U.S. patent application number 12/855299 was filed with the patent office on 2011-04-07 for methods for detecting fluorescent signals in a biological sample.
This patent application is currently assigned to Ikonisys, Inc.. Invention is credited to Yash AGARWAL, Aaron ARMSTRONG, Robert BORGERDING, llia ICHETOVKIN, Michael KILPATRICK, Young Min KIM, Andrew MACGINITIE, Antti SEPPO, Triantafyllos TAFAS, Petros TSIPOURAS, Xiuzhong WANG, Yanning ZHU.
Application Number | 20110079640 12/855299 |
Document ID | / |
Family ID | 39033596 |
Filed Date | 2011-04-07 |
United States Patent
Application |
20110079640 |
Kind Code |
A1 |
KIM; Young Min ; et
al. |
April 7, 2011 |
METHODS FOR DETECTING FLUORESCENT SIGNALS IN A BIOLOGICAL
SAMPLE
Abstract
A method for automated microscopic analysis wherein the test
protocol is obtained from interrogatable data affixed to each
microscope slide. The method further comprises the algorithms that
implement the test protocol.
Inventors: |
KIM; Young Min;
(Wallingford, CT) ; ZHU; Yanning; (Hamden, CT)
; AGARWAL; Yash; (New Haven, CT) ; WANG;
Xiuzhong; (Hamden, CT) ; ARMSTRONG; Aaron;
(Norwalk, CT) ; BORGERDING; Robert; (New Haven,
CT) ; MACGINITIE; Andrew; (Roxbury, CT) ;
SEPPO; Antti; (New York, NY) ; ICHETOVKIN; llia;
(Costa Mesa, CA) ; KILPATRICK; Michael; (West
Hartford, CT) ; TSIPOURAS; Petros; (Madison, CT)
; TAFAS; Triantafyllos; (Rocky Hill, CT) |
Assignee: |
Ikonisys, Inc.
New Haven
CT
|
Family ID: |
39033596 |
Appl. No.: |
12/855299 |
Filed: |
August 12, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11833849 |
Aug 3, 2007 |
7829868 |
|
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12855299 |
|
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60821557 |
Aug 4, 2006 |
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Current U.S.
Class: |
235/375 |
Current CPC
Class: |
G01J 3/2803 20130101;
G01N 2021/6421 20130101; G01N 21/6428 20130101; G01J 3/4406
20130101; G01N 2021/6441 20130101; G01N 2201/12 20130101; G01N
2021/6419 20130101; G01N 21/6458 20130101; G01N 2021/6482 20130101;
G01N 35/00732 20130101; G01N 2021/6439 20130101; G01N 2201/06113
20130101; G01N 2035/00851 20130101 |
Class at
Publication: |
235/375 |
International
Class: |
G06F 17/00 20060101
G06F017/00; G02B 21/34 20060101 G02B021/34 |
Claims
1-24. (canceled)
25. A method performed by an apparatus, the method comprising:
reading electronically-interrogatable data from each slide of one
or more slides, wherein said each slide comprises a sample and said
electronically-interrogatable data pertaining to said sample;
determining from said electronically-interrogatable data for said
each slide of the one or more slides information on how said sample
is to be analyzed; and analyzing said sample situated on said each
slide of said one or more slides using said information determined
from said electronically-interrogatable data.
26. The method of claim 25, wherein said
electronically-interrogatable data is encoded on said each slide of
one or more slides as a barcode or a barred array.
27. The method of claim 25, wherein sample is a biological
sample.
28. The method of claim 25, wherein said apparatus is a microscope
or an automated microscope.
29. The method of claim 25, wherein said analyzing comprises
scanning said slide according to an analytical protocol comprised
in said information.
30. The method of claim 25, wherein said determining comprises
obtaining an analytical protocol for said analyzing from a database
using said information.
31. The method of claim 25, wherein said analyzing comprises
obtaining an image of the sample or a plurality of images at
different magnifications of said sample or of a portion of said
sample.
32. The method of claim 25, wherein said one or more slides
comprise a plurality of slides and said plurality of slides is
contained in one or more slide cassettes.
33. The method of claim 32, wherein before said reading, the method
comprising: sequentially transporting each cassette of said one or
more slide cassettes into a position suitable for loading
corresponding one or more slides of said plurality of the slides
comprised in said each slide cassette to a corresponding stage of
said apparatus; and for said each cassette of the sequentially
transported slide cassettes, sequentially transporting said one or
more slides of said plurality of the slides comprised in said each
slide cassette to said corresponding stage of said apparatus for
said reading, determining and analyzing.
34. A computer readable medium encoded with a computer program
comprising computer readable instructions recorded thereon for
execution a method comprising: reading
electronically-interrogatable data from each slide of one or more
slides, wherein said each slide comprises a sample and said
electronically-interrogatable data pertaining to said sample;
determining from said electronically-interrogatable data for said
each slide of the one or more slides information on how said sample
is to be analyzed; and analyzing said sample situated on said each
slide of said one or more slides using said information determined
from said electronically-interrogatable data.
35. The computer readable medium of claim 34, wherein said
analyzing comprises scanning said slide according to an analytical
protocol comprised in said information.
36. The computer readable medium of claim 34, wherein said
determining comprises obtaining an analytical protocol for said
analyzing from a database using said information.
37. The computer readable medium of claim 34, wherein said
analyzing comprises obtaining an image of the sample or a plurality
of images at different magnifications of said sample or of a
portion of said sample.
38. An apparatus, comprising: a reading device, configured to read
electronically-interrogatable data from each slide of one or more
slides, wherein said each slide comprises a sample and
electronically-interrogatable data pertaining to said sample; a
controller, configured to receive and determine from said
electronically-interrogatable data for said each slide of the one
or more slides information on how said sample is to be analyzed,
and to provide instructions for performing a corresponding analysis
based on said information; and an analyzing module, responsive to
said instructions from said controller, configured to analyze said
sample situated on said each slide of said one or more slides using
said instructions from said controller.
39. The apparatus of claim 38, further comprising: a stage
configured to hold said each slide of the one or more slides in
order to read electronically-interrogatable data and to analyze
said sample using said instructions, and wherein said corresponding
analysis comprises scanning said slide according to an analytical
protocol comprised in said information, and said analyzing module
comprises a scanning device configured to provide said
scanning.
40. The apparatus of claim 38, wherein said apparatus comprises a
database, such that said controller is configured to determine said
information on how said sample is to be analyzed from said database
where said information is stored.
41. The apparatus of claim 38, further comprises one or more
objective lens components, such that said said apparatus comprises
a focusing module configured to focus each of said one or more
objective lens components on said sample or on a focal plane
comprised within the sample while analyzing said sample according
to said instructions.
42. The apparatus of claim 41, wherein said one or more objective
lens components comprises two or more objective lens components,
such that said apparatus is configured to obtain a plurality of
images at different magnifications of said sample or of a portion
of the sample using said two or more objective lens components.
43. The apparatus of claim 38, wherein said apparatus comprises an
image capture device configured to capture an image of said sample
or a portion of said sample while analyzing said sample according
to said instructions.
44. The apparatus of claim 38, wherein said apparatus is a part of
a system which comprises other one or more components selected from
the following components: a stage configured to hold said each
slide of the one or more slides in order to read
electronically-interrogatable data and to analyze said sample using
said instructions; a database, such that said controller is
configured to determine said information on how said sample is to
be analyzed from said database where said information is stored;
one or more objective lens components and a focusing module
configured to focus each of said one or more objective lens
components on said sample or on a focal plane comprised within the
sample while analyzing said sample according to said instructions;
and an image capture device configured to capture an image of said
sample or a portion of said sample while analyzing said sample
according to said instructions.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of the
Non-provisional patent application Ser. No. 11/833,849 filed Aug.
3, 2007 (Publication Number US-2009-0250629), which claims priority
to the U.S. Provisional Patent Application No. 60/821,557, filed
Aug. 4, 2006, which is incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0002] 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. Incorporation by reference to
non-patent publications is only for nonessential material.
[0003] 1. Field of the Invention
[0004] The present invention generally relates to the automated
microscopic detection of biological structures using fluorescent
tags directed to such biological structures.
[0005] 2. Description of the Related Art
[0006] Conventional optical microscopy generally employs a
microscope slide to which a biological sample has been affixed, and
a single objective lens that is used to focus on discrete areas of
the biological sample in a search for structures of interest, such
as cells, nuclei, etc. Dimensions of the image seen through the
objective lens depend on the magnification and numerical aperture
of the objective lens. The specimen on the microscope slide is
manually moved with respect to the objective lens resulting in a
plurality of fields of view. Structures of interest seen through
the objective in each field of view are analyzed with image details
recorded. Images may be stored by means of acquisition by a camera.
The multiple field of view are used to characterize the sample as a
whole. Of course, such process may be slow for any application that
requires a complete view of the specimen.
[0007] Numerous factors must be dealt with in microscopy, including
resolution, contrast, depth of focus, working distance,
magnification, parfocality, and parcentricity. Resolution is the
ability to distinguish in an image two points as two points.
Resolution is important to determine differentiate features in a
sample. Resolution may decrease with magnification, and is
typically related to the numerical aperture of the objective.
Contrast is also necessary in the evaluation of an image. Contrast
is the difference between the brightest point in an image and the
darkest point in the image, or the relative intensity of the zero
order versus the diffracted orders. Without sufficient contrast an
image may appear "flat" at best, or invisible at worst. Contrast is
conventionally controlled in a manual microscope by way of a
condenser diaphragm. Depth of focus refers to the depth of the
image in focus. Depth of focus changes as the numerical aperture of
the objective changes, and the working distance of the objective
changes (as the working distance of the objective is increased the
depth of focus increases). The depth of focus is important in that
objects within the specimen that are outside the depth of focus are
not detected. Working distance refers to the distance from the
front of the objective to the specimen plane. When objectives are
changed working distance (particularly when the objective has a
different numerical aperture) may change as well as focus. It is
generally important to keep the working distance sufficient so as
not to have the objective interfered by the specimen proper.
Parfocality, that is the specimen staying in focus when the
objective is changed, and parcentricity, that is, an object in the
center of the field staying in the center of the field no matter
which objective is being used, are also generally desirable.
[0008] Many methods are known to aid in the microscopic analysis of
samples. For example, without limitation, it is known that certain
dyes have an affinity for certain cellular structures. Such dyes
may therefore be used to aid in analysis by helping to further
elucidate such structures.
[0009] Fluorescence microscopy of cells and tissues is well known
in the art. Treating cells with fluorescent reagents and imaging
the cells is well known in the art. Methods have been developed to
image fluorescent cells in a microscope and extract information
about the spatial distribution and temporal changes occurring in
these cells. Some of these methods and their applications are
described in an article by Taylor, et al. in American Scientist 80
(1992), p. 322-335. These methods have been designed and optimized
for the preparation of a few specimens for high spatial and
temporal resolution imaging measurements of distribution, amount
and biochemical environment of the fluorescent reporter molecules
in the cells. Detection of fluorescent signals may be by way of an
epifluorescent microscope which uses emitted fluorescent light to
form an image (whereas a conventional reflecting microscope uses
scattered illumination light to form an image). The excitation
light of a epifluorescence microscope is used to excite a
fluorescent tag in the sample causing the fluorescent tag to emit
fluorescent light. The advantage of an epifluorescence microscope
is that the sample may be prepared such that the fluorescent
molecules are preferentially attached to the biological structures
of interest thereby allowing identification of such biological
structures of interest.
[0010] One fluroescent dye used in flouresence microscopy is DAPI
or 4',6-diamidino-2-phenylindole [CAS number: [28718-90-3]; SMILES
structure: NC(C2=CC1=C(C=C2) C=C(C3=CC=C(C(N)=N)C=C3)N1)=N], 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.
[0011] 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.
[0012] In a similar vein, the UroVysion 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 sequences 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 SpectrumAqua and Locus Specific Identifier
(LSI 9p21) SpectrumGold.
[0013] To overcome the laborious process of manual microscopy, a
number of researchers, including the present inventors, have
proposed automated microscopy systems for capturing and analyzing
multiple image views of a biological sample on a microscope slide
or other sample retaining device (such as a multiple well plate).
Such systems have the potential to greatly improving the efficiency
of microscopic analysis and to remove some of the subjective inputs
that affect microscopic analysis of a sample.
[0014] A number of difficulties are associated with automated
microscopy. For example, many of the functions performed in manual
microscopy are dictated by undefined methodologies under the
control of the human eye and brain. Each of these functions needs
to be addressed to allow for the slide to be reviewed with the
required clarity. Further, much of the analysis undertaken in
traditional manual microscopy involves human reasoning based upon a
prior experiences. For example, microscopists are often able to
discern an artifact or mistreated sample portion from an actual
biological structure, yet have difficult expressing the basis for
such decision when asked to set forth the same in words. Further
automated microscopy entails the automated device having the
ability to handle the slide, interpret the biological structure
which is to be investigated and the protocol by which
interpretation is to be performed, adjust the slide with respect to
the objective, search numerous areas on the slide for such
biological structure, determine areas on the slide in which
structures of interest reside, process desired signals from
structure from extraneous signals, interpret signals, etc.
[0015] The present inventors have recognized these and related
needs in implementing automated microscopy of a plurality of
samples, such as may be used in high throughput microscopic
analysis, and addressed these needs herein.
SUMMARY OF INVENTION
[0016] In embodiments there is included:
[0017] First, a method of microscopic analysis comprising [0018]
(a) providing an automated microscope comprising a slide stage, at
least one objective lens, image capturing means, programmable means
for operating the microscope according to a protocol, and
programmable means for providing an analytical outcome; [0019] (b)
providing a microscope slide containing a sample and interrogatable
data thereon, wherein the interrogatable data provide information
related to a protocol for analysis of said sample; [0020] (c)
interrogating the data; [0021] (d) positioning the slide on the
slide stage; [0022] (e) causing the microscope to analyze the
sample in accordance with the analytical protocol encoded in the
interrogatable data; and [0023] (f) causing the microscope to
provide an analytical outcome representing the sample.
[0024] Second, a method for high throughput microscopic analysis
comprising [0025] (a) providing an automated microscope comprising
a slide stage, at least one objective lens, at least one slide
cassette containing at least one microscope slide therein,
programmable means for operating the microscope according to a
protocol, and programmable means for providing an analytical
outcome; [0026] (b) providing a plurality of microscope slides each
containing a sample and interrogatable data thereon, wherein the
plurality of slides is contained in one or more of said slide
cassettes, wherein the interrogatable data provide information
related to a protocol for analysis of said sample; [0027] (c)
transporting a first cassette into a position suitable for
transporting a slide to said microscope stage; [0028] (d)
transporting a first slide from the first cassette to said
microscope stage; [0029] (e) interrogating the data found on said
first slide; [0030] (f) positioning said first slide on the slide
stage; [0031] (g) causing the microscope to analyze the sample on
said first slide in accordance with the analytical protocol encoded
in the interrogatable data; [0032] (h) causing the microscope to
provide an analytical outcome representing the sample on said first
slide; [0033] (i) if there remains another slide to be analyzed in
said first cassette repeating steps (d) to (h); and [0034] (j) if
there remains another cassette repeating steps (c) to (i).
[0035] Third, a computer-readable storage medium tangibly embodying
a program of instructions executable by a computer for a method of
microscopic analysis using an automated microscope comprising a
slide stage, at least one objective lens, image capturing means,
programmable means for operating the microscope according to a
protocol, and programmable means for providing an analytical
outcome;
wherein the program comprises [0036] a) a set of instructions for
interrogating data on a microscope slide wherein the interrogatable
data provide information related to a protocol for analysis of a
sample included on said slide; [0037] b) a set of instructions for
positioning the slide on the slide stage; [0038] c) an analyzing
set of instructions for causing the microscope to analyze the
sample in accordance with the analytical protocol encoded in the
interrogatable data; and [0039] d) a set of instructions for
causing the microscope to provide an analytical outcome
representing the sample.
[0040] Fourth, a computer-readable storage medium tangibly
embodying a program of instructions executable by a computer for a
method of high throughput microscopic analysis wherein the method
uses an automated microscope comprising a slide stage, at least one
objective lens, at least one slide cassette containing at least one
microscope slide therein, programmable means for operating the
microscope according to a protocol, and programmable means for
providing an analytical outcome;
wherein the program comprises [0041] a) a set of instructions for
transporting a first cassette into a position suitable for
transporting a slide to said microscope stage; [0042] b) a set of
instructions for transporting a first slide from the first cassette
to said microscope stage; [0043] c) a set of instructions for
interrogating data on a microscope slide wherein the interrogatable
data provide information related to a protocol for analysis of a
sample included on said slide; [0044] d) a set of instructions for
positioning the slide on a slide stage; [0045] e) an analyzing set
of instructions for causing the microscope to analyze the sample in
accordance with the analytical protocol encoded in the
interrogatable data; [0046] f) a set of instructions for causing
the microscope to provide an analytical outcome representing the
sample; [0047] g) a set of instructions for determining whether
there remains another slide to be analyzed in said first cassette
and if so repeating the instructions in (b) to (f); and [0048] h) a
set of instructions for determining whether there remains another
cassette and if so repeating instructions in (a) to (g).
[0049] Fifth, a method comprising obtaining a slide containing
electronically interrogtable data recorded therewith and having a
biological sample thereon; [0050] reading said
electronically-interrogatbele data from said slide; [0051]
determining from said electronically-interrogatbale data how said
biological sample is to be scanned by an automated microscope;
[0052] scanning with a automated microscope said slide in the
manner dictated by the electronically interogatable data recorded
therewith; and [0053] determining from said scans a testoutcome
indicative of a state of said biological sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0054] FIG. 1 provides a flow chart giving an overview of steps in
an embodiment of the invention.
[0055] FIG. 2 provides a flow chart giving details of steps in an
embodiment of the invention.
[0056] FIG. 3 provides a flow chart giving details of steps in an
embodiment of the invention.
[0057] FIG. 4 provides a flow chart giving details of steps in an
embodiment of the invention.
[0058] FIG. 5 provides a flow chart giving details of steps in an
embodiment of the invention.
[0059] FIG. 6 provides a flow chart giving details of steps in an
embodiment of the invention.
[0060] FIG. 7 provides a flow chart giving details of steps in an
embodiment of the invention.
[0061] FIG. 8 provides a flow chart giving details of steps in an
embodiment of the invention.
[0062] FIG. 9 provides a flow chart giving details of steps in an
embodiment of the invention.
[0063] FIG. 10 provides a flow chart giving details of steps in an
embodiment of the invention.
[0064] FIG. 11 provides a flow chart giving details of steps in an
embodiment of the invention.
[0065] FIG. 12 provides a flow chart giving details of steps in an
embodiment of the invention.
[0066] FIG. 13 provides a flow chart giving details of steps in an
embodiment of the invention.
[0067] FIG. 14 is a schematic representation of a scanned area
using a spiral scanning technique, according to an embodiment
related to FIG. 8.
DETAILED DESCRIPTION OF THE INVENTION
[0068] Turning to FIG. 1, there is disclosed a master diagrammatic
flow chart of an embodiment of the present invention. FIG. 1
presents an overview of the various computational modules that
together implement the automatic retrieval and analysis of samples
on multiple slides. Such a collection of slides may arise in a
research setting or in a diagnostic setting. Large numbers of
slides are advantageously examined and analyzed by the automated
methods disclosed herein. Biological specimens, cellular or tissue
preparations, and similar subjects of investigation constitute
nonlimiting examples of subjects for microscopic analysis by
methods of the invention. These are generally termed "samples" or
"specimens" herein. Commonly the samples include labels to assist
in microscopic analysis. Frequently such labels are fluorescent
labels. A sample may furthermore include more than one fluorescent
labels, wherein each label has particular and distinguishable
fluorescent properties, esp. distinguishable excitation and
emission wavelengths. In order to conduct suitable microscopic
analysis of such samples, appropriate excitation filters are placed
in the light beam illuminating the sample, or one of a plurality of
laser sources of differing wavelengths is chosen, and corresponding
emission filters are placed between the sample and an image capture
device such as a camera or charge coupled detector (CCD). In a
procedure governing automated microscopic analysis of such samples,
a computer or similar controlling device must have available
information describing the nature of the probes to be examined.
Sample identification including this requisite information, as well
as additional sample identifiers, may be encoded on each slide
using an interrogatable coding means, such as a barcode or barred
array. The interrogatable coding is read as a slide is positioned
in the microscope, and the corresponding information is
communicated to the computer or controlling device.
[0069] As seen in FIG. 1, the analysis for a particular slide, once
loaded in place onto the stage of a microscope (15), begins by
reading a barcode present on the slide (20). The barcode include
information designating the nature of the microscopic analysis to
be carried out. The details for the diverse analytical protocols
are stored in a database for reference by the computer or
controlling device. Once the slide barcode is read, the correct
experimental protocol is identified in a database (DB) according to
the information encoded in the barcode (25). With this information
now available to control the operation of the microscope, a
concatenated series of operations that regulate the focusing,
optimize the region on the slide to be scanned to provide a
suitable image, including adjustments for low magnification to
start with, and moving to a higher magnification for the actual
analysis, is carried out (see steps 30, 35, 40, 45, and 50). A
successful implementation of the various modules involved in this
protocol provides results, designated a "Testoutcome" in FIG. 1
(55). The remaining loops illustrated in FIG. 1 relate to
determining whether, in a given cassette, the last slide in the
cassette has been examined (65 and 85); and whether slides in the
last cassette have been analyzed (70 and 80). When the last
cassette has been examined, the operation of the microscope ceases
(75).
[0070] As indicated at FIG. 1, the databridge application is
started (step 5) to run as a system service for file handling in
parallel with other process that may be running Such service may be
a method such as shown at FIG. 13, wherein the service is started
(step 300) which might include setting parameters and the
environment in which the application will run. In the method of
FIG. 13, a configuration file is read (step 310) such as may be
provided by IKoDataBridge.exe.config (step 305). If preconditions
are not met an error is recorded in a file, such as an application
event log (step 320) and the process shut down (step 325). If
preconditions are meet (step 315) such as the existence of source
folders, a loop is performed (step 335) until a shut down is
requested. Starting the loop a log file is queried for a list of
files (step 340), for example ".txt" files. If files are found
(step 345) another loop is started (step 350) wherein a further
check is performed for a corresponding file, such as a ".nvc" type
file. Existence of the corresponding file would then lead to a read
of success counts within such a ".nvc" file and cause a skip of
entries in the original file (step 360). After reading of the entry
from the original file, for example the ".txt" file (step 365) a
query is performed as to whether the complete marker is found (step
370), whereupon the text file would be removed (step 375).
Interrogation of more files is made (step 380), resulting in a
return and continuation of the loop initiated for each file found,
such as a ".txt" file (step 350). If more files are not found (step
380) the system, as illustrated by the alternative path (step 385,
385'), is put to sleep based on the time specified, for example in
a configuration file such as ".config" (step 330). Completion of
the sleep period (step 330) results in return and continuation of
the shutdown loop starting (step 335). Failure of finding the
complete marker in step 370 will trigger a specific command in step
425 to execute. If the execution is successful (step 405) the
reading of an entry from, for example, a ".txt file" is resumed as
seen in step 365. Non success at step 405 in executing the command
of step 425 records an entry into a log file, such as an
application event log (step 410), query of the error type and count
(step 415) and possible increment of a retry count at step 430,
returning to the execution step of 425. A sufficient error or retry
count of commands, as tested at step 415 may result in a
notification to a scanner application as in step 420 and return to
step 350 for continue to loop for another file, such as ".txt"
file. In the event a corresponding file, such as a ".nvc" file does
not exist (step 355), a file will be created containing a zero
(step 400), where after the process will occur as performed above
continuing from step 365. The absence of found files at step 345
would cause a retrieval of a file list from a folder, for example a
databaselog folder (step 390), and query of the list in step 395
for files. If no files are found the service would be placed in
sleep mode as shown in step 330, or if files were found the process
would return to the file loop at step 350.
[0071] Turning back to FIG. 1, slides having bar coded or other
electronically-readable indicia are loaded into a cassette (step
10) having multiple slots from which such slides may be obtained. A
slide for analysis is then loaded (step 15) into an automated
microscope. The barcode or other electronically-readable indicia is
read (step 20) to determine the type of processing demanded (e.g.,
type of application demanded) on the slide by reference to a
database (step 25). The automated microscope then seeks to execute
a number of steps to detect objects of interest in the sample based
on the processing demand.
[0072] First the sample is focused with respect to the objective.
Focusing may be transacted by using a known reference point, such
as the slide edge (step 30) from which focus may be effectuated.
Such focusing may be a method such as shown at FIG. 7 wherein depth
of focus in the z range is redefined if certain parameters raise a
flag of out-of-focus situation (step 11) or not (step 19
termination). In the method described at FIG. 7, the slide is
exposed to an interrogation for a period of time, for example 100
msec (step 12), with the binning mode being set to cover a
substantial area, for example set to 4.times.4 (step 13). The
interrogation spot is then set to a reference point on the slide
edge, such as the top middle slide edge (step 14). Autofocus is
then performed to determine a Zbase (step 16), that is, a base
point along the Z axis, such as at the top surface of the slide
edge. From the Zbase a z-focus upper limit is defined (step 17),
such as 25 times the depth of focus from the Zbase, and a z-focus
lower limit is defined (step 18)
[0073] Returning to FIG. 1, after focusing, the scan area is
determined (step 35) based upon a predetermined algorithm. For
example, FIG. 2 shows two different schemes for scan area
definition based upon two different FISH-based tests, AneuVysion
(22) and UroVysion (23) based on bar coded or other
electronically-readable indicia on the slides (step 21). Such tests
differ in the manner of applying the sample, with the AneuVysion
sample being placed in smear on the slide, and the sample applied
to a UroVysion Slide a dropped blob.
[0074] As illustrated at FIG. 2, if an AneuVysion test (22) is
indicated, the scanned area is defined at step 24 as being the
entire scannable area on the slide to determine the position of a
smear on the slide. As illustrated, low magnification field visits
("survey visits") are made for rapid detection of possible
candidates according to a sequence along the vertical axis of the
slide (step 26), for example, in a pattern as set forth at 27.
Query of isolated possible candidates may then be performed by high
magnification ("investigation mode").
[0075] As further shown in FIG. 2, with respect to UroVysion slide
28 investigation of possible candidate may employ numerous steps.
At step 29, a filter is set to selectively determine fluorescent
signals from a label such as DAPI interacting with the sample.
Exposure value is set to a predefined value at step 31, and the
binning mode (merging of distinct pixels) of the camera set to a
predefined level, such as 4.times.4 (step 32), to allow for
expeditious scanning of the slide. The Z-motor is then positioned
to allow for fixed z-position reading of locations on the slide,
for example, set to the middle of the entire z-movement range (step
33). Read is made of pre-recorded positions on the Urovyision Slide
28, for example, as illustrated 2, 8, 11, and 5 of the registry
(step 34). Interrogation is made of pre-programmed location field
on slide 28, such location field for example, encompassing
positions 1, 2 and 3 (36), with imaging being made of the DAPI
signals at such pre-programmed filed and a mean pixel value at each
position being determined at step 36). At step 37 the position with
the largest mean pixel value (upper bound) is selected for each
pre-programmed location field, as reiterated at steps 38/39, 41/42
and 43/44. Using the positions identified as having the largest
mean pixel value, an enclosed boundary is defined (step 46). Within
such defined enclosed boundary there is then assigned a low
magnification yield visit sequence starting form the center of the
defined boundary (for example, circle) with the sequence number
increasing as one spirals out (step 47).
[0076] Turning back to FIG. 1, a low magnification scan is then
performed at step 40. Such low magnification scan may entail
discrete steps as set forth at FIG. 3. At step 49 magnification is
set to a low value, for example, to an objective lens having
10.times. magnification. Quality control measures, such as
Objective repeatability, or other forms of quality checks may then
be determined at step 51, using methodology, for example, as set
forth at FIG. 5.
[0077] Objective repeatability may be determined using the
embodiment methodology as shown at FIG. 5. First, binning mode is
set for each magnification level (for example, 10.times. or
100.times. as set forth at 139) which will be used to scan the scan
area. For example, binning mode may be set to 2.times.2 (141) or
alternatively 4.times.4 (142) as shown in FIG. 5. With the
objective set to the appropriate magnification, e.g., 10.times. as
set forth at 143, the interrogation is sent to a predefined
position that has been determined to include some features of
potential interest 144. Autofocus and autoexposure are performed
(step 146) with one image grabbed and at least one feature is
identified as, for example, by determining a gradient, such as an
optical gradient (step 147). If a feature is not determined at step
148 the low magnetic field is lowered more and autofocus and
autoexposure of step 146 is repeated. If a feature is determined at
step 148 the magnification is verified at step 149, features of
interest are centered applying a pre-defined parfocality offset
(step 152) and the objective magnification changed, as for example,
to 100.times. as at step 153. Again, autofocus and autoexposure are
performed (step 154) and a gradient used to find the feature of
interest (step 155). A template may then be generated around the
feature isolated for correlation matching (step 157). The objective
is then changed once more to the original objective and position,
the image is grabbed and the offset determined from the previous
image based on correlation (step 159). If the offset is acceptable
(step 161) and offset is acceptable multiple consecutive times,
such as, three times (step 162) the objective repeatability test is
terminated (step 164). If acceptability does not reach offset
acceptability in a consecutive predetermined maximum number of
attempts (step 163) then there is change of the objective back to
the original position (step 158). If a feature is not found at 148,
then there may be a move down of one low magnification field (151)
and the path continued at step 146.
[0078] Turning back to FIG. 3, after objective repeatability is
confirmed at step 51, an image processing thread is created (step
52). As a simultaneous process, the image processing thread is
first initialized (step 73), and images saved (step 76) after
waiting for image processing jobs in the queue (step 74). The
images are then processed and in accord with an algorithm candidate
nuclei are selected and x-y positions of each candidate nuclei
target are determined (step 77). From the x-y positions determined,
the interrogation strategy is set based on the high magnification
to be used, so as to maximize the number of nuclei per field and
minimize the total number of high magnification fields necessary to
visit such nuclei candidates (step 78). A determination is made
upon receipt of images whether the thread should be terminated
(step 79), if not image processing continues (step 74), and if
termination is determined (step 81), then based on the test
screening protocol, for example, as illustrated, AneuVysion or
UroVysion (step 83), the fields are sorted in a manner to provide
required information. For example, with respect to an AneuVysion
test (step 82), the list of high magnification fields may be sorted
based on a number of nuclei in the filed (step 86), and with
respect to a UroVysion test (step 84), the list of high
magnification fields may be sorted on largest nucleus size in the
field (step 87), followed by termination (step 88).
[0079] Now turning to step 53 of FIG. 3, after creating the image
processing thread (step 52) as discussed above, the system is set
for acquiring images. First parameters necessary for imaging are
checked, for example, disk space and activating source (e.g.,
lamp). The sample is then visited with a low magnification field
search in the pre-determined visit sequence order (step 54). In
conjunction, filters may be effectuated, for example a DAPI filter
for determining nuclear tags, and the binning mode adjusted for
appropriate resolution (step 56). The low magnification objective
lens is then adjusted for focus (step 57), for example, by a
methodology such as described at FIG. 10.
[0080] In FIG. 10, there is shown a method for adjusting low
magnification focus. First there is a determination of whether the
low magnification field is the first low magnification field in the
sequence order (step 232). If the low magnification field is the
first low magnification field in the sequence order at step 236 the
z-range at the low magnification field is recalculated by
interpolation using database(s) incorporating z-focus range found
from the "find focus on slide edge" (233) and z-difference from the
top edge to bottom edge (234) if possible if not (step 237) there
is termination (step 186). If the low magnification field is not
the first low magnification field in the sequence order, then the
neighborhood of potential structures of interest is set to a
defined number (step 239) and each neighborhood is inquired in low
magnification (step 241) to determine if there is one or more
neighborhoods with a valid z focus value (step 244), and if so, the
average of all the z focus values is taken (step 247), and if not,
the number or size of neighborhoods are expanded (step 243) until
there are no more neighbors to expand (243), and a flag (237) is
sent to complete (186) the string.
[0081] Returning back to FIG. 3, at step 58 autofocus and
autoexposure are performed The binning mode may then be changed
(step 59), for example, to 1.times.1 as illustrated, an image, for
example a DAPI image (step 71), acquired. Depending on the test
used to elucidate objects of interest, such as, for example, an
Aneuvyision test (72), one may need to alter other microscopic
parameters to elucidate such objects. For example, there may be
need to alter filtering (step 61) of emanating signals from the
sample, and change the exposure value of the sample (step 62). Once
an image is acquired (step 63) it may be processed using the
processing thread discussed supra (step 64) and once all candidates
are located (step 66), and each of the fields interrogated (step
67), the imaging process thread is terminated (step 81).
[0082] Depending upon the test protocol used (e.g., AneuVysion or
UroVysion 82, 83, 84), the processed images are handled in a
predetermined manner, for example, with respect to an AneuVysion
test by sorting the list of high magnification fields based on the
number of nuclei in a field (step 86) and with respect to a
UroVysion test, sorting the list of high magnification fields on
the basis of the largest nucleus size in the field (step 87). If
all candidates are not located (step 66), and each of the fields is
not interrogated (step 67), and the scan area may be redefined
(steps 68, 69).
[0083] Redefinition of the scanner area may be by the methodology
of FIG. 8 wherein a central point is selected from which spiral
scanning techniques such as in the order set forth in FIG. 14 are
performed. Such spiral scanning may be defined by the equation of
step 181. In such methodology, at step 179, obtain the number of
nuclei, Ny, in each field scanned along the vertical central line.
At step 182, calculate the y-coordinate of the center, Cy, using
weighted average. Subsequently at step 183, calculate the
x-coordinate, Cx, where the vertical central axis of the slide
lies. Then at step 184, define the scanning area centered around
(Cx, Cy) with its diameter about the width of the slide. Finally at
step 185, before termination (step 187), assign scanning sequence
number for each low mag field inside the circle. Sequence number
starts from the center of the area and increases as it spirals out.
It should skip the area which was scanned already.
[0084] Once the low magnification scan area is defined (step 35 of
FIG. 1) and the sample is scanned at low magnification (step 40 of
FIG. 1), a scan at high magnification may be performed (step 45 of
FIG. 1).
[0085] High magnification scanning may employ a methodology such as
portrayed at FIG. 4. The objective is set to high magnification,
and camera gain set to highest gain (step 89). The imaging
processing thread for high magnification is then created (step 91)
by first initialization (step 129), waiting for image processing
jobs in the queue (step 131), saving the image (step 132),
processing image stacks (step 133) (such as DAPI and FISH images),
updating the high magnification field probability map (step 134),
classifying the targets of interest (step 136), e.g., nuclei, and
finally ending the thread if appropriate (steps 137/124) and
continuing at 126. The updating of the high magnification field
probability map of step 134 may be by a method as set forth in the
flow chart set forth at FIG. 12.
[0086] As shown, at step 300, there is provided input as to the
probability that a object (such as a DAPI object) has other objects
of interest associated (such as FISH objects) and input pertaining
to the number of objects for each high magnification field. Next
there is calculation of the expected value of the number of signals
of interest having other objects of interest associated therewith
(step 305) such as DAPI objects having Fish Signals, in each high
magnification field. The high magnification fields are then sorted
(step 310) according to the number of useful objects, such as DAPI
objects. (step 310), the high magnification fields with the largest
number of useful objects, such as DAPI objects, are scanned and the
probability of useful objects, such as DAPI objects, for the low
magnification fields are adjusted (step 315). The expected valve of
the number of objects having a desired signal (e.g. DAPI objects
having FISH signals) in each of the high magnification fields are
calculated at step 320.
[0087] For example, the high magnification field probability map
with respect to DAPI objects having FISH signals may be determined.
DAPI objects for high magnification scanning may be sorted based on
the number of objects contained in the high magnification field in
order to reduce the number of fields to be scanned to find enough
useful DAPI objects within the least amount of time. DAPI objects
having good FISH signals (i.e. objects containing the most number
of useful DAPI objects) may be further sorted to reduce the time
necessary of high magnification analysis. Assuming the probability
for a high magnification field being properly processed to have
FISH objects to be p=m/n, every time a DAPI object is found to
contain FISH objects, the probability can be addressed to be
p=(m+1)/(n+1). Every time a DAPI object is found to contain FISH
objects, adjust the probability to be p=m/(n+1). The expected value
of the number of useful objects in each high magnification field is
then the multiplication of the number of DAPI objects and the
probability. The high magnification field with the largest expected
value of the number of objects may be chosen to be scanned. Note
that, the value of p can be obtained statistically by experiments
on typical slides. With a fixed p, the value of m (or n) needs to
be carefully chosen so that each object, no matter it has FISH
signals or not, can have a proper impact factor on the probability
adjustment.
[0088] The pseudo code of an algorithm for a DAPI/FISH system that
may be used is set forth below: [0089] 1. Let the initial lowmag
field quality indicator be p.sub.i=m.sub.i/n.sub.i=p=m/n. [0090] 2.
Calculate the expected value of the number of objects in each himag
field and sort them. [0091] 3. Choose the himag field with the
largest expected number of objects. [0092] 4. If the expected
number of objects is less than N.sub.min, stop. [0093] 5. Scan and
analyze the himag field chosen. [0094] 6. For each object in the
himag field, decide if it contains FISH signals. Let
n.sub.i=n.sub.i+1. If the object contains FISH signals, then
m.sub.i=m.sub.i+1. [0095] 7. If enough useful DAPI objects have
been found, stop. [0096] 8. Calculate the new field quality
indicator p.sub.i=m.sub.i/n.sub.i. [0097] 9. Update the expected
value of the number of objects based on the field quality indicator
in the remaining himag fields within the current lowmag field.
[0098] 10. Sort the remaining himag fields and go to 3.
[0099] By choosing appropriate values from m and n, one can achieve
a large variety of scanning strategies. For high magnification
scanning application, it may be desired that the algorithm be able
to abandon the field where there are objects without FISH signals.
To do so, one may choose small values for m and n (for example,
m=1, n=2; or if one wants to abandon fields faster, m=0.5, n=1).
The N.sub.min may be chosen, for example, to=0.2.about.0.3.
[0100] In respect of the classification of nuclei at step 136,
classification may be directed by the particular testing protocol
being employed, such as, for example, AneuVysion/UroVysion (209,
211, 212) of FIG. 11. For example, when nuclei on a AneuVysion test
slide are being counted, a simple determination of whether the dot
count in any of the FISH channels does not contain a countable flag
(step 213) may be used to determine whether the proposed nuclei dot
should be counted (216) or not counted (214). Similarly, when
nuclei on an UroVysion test slide are being counted, channel count
may be used in respect to classification of the nuclei. For
example, if two or more channels in a plurality of channels, for
example three channels, have more than two dots (217), then an
abnormal classification (223) may be given, or the first three
channels have two dots and the last (e.g. gold) channels has zero
dots (219), a classification of abnormal (226) may be given, while
if the first three channels have two dots and the last (e.g., gold)
channel has two dots (221), then a classification of normal (227)
may ensue. If only one channel in the first of the plurality of
channels has more than two dots (218) then the classification may
be singlegain (224), while if at least two channels in the first
three channels has more than one dot and zero dot in gold (222),
then a classification of zerogold (228) or unclassified (229) may
be rendered. Upon classification of each nuclei the classification
process may be terminated (231).
[0101] A scan at high magnification (step 45 of FIG. 1) employing
the methodology as set forth at FIG. 4, after creation of the image
processing thread (step 91) may transact an object repeatability
test (92), for example, as discussed with respect to FIG. 5 supra.
Again parameters of the microscope such as disk space and lamp
(step 93) may be performed and the stop condition checked (94).
[0102] Stop condition checking (94) may depend on the particular
testing protocol being employed, for example, AneuVysion or
UroVysion (166, 167, 168; see FIG. 6).
[0103] If AneuVysion (167), for example, a determination may be
made if the total scanning area has been scanned (169) and if it is
so having the stop condition being set (173) and the process
terminated (174). On the other hand, if a determination is made
that the total scanning area has not been made (169), then the
total nuclei collected at high magnification may be compared to a
threshold, such as equal to or greater than 500 (171). If this
threshold has been met, the stop condition may be determined to be
met (173). If the threshold has not been found to be met, and the
highest nuclei number in all the cell categories is determined to
be above a predetermined minimum threshold (such as equal to or
greater than 50) (172), the stop condition may also be determined
to have been met (173). If it is below the predetermined minimum
threshold, the stop condition may be determined not to have been
met (176).
[0104] If UroVysion is the particular protocol employed (168), a
determination may be made if the total scanning area has been
scanned (177), and if so the stop condition being met, and if not
another parameter being sued to meet the stop condition (173). For
example, one might make as a condition of a stop condition being
met (173) that the total nuclei collected at high magnification be
equal to or greater than the value the user specified (178) (if not
the stop condition is not met 176).
[0105] Turning back to FIG. 4, the type of test performed on the
sample (for example, AneuVysion (step 96)) may influence the step
of high magnification scanning (step 45 of FIG. 1). For example, if
AneuVysion is the test (step 96) one might choose the high
magnification field with the next highest expected number of nuclei
(step 138) for scanning, while if such test was not employed, the
next high magnification field in the list (step 97) might be
scanned. It may be necessary in the process to periodically adjust
parameters of the microscope, for example, resenting the lamp timer
at every 50th high magnification field (step 98). Before taking an
image it is advantageous to confirm that the image processing queue
is available (step 99). Appropriate filters (step 102) may need to
be set, the shutter set to on (step 103) and the high magnification
field entered (step 101). The exposure time to an appropriate
interrogation wavelength may then be estimated with a setting of a
binning mode (step 104). After adjusting autoexposure and autofocus
(step 106), an image, such as a DAPI image, may be taken at the
focus position and the exposure values found (step 107).
Parcentricity should be confirmed by determining parcentricity
offset (step 108) and if the offset is too much (step 109) the
objective turned between low and high magnification (step 127), the
check process repeated, or if there is a determination that the
last high magnification field has been reached (step 123) the image
processing thread terminated (step 124). If the offset is not too
much, then other mask may be employed, such as a DAPI mask and the
parcentricity offset updated (step 111). After requiring a stack of
images, for example nine slices, the best focused plane may be
determined (step 112), further filters set (step 113), such as a
filter for detecting FISH signals, and exposure time recalculated
and binning mode set (step 114). Autoexposure on the best focused
plane may be effected (step 116) followed by resetting of the
binning mode to a new value and applying exposure (step 117) to
obtain a stack of images of the signals to which the filter has
been set (step 118), for example FISH signals, until the desired
number of filters to produce the stack has been completed (step
119). The shutter of the image obtaining device may then be set to
off (step 121), the images obtained sent to the image processing
thread (step 122) with the image processing thread being terminated
(step 124) after determining the last high magnification field has
been queried (step 123). Finishing of the high magnification scan
(step 126) upon a stop condition check (step 50 of FIG. 1)--such as
described above with respect to FIG. 6, may prompt the automated
microscope to generate a testoutcome (step 55 of FIG. 1).
[0106] A exemplary automated method for determining a testoutcome
(step 55 of FIG. 1) with respect to a Aneuvyision or UroVysion test
(188, 189, 191) is set forth at FIG. 9. As depicted with respect to
a AneuVysion test (189) each fluorescent taggant (CEP v. LSI) (192)
is analyzed with respect to binding with the target chromosomal
regions for such taggants. For example, with respect to CEP (193)
the X, Y and 18 dotcounts are determined (step 196), and with
respect to LSI (194) the dotcounts with respect to chromosomes 13
and 21 are obtained (step 197). The dotcounts determined are then
matched (step 198) against a database of possible outcomes for CEP
labeling (201) or LSI labeling (202). If the dotcount obtained
matches a possible dotcount outcome for valid CEP labeling (201)
then the output matched is sent as the testoutcome. However if the
dotcount obtained does not match with a possible dotcount outcome
for valid CEP labeling (201), then there is a determination if the
reason for the failure of the match is due to the analysis of too
few nuclei (step 199), and if yes the testoutcome output is sent as
"less than 50 nuclei images" (206), and if no the testoutcome is
output as "review recommended" (204). Testoutcome is terminated at
208.
[0107] Turning back to FIG. 1, after generation of a testoutcome
(step 55), the slide having been interrogated is unloaded (step 60)
and a new slide from the cassette is loaded (step 85) if the slide
is not the last slide in the cassette (steps 65, 70). If it is the
last slide in the cassette (step 70) then the next cassette may be
loaded if such is available (step 80), or if not the run may be
terminated (step 75).
STATEMENT REGARDING PREFERRED EMBODIMENTS
[0108] 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.
* * * * *