hybridization reaction.
[0012] Briefly, fluorescence in situ hybridization involves fixing
the sample to a solid support and preserving the structural
integrity of the components contained therein by contacting the
sample with a medium containing at least a precipitating agent
and/or a cross-linking agent. Exemplary agents useful for "fixing"
the sample are well known to those of ordinary skill in the art and
are described, for example, in the above-noted patents and/or
patent publications.
[0013] One fluorescent dye used in fluorescence microscopy is DAPI
or 4',6-diamidino-2-phenylindole [CAS number: [28718-90-3], a
fluorescent stain that binds strongly to DNA. Since DAPI will pass
through an intact cell membrane, it may be used to stain live and
fixed cells. DAPI is excited with ultraviolet light. When bound to
double-stranded DNA its absorption maximum may be about 358 nm and
its emission maximum may be about 461 nm. DAPI will also bind to
RNA, though it is not as strongly fluorescent. Its emission shifts
to about 400 nm when bound to RNA. DAPI's blue emission is
convenient for microscopists who wish to use multiple fluorescent
stains in a single sample. There is very little fluorescence
overlap, for example, between DAPI and green-fluorescent molecules
like fluorescein and green fluorescent protein (GFP), or
red-fluorescent stains like Texas Red. Other fluorescent dyes are
used to detect other biological structures.
[0014] Other types of fluorescing materials are used in
fluorescence in situ hybridization (FISH). The FISH method uses
fluorescent tags to detect chromosomal structure. Such tags may
directed to specific chromosomes and specific chromosome regions.
Such technique may be used for identifying chromosomal
abnormalities and gene mapping. For example, a FISH probe to
chromosome 21 permits one to identify cells with trisomy 21, i.e.,
cells with an extra chromosome 21, the cause of Down syndrome. FISH
kits comprising multicolor DNA probes are commercially available.
For example, AneuVysion.RTM. Multicolor DNA Probe Kit sold by the
Vysis division of Abbott Laboratories, is designed for in vitro
diagnostic testing for abnormalities of chromosomes 13, 18, 21, X
and Y in amniotic fluid samples via fluorescence in situ
hybridization (FISH) in metaphase cells and interphase nuclei. The
AneuVysion.RTM. Assay (CEP 18, X, Y-alpha satellite, LSI 13 and 21)
Multi-color Probe Panel uses CEP 18/X/Y probe to detect alpha
satellite sequences in the centromere regions of chromosomes 18, X
and Y and LSI 13/21 probe to detect the 13q14 region and the
21q22.13 to 21q22.2 region. The AneuVysion kit is useful for
identifying and enumerating chromosomes 13, 18, 21, X and Y via
fluorescence in situ hybridization in metaphase cells and
interphase nuclei obtained from amniotic fluid in subjects with
presumed high risk pregnancies. The combination of colors emitted
by the tags is used to determine whether there is a normal
chromosome numbers or trisomy.
[0015] In a similar vein, the UroVysion.RTM. kit by the Vysis
division of Abbott Laboratories designed to detect chromosomal
abnormalities associated with the development and progression of
bladder cancer by detecting aneuploidy for chromosomes 3, 7, 17,
and loss of the 9p21 locus via fluorescence in situ hybridization
in urine specimens from persons with hematuria suspected of having
bladder cancer. The UroVysion Kit consists of a four-color,
four-probe mixture of DNA probe homologous to specific regions on
chromosomes 3, 7, 9, and 17. The UroVysion probe mixture consists
of Chromosome Enumeration Probe (CEP) CEP 3 SpectrumRed, CEP 7
SpectrumGreen, CEP 17 SpectrtimAqua and Locus Specific Identifier
(LSI 9p21) SpectrumGold.
[0016] Despite the above-described advances in the development of
fluorescent in situ hybridization methods for the diagnosis of
genetic abnormalities, the analysis of the fluorophore-labeled
sample remains labor-intensive and involves a significant level of
subjectivity. This is particularly true in connection with the
prenatal diagnosis of genetic abnormalities in which fetal cells
must either be isolated from maternal cells or visually
distinguished therefrom prior to assessment for genetic
abnormalities. Thus, for example, a laboratory technician must
manually prepare and sequentially stain the sample (first, with a
histochemical stain to phenotype the cells, second, with a
hybridization probe to genotype the cell); visually select fetal
cells from other cells in the optical field (using, for example,
the above-mentioned histochemical staining procedure); assess the
relative distribution of fluorescent color that is attributable to
hybridization of the fluorophore-tagged probe; and compare the
visually-perceived distribution to that observed in control samples
containing a normal human chromosome complement. As will be readily
apparent, the above-described procedure is quite time-consuming.
Moreover, because the results are visually-perceived, the frequency
of erroneous results can vary from one experiment to the next, as
well as from one observer to the next.
[0017] The invention disclosed in co-owned U.S. Pat. No. 6,221,607,
"Automated fluorescence in situ hybridization detection of genetic
abnormalities," discloses computer-implemented methods for
determining a genetic abnormality such as trisomy 21 which
eliminate subjective analysis of selectively stained chromosomes.
More specifically, the patent provides a method for detecting
whether a genetic abnormality is present in a fixed sample
containing at least one target nucleic acid. The method is useful
for diagnosing genetic abnormalities associated with an aberration
in chromosomal number and/or arrangement, such as, for example,
chromosomal additions, deletions, amplifications, translocations
and rearrangements.
SUMMARY OF THE INVENTION
[0018] Embodiments are disclosed which perform various image
processing functions which may be employed to implement the
automated fluorescence in situ hybridization method. The
embodiments include an auto-exposure method for acceptably imaging
all regions of the sample over an intensity range exceeding the
dynamic range of the digital electronics; a method for fluorescence
in situ hybridization (FISH) object of interest enumeration which
locates targets within the sample; nuclei identification which is a
method for classifying and characterizing the objects-of-interest
enumerated; segmenting nuclei which, is a method for defining the
shape of an identified object of interest. Embodiments of the
method are useful to characterize cell nuclei or to enumerate a
chromosome. An embodiment of the method is adapted for conducting
an AneuVysion.TM. assay (Vysis, Inc., Downers Grove, Ill.).
[0019] In an embodiment, there is disclosed:
[0020] An image processing method for analyzing a fluorescence in
situ hybridization image of a fluorescently-hybridized specimen
using a microscope system having a fluorescence exciting light
source and an electronic imaging device, the method comprising the
steps of:
[0021] illuminating the specimen with fluorescence exciting
illumination;
[0022] adjusting exposure parameters of the electronic imaging
device for capturing an image of the specimen at a depth of focus
within an illuminated field;
[0023] enumerating objects of interest in a captured image of the
specimen;
[0024] identifying a nucleus in the image;
[0025] segmenting a nucleus in the image;
[0026] counting and characterizing a fluorescent signal occurring
within the nucleus; and
[0027] interpreting and reporting results.
BRIEF DESCRIPTION OF THE FIGURES
[0028] FIG. 1 is a flow chart representing an overview of an
embodiment of a computer program for carrying out the automated
analysis of the invention.
[0029] FIG. 2 is a flow chart representing an embodiment of a
program module for adjusting exposure parameters.
[0030] FIG. 3 is a flow chart representing an embodiment of a
program module for enumerating objects of interest.
[0031] FIG. 4 is a flow chart representing an embodiment of a
program module for identifying nuclei.
[0032] FIG. 5 is a flow chart representing an embodiment of a
program module for segmenting nuclei.
[0033] FIG. 6 is a flow chart representing an embodiment of a
program module for characterizing signals and reporting
results.
DETAILED DESCRIPTION OF THE INVENTION
[0034] Auto-Exposure: The acquisition of the digital image
typically requires proper exposure of all of the regions of the
specimen being examined. The electronic imaging device may be a
multi-pixel planar array of light-sensitive detectors in a charge
coupled device (CCD), or complementary metal oxide semiconductor
(CMOS) elements, or any other technology suitable for converting an
optical image into electrical signals. Exemplary CCD cameras
include intensified CCD cameras utilizing gating techniques to
achieve gate speeds of less than about nine nanoseconds with
improved quantum efficiency (e.g., such as Princeton Instruments
(Trenton, N.J.) PI MAX.sub.MG which support a full range of 16-bit
scientific-grade CCDs), allowing for a very fast response limited
by the time constant of the output phosphor, and electron-bombarded
CCD (EBCCD) wherein photons are detected by a photocathode and
released electrons are accelerated across a gap and impact on the
back side of a CCD (allowing for additional gain and accompanying
speed). CMOS cameras in particular may find use in fluorescence
microscopy. CMOS cameras have an amplifier and digitizer associated
with each photodiode in an integrated on-chip format. Recent CMOS
sensors have a greatly reduced residual noise, and provide an
extraordinary dynamic range.
[0035] Low intensity detection can be enhanced through the
employment of an image intensifier and similar technologies. The
correct exposure time may be calculated using an algorithm which
takes into account conditions such as (i) the range of mean image
intensity value within certain areas (nuclei), (ii) the range of
highest image intensity, (iii) the maximum allowed exposure time,
and (iv) independently provided exposure conditions.
[0036] Each sensor picture element or pixel typically accumulates
an electrical charge which is in proportion to the number of
photons (amount of light) falling on the element. As there is
usually an inverse linear relationship between required exposure
time and intensity of the light impinging on the pixel, dimmer
areas may be imaged in separate exposures by increasing exposure
time. After exposure, the pixels of the imaging arrays can be
individually measured in a sequential scanning pattern. Each of the
element measurements can then be digitized by means of an analog to
digital converter (A/D) or other means of digitizing the data. The
resulting stream of digitized measurements can be stored in a
memory upon which image analysis procedures may be performed.
[0037] In most cases, however, a single exposure time does not
adequately image all portions of the specimen since the dynamic
range of the image sensor and associated electronics is typically
less than the range of intensities emanating from various locations
of a single specimen. For example, the dynamic range of an 8 bit
D/A converter is 256:1, which may be inadequate for this
application. For that situation, with a single exposure time,
either bright objects or nuclei will be lost if the dimmest objects
are properly exposed or, alternatively, dim objects will be lost
when the brightest objects are properly exposed.
[0038] When the intensity dynamic range of the imaging field is too
large for a single exposure duration, a multiple exposure strategy
can be employed. The portions of the specimen or nuclei with the
highest intensities are exposed first using a relatively shorter
exposure time, and analyzed. For that exposure, the dimmer areas of
the image may be next analyzed using edge detection or entropy
measurement to determine whether a longer exposure time is
necessary to adequately image those areas. If so, an additional
exposure can be made for an appropriately longer exposure time,
with the pixel measurements corresponding to the high intensity
nuclei excluded or masked from the image. The process may be
repeated, for longer exposure times, until no more structures of
interest are found. As an alternative to a longer exposure time,
multiple shorter exposures may be computationally combined to
synthesize a single longer exposure. Alternatively, the exposure
may be varied by changing the effective microscope aperture or
irradiating intensity.
[0039] "Dot" Enumeration: The specimen has finite thickness with
objects of interest dispersed throughout the depth of the sample.
As used herein, an "object of interest" relates to any feature in a
microscope field that has been identified as a result of labeling
with a FISH probe. Nonlimiting examples of an object of interest
include the plasma membrane or portion thereof, a cytoplasmic
organelle or structure, a ribosome, a mitochondrion or portion
thereof, a mitochondrial nucleic acid, a Golgi membrane,
endoplasmic reticulum or portion thereof, an endosome, a nucleus, a
nucleolus, a nuclear membrane or portion thereof, a chromosome or
portion thereof, and a portion of a DNA molecule. Imaging,
therefore, requires that the optical system be individually focused
to form well resolved images of each of the objects. Alternatively,
a series of exposures may be taken at spaced focal planes selected
at sufficiently small intervals so that all objects of interest are
acceptably focused. The required number and separation of the focal
planes can readily be determined from the thickness of the specimen
and the depth-of-field of the optical system.
[0040] Once the properly focused exposures have been obtained, the
images can be processed to identify and separate the objects of
interest or fluorescence in situ hybridization "dots." These "dots"
are revealed by the fluorescent light that they emit, and the
properties of the emitted light vary in accordance with the
characteristics of the object. In particular, nonlimiting examples
of fluorescent properties emanating from an object of interest
include optical properties of the fluorescent label, and the
intensity of hybridization of the FISH probe to the object.
[0041] Having obtained the images, an enumeration algorithm may be
employed to enumerate the fluorescence in situ hybridization
objects of interest ("dots"). The first step of the algorithm can
be segmentation of the 4',6-diamidino-2-phenylindole (DAPI, a
double-stranded DNA staining fluorescent probe) stained image, in
accordance with intensity, effectively defining intensity contours
of brightness across each of the image focal planes. The raw
fluorescence in situ hybridization channel images may be
computationally converted into contrast images. Contrast images are
a mathematical transformation of the original image where the
intensity of each transformed pixel represents the change in
intensity relative to the adjoining pixels in the original image.
Objects within the nuclei may be resolved by successively lowering
the contrast threshold from the highest possible value to a preset
low value. For each object, the highest contrast can be compared to
the moving average and standard deviation of the previous objects.
If a significant jump in contrast is detected, all the objects with
higher contrast can be marked as potential fluorescence in situ
hybridization "dots." In addition, if two potential fluorescence in
situ hybridization "dots" are positioned closer than a preset
threshold value, they may be merged to form a single "dot." The
relative contrasts and sizes of the identified potential
fluorescence in situ hybridization "dots" can be compared, and the
final fluorescence in situ hybridization "dots" can be
characterized and logged in a data base.
[0042] Nuclei Identification: Once the potential objects of
interest or fluorescence in situ hybridization "dots" are
identified, automatic pattern recognition techniques may be
employed to classify and characterize each of the objects. Each of
the fluorescence in situ hybridization "dot" sites determined by
the fluorescence in situ hybridization "dot" enumeration analysis
can be analyzed to develop an elliptic Fourier shape descriptor of
the object that is invariant to translation, rotation and scaling.
Other characterizations, including but not limited to object size
and emitted intensity distribution within the nucleus, may also be
employed to describe the object. A pattern recognition algorithm
can be employed to identify and categorize the object of interest
based on these characterizations. Initially, the pattern
recognition algorithm may be trained by employing an expert human
observer to classify the object and input his result into the
pattern recognition data base. After the initial learning period,
the algorithm can be performed automatically and the pattern
recognition data base continually updated.
[0043] Segmenting Nuclei: For each nucleus identified by the
pattern recognition algorithm, contours of constant intensity
starting at maximum brightness can be determined. At each point on
the respective contour, the gradient may also be computed. The size
of the nucleus can be determined, by way of nonlimiting example, as
the contour corresponding to the greatest average gradient.
[0044] AneuVysion.TM. Scanning Method: The AneuVysion.TM. assay is
an FDA cleared test for prenatal diagnosis which allows for rapid
detection of the most common abnormalities of chromosome number
using fluorescence in situ hybridization. It utilizes molecular
genetic techniques to create a fluorescent DNA probe that produces
a bright microscopic signal when it selectively attaches to one
specific part of a particular chromosome. The DNA probes are able
to attach to the appropriate chromosomes in non-dividing cells. The
signals are different colors for different chromosomes. By counting
the number of signals within a cell, the cytogenetic technologist
knows whether either the normal number or a trisomy, monosomy or
other aneusomy of the detectable chromosomes is present in the
fetus.
[0045] The disclosed embodiments can be employed to effectively
automate the AneuVysion.TM. assay utilizing the following method. A
computational histogram may be organized where each bin in the
histogram represents each possible combination of chromosomal
constituency. The AneuVysion.TM. CEP (Chromosome Enumeration Probe)
analysis results utilize a histogram structure including three
coefficients representing the X chromosome, the Y chromosome and
chromosome 18. A second histogram structure corresponding to the
LSI probe includes two coefficients representing chromosome 12 and
chromosome 13. The automated fluorescence microscope system finds a
specified number (N) of nuclei using low magnification. In
nonlimiting examples, the value of N is specified to be an integer
such as 10, or 20, or 30, or 40, or 50, or 60, or 80, or 100, or
125, or 150, or 200, or even more. Furthermore, N may be specified
to be an integer anywhere between the values identified herein. The
system then individually images each nucleus using high
magnification. The fluorescence in situ hybridization light points
are counted for all channels and the results are organized into the
above described histogram formulation. The measurement process
continues until one bin count reaches a predetermined number (M).
For example, the number M may be 50, corresponding to current
government guidelines. More generally, in nonlimiting examples, the
value of M is predetermined to be an integer such as 10, or 20, or
30, or 40, or 50, or 60, or 80, or 100, or 125, or 150, or 200, or
even more. Furthermore, M may be predetermined to be an integer
anywhere between the values identified herein. If, when the N
nuclei have been measured, no bin contains the required M quantity,
the system may search for additional nuclei at low magnification
and continue the measurement at high magnification until one bin
reaches the quantity N. When N is reached, the measurement stops
and the highest bin count may be compared with the next highest bin
count. If the highest bin count is some predetermined percentage
greater than the next greatest, then the result corresponding to
that bin can be reported as the clinical result. If it is does not
satisfy the condition, then an inconclusive result can be reported.
In various nonlimiting examples, the predetermined percentage for
comparing the highest bin count with the next highest bin count may
be 5%, or 10%, or 15%, or 20%, or 25%, or 30%, or 35%, or 40%, or
45%, or 50%, or 60%, or 70%, or 80%, or 90%, or 100%, or 125%, or
150%, or 175%, or 200%, or even a higher percent value.
Furthermore, the predetermined percentage may be set at any
integral or nonintegral value between those identified herein.
[0046] An embodiment of the image processing method for analyzing
fluorescence in situ hybridization images of the present invention
is schematically portrayed in FIG. 1. A microscope slide containing
a specimen which has been suitably treated to hybridize it in situ
to a fluorescent probe is placed 400 in the field of view of a
microscope. In a nonlimiting embodiment the basic elements of a
microscope that may be used in the present method include an X-Y
stage, a mercury or equivalent light source suitable to excite
fluorescence of the labels, a fluorescence microscope, a color
detecting CCD image detecting device, a computer, and one or more
monitors. The individual elements of the system may be custom-built
or purchased off-the-shelf as standard components. Nonlimiting
examples of specimens include cells, blood cells, epithelial cells,
tissues, disrupted tissues, tissue slices, biopsy samples, excised
surgical samples, and the like.
[0047] The microscope is adjusted 410 to bring the specimen into
focus at a focal plane of choice within the depth of the specimen.
The specimen is irradiated 420 with fluorescence exciting
illumination causing various loci of the sample having a labeled
probe bound to fluoresce. The exposure parameters of the electronic
imaging device are adjusted to properly expose these areas 430. As
is well known in the art, the exposure parameters may be varied by
changing exposure time, and/or aperture, and/or illumination level.
An image is captured 440.
[0048] In many cases, the intensity range in a field under scrutiny
and provided in a captured image (FIG. 1, 440) will exceed the
dynamic range capabilities of the electronic imaging device and/or
its supporting electronics. For those situations, referring to FIG.
2, the exposure is adjusted using a sequence of images. The
brightest spots are first identified 500 and the exposure
parameters are set 510 so that the brightest spots are properly
exposed, corresponding to the top end of the electronic imaging
device's dynamic range. An image is captured 520 under these
conditions. The less bright portions of the captured image are
analyzed 530 to determine if detail was lost due to underexposure.
Underexposure of these areas may not detect dimmer fluorescent
spots. If there is a lack of detail in these dimmer regions 540,
the exposure parameters are increased 550 to extend the electronic
imaging device's dynamic range into the region below the initial
dynamic range. The range of the electronic imaging device's
supporting electronics is adjusted to correspond to the lower
intensity levels to be imaged; the output of the image sensor's
pixels corresponding to the initial brightest areas are masked or
bypassed 560 so as not to overload the supporting electronics. A
new image is captured 520 and similarly analyzed 530. This process
is repeated until the entire image has been acceptably imaged 570.
The objects-of-interest are next enumerated from the set of
captured images (FIG. 1, 450).
[0049] Referring to FIG. 3, a schematic presentation of an
enumeration algorithm, for a given level in the depth of the field
of scrutiny the captured images are low pass filtered 600. The
images may be mathematically re-expressed as contrast images 610 by
dividing each of the original images by the corresponding low pass
filtered version. Objects-of-interest and attributes may be
identified by creating contours of constant contrast. Contours of
successively lower constant contrast may be resolved 620 by
sequentially lowering the contrast threshold from the highest
observed value to a selected low value. For each object, the
highest contrast may be compared 630 to the moving average and the
standard deviation of the previous objects. If a significant jump
in contrast is detected 640, all the objects with higher contrast
can be marked as potential objects of interest ("dots"). A "dot" is
considered an outlier 650 if it has significantly lower contrast or
lesser size and is excluded from further consideration. In
addition, if two potential fluorescence in situ hybridization
"dots" are positioned closer than a preset threshold value, they
may be merged 660 to form a single "dot." The relative contrasts
and sizes of the identified potential fluorescence in situ
hybridization "dots" can be compared, 670 and the final
fluorescence in situ hybridization "dots" can be characterized and
logged in a data base 680. The process is repeated (FIG. 1, 455)
for additional planes of focus required to perform the analysis
throughout the depth of the sample.
[0050] Having enumerated the objects-of-interest, the nuclei are
then identified (FIG. 1, 460). As schematically shown in FIG. 4, a
properly focused and exposed DAPI image is acquired at low
magnification 700. Contours of constant intensity are
mathematically generated to segment the image, thereby identifying
the objects 710. For each of the objects enumerated 450, a shape
characterization such as an elliptic Fourier shape descriptor that
is invariant to translation, rotation and scaling is computed 720.
The granulometry, or size distribution, of the constituent features
of each object is computed 730. The combination of all of the
variables used to classify the object and characterizations is
employed to fully describe the nuclei. A pattern recognition
algorithm, in conjunction with an experience based pattern data
base, is used to identify and categorize the object-of-interest
740.
[0051] The nuclei thus identified are next segmented (FIG. 1, 470)
as schematically shown in FIG. 5. A contour of constant intensity,
corresponding to the highest intensity, is first computed 800. The
characteristics of the objects thereby identified are recorded 810.
The threshold intensity level is successively reduced and new
contours are computed 820. As the intensity threshold is lowered,
the contour associated with each of the objects will expand 830,
and new objects may become visible 840. In some cases, objects
which are separated at higher intensity levels may merge as the
threshold is reduced 850. The computation of additional contours is
terminated once the intensity threshold level has reached a
sufficiently low level 860. A threshold level determination
generally uses instrumental or system parameters that are
particular to the microscope system being used. Thus establishing a
threshold is a procedure particular to the installation. By way of
nonlimiting example, a microscope system may provide intensity
measurements as photon counts, or current, or charge accumulation.
A worker of skill in the field of the invention understands and can
implement evaluations of threshold levels that distinguish from
overall background, on the one hand, and significant contour
intensity levels on the other. At that point, objects which do not
conform to the valid size range may be eliminated from further
consideration 870. The contours of constant brightness which have
already been computed are used to determine the edges of each of
the objects 880. At each point along each contour, the intensity
gradient is computed and averaged. The boundary of each nucleus is
defined as the contour having the greatest average gradient. The
objects are thenceforth defined only within their edges 890.
[0052] Having thus defined and segmented the nuclei (FIG. 1, 470),
the fluorescent light signals within each nucleus are counted and
characterized (FIG. 1, 480), then interpreted and the results
reported (FIG. 1, 490). An embodiment of this final step is shown
in FIG. 6 and is suitable for use, for example, with an
AneuVysion.TM. assay. As previously described, the AneuVysion.TM.
assay is a test for prenatal diagnosis. The disclosed embodiment
described in this application is equally valid for other
fluorescence in situ hybridization assays. A mathematical histogram
structure is created 900 so that each bin corresponds to a possible
combination of fluorescent light spot quantities and colors
contained in a nucleus. Each of the first group of N fluorescent
light spots is counted 910, its color is characterized, and the
results are added to the appropriate bin of the histogram. After
the first N spots have been counted, the maximum count in any of
the histogram bins is determined 920. If that maximum is less than
N 930, additional nuclei are located 940. Nonlimiting examples for
values of N have been disclosed above. If no new nuclei can be
located 945, an inconclusive result is reported. The number M is
predetermined and may correspond to regulatory requirements. A
exemplary value for M is 50. Nonlimiting examples for values of M
have been disclosed above. The associated additional light spots
are counted and characterized 950, and the results are added to the
existing bin contents. This process continues until the maximum bin
count is greater than or equal to N 930. When the maximum number in
any bin has reached M, the number in the next greatest histogram
bin is determined 970. If M exceeds the next greatest number by a
predetermined Z percent, the chromosomal constituency is reported
out 980 as a valid test result. Nonlimiting examples for values of
a predetermined percent have been disclosed above. If, on the other
hand, M does not exceed the next greatest number by Z percent, the
test is reported out as inconclusive 960.
STATEMENT REGARDING PREFERRED EMBODIMENTS
[0053] While the invention has been described with respect to
preferred embodiments, those skilled in the art will readily
appreciate that various changes and/or modifications can be made to
the invention without departing from the spirit or scope of the
invention as defined by the appended claims. All documents cited
herein are incorporated by reference herein where appropriate for
teachings of additional or alternative details, features and/or
technical background.
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