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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6841—In situ hybridisation
• • C—CHEMISTRY; METALLURGY
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6816—Hybridisation assays characterised by the detection means

Definitions

• the present invention relates to nucleic acid chemistry, and more specifically to reagents and methods for accomplishing multiplex image analysis of chromosomes and chromosomal fragments.
• the invention may be used to diagnose chromosomal abnormalities, infectious agents, etc.
• chromosome identification has involved the use of labeled chromosome-specific oligonucleotide probes to label repetitive sequences of interphase chromosomes (Cremer, T. et al, Hum. Genet. 74:346-352 (1986); Cremer, T. et A/., Exper. Cell Res. 176:119-220 (1988)).
• Such methods have been shown to be useful in the prenatal diagnosis of Down's Syndrome, as well as in the detection of chromosomal abnormalities associated with tumor cell lines.
• Chromosome-specific probes of repetitive DNA that localize to discrete sub-regions of a chromosome are, however, unsuitable for analyses of many types of chromosomal abnormalities (e.g., translocations or deletions).
• Ward, D.C. et al. discloses a chromosomal in situ suppression ("CISS") hybridization method for specifically labeling selected mammalian chromosomes in a manner that permits the recognition of chromosomal aberrations.
• CISS chromosomal in situ suppression
• sample DNA is denatured and permitted to hybridize with a mixture of fluorescently labeled chromosome-specific probes having high genetic complexity and unlabeled non-specific competitor probes.
• Chromosomal images were obtained as described by Manuelidis, L. et al. (Chromosoma 96:397-410 (1988), herein incorporated by reference).
• the method provides a rapid and highly specific assessment of individual mammalian chromosomes.
• the method permits, by judicious selection of appropriate probes and/or labels, the visualization of sub- regions of some or all of the chromosomes in a preparation. For example, by using more than one probe, each specific for a sub-region of a target chromosome, the method permits the simultaneous analysis of several sub- regions on that chromosome.
• the number of available fluorophores limits the number of chromosomes or chromosomal sub-regions that can be simultaneously visualized.
• a "combinatorial" variation of the CISS method can be employed.
• two fluors permit three different chromosomes or chromosomal sub-regions to be simultaneously visualized.
• a hybridization probe mixture is made from a single set of probe sequences composed of two halves, each separately labeled with a different fluorophore.
• the two fluorophores Upon hybridization, the two fluorophores produce a third fluorescence signal that is optically distinguishable from the color of the individual fluorophores.
• Extension of this approach to Boolean combinations of n fluorophores permits the labeling of 2 W -1 chromosomes.
• the invention concerns reagents and methods for combinatorial labeling of nucleic acid probes sufficient to permit the visualization and simultaneous identification of all 22 autosomal human chromosomes and the human X and Y chromosomes, or defined sub-regions thereof. Such specific labeling of entire chromosomes or defined sub-regions thereof is referred to as "painting.”
• the invention provides a method of simultaneously identifying and distinguishing the individual autosomal and sex chromosomes of a human karyotype which comprises the steps:
• step (b) repeating step (b) until each autosomal and sex chromosome of the human karyotype has been identified in the preparation.
• the invention also concerns the embodiment of the above method, wherein, in step (b), an interferogram is produced from the interferometer, and the interferogram is Fourier transformed to recover the spectral signature of the predetermined label of the hybridized probe member, and more preferably, wherein the spectral signature of the predetermined label of the hybridized probe member in step (b) is recovered by comparing the interferogram to a library or lookup table of previously determined interferograms.
• the invention also concerns an interferometer (preferably a common path interferometer, and most preferably a Sagnac common path interferometer) that simultaneously identifies and distinguishes the individual autosomal and sex chromosomes of a human karyotype which comprises means for:
• the invention also provides such an interferometer, wherein, in step (b), an interferogram is produced from the interferometer, and the interferogram is Fourier transformed to recover the spectral signature of the predetermined label of the hybridized probe member, and more preferably, wherein the spectral signature of the predetermined label of the hybridized probe member in step (b) is recovered by comparing the interferogram to a library or lookup table of previously determined interferograms.
• FIGURES Figure 1 provides a schematic illustration of a CCD camera and microscope employed in accordance with the present methods.
• Figure 2 shows the raw data from a karyotypic analysis of chromosomes from a bone marrow patient (BM2486). Adjacent to each source image is a chromosome "mask" generated by the software program.
• panels A and B are the DAPI image and mask
• panels C and D are FITC image and mask
• panels E and F are Cy3 image and mask
• panels G and H are Cy3.5 image and mask
• panels I and J are Cy5 image and mask
• panels K and L are Cy7 image and mask.
• Figures 3A and 3B show the identification of individual chromosomes by spectral signature of patient BM2486.
• Figure 2 is the same photograph as Figure 3A, except that it is gray scale pseudocolored.
• Figure 3B displays the karyotypic array of the chromosomes.
• Fluorescence in situ hybridization is used in a variety of areas of research and clinical diagnostics (Gray, J.W. et al., Curr Opin Biotech 3:623-631 (1992); Xing, Y. et al., In: The Causes and Consequences of Chromosomal Aberrations. I.R. Kirsch Ed. CRC Press, Boca Raton, pages 3-28 (1993)).
• FISH Fluorescence in situ hybridization
• FISH FISH
• the present invention results, in part, from the realization of multiparametric fluorescence in situ hybridization to achieve the simultaneous visualization of 24 different genetic targets with a combinatorial labeling strategy.
• This strategy permits discrimination between many more target sequences than there are spectrally distinguishable labels.
• the simplest way to implement such labeling is using a simple "Boolean" combination, i.e., a fluor is either completely absent (i.e. the value of "0" will be assigned) or present in unit amount (value of 1).
• the invention concerns a set of combinatorially labeled oligonucleotide probes, each member thereof: (i) having a predetermined label distinguishable from the label of any other member of the set, and (ii) being capable of specifically hybridizing with one predetermined autosomal or sex chromosome of a human karyotype.
• the set will have a sufficient number of members to be capable of specifically and distinguishably hybridizing each autosomal or sex chromosome of said human karyotype to at least one member.
• the term "karyotype" denotes the compliment of chromosomes found in a normal or aberrant cell.
• the number of chromosomes is 46, comprising 22 pairs of autosomal chromosomes and 2 sex chromosomes (either 2 X chromosomes (if female) or an X and Y chromosome (if male)).
• the labels are said to be distinguishable in that the particular label of any one member of the set (and the identity of that member) differ from the particular label and identity of any other member of the set.
• each probe member is capable of specifically hybridizing to only one chromosome (or sub- chromosomal region) and since the identity of the label and probe are known in advance, the detection of a particular label associated with an unidentified chromosomal region means that the probe bearing that label has become hybridized to the unidentified chromosomal region. Since the chromosome to which that probe specifically hybridizes is known, the detection of a distinguishable label permits the identification of the chromosomal region.
• the invention concerns fluors that can be used to label oligonucleotide probes so that such probes may be used in multiparametric fluorescence in situ hybridization.
• a "fluor” or “fluorophore” is a reagent capable of emitting a detectable fluorescent signal upon excitation.
• the fluor is conjugated to a ligand capable of binding to a modified nucleotide residue.
• the most preferred ligands for this purpose are avidin, streptavidin, biotin-binding antibodies and digoxigenin-binding antibodies. Methods for performing such conjugation are described by Pinkel, D. et al, Proc. Nat'l Acad. Sci.
• the fluor may be coupled directly to the pyrimidine or purine ring of the nucleotides of the probe (Ried, T. et al. (Proc. Natl. Acad. Sci. (U.S.A.) 89:1388-1392 (1992), herein incorporated by reference; U.S. Patent Nos. 4,687,732; 4,711,955; 5,328,824; and 5,449,767, each herein incorporated by reference.
• multiparametric fluorescence denotes the combinatorial use of multiple fluors to simultaneously label the same chromosome or sub-chromosomal fragment, and their detection and characterization.
• Chromosomes or sub-chromosomal fragments are said to be simultaneously labeled if they are exposed to more than a single chromosome-specific probe under conditions sufficient to permit each chromosome-specific probe to independently hybridize to its target chromosome. As used herein, it is thus unnecessary for all such hybridization reactions to commence and conclude at the same instant.
• the simultaneous labeling permitted by the present invention is thus in contrast to protocols in which chromosomes are exposed to only a single chromosome-specific probe at a time.
• the simultaneous detection and characterization permitted by the present invention denotes an ability to detect multiple (and most preferably all) of the autosomal and/or sex chromosomes in a sample, without any need to add further reagent, or probe after the detection of the first chromosome.
• digital images of the chromosomes are obtained for each fluorophore employed, thereby providing a series of gray scale fluorescence intensities associated with each fluorophore and each chromosome.
• the final image is obtained by pseudocoloring the blended gray scale intensities for each chromosome.
• the invention thus provides a method of simultaneously identifying and distinguishing the individual autosomal and sex chromosomes of a human karyotype which comprises contacting a preparation of chromosomes, that has been previously treated to render it in single- stranded form, with the above-described set of combinatorially labeled oligonucleotide probes, under conditions sufficient to permit nucleic acid hybridization to occur.
• Such treatment causes at least one of each autosomal or sex chromosome of the preparation to become hybridized to at least one member of said set of probes.
• each chromosome of the preparation hybridized to a member of the set of probes one next detects and identifies the predetermined label of that member and correlates the identity of the label of that member with the identity of the autosomal or sex chromosome of said human karyotype with which that member specifically hybridizes. This process identifies the chromosome hybridized to the member. This last step is repeated until each or a desired number of autosomal and sex chromosome of the human karyotype has been identified in the preparation.
• oligonucleotide probes used in accordance with the methods of the present invention are of either of two general characteristics.
• such probes are chromosome or sub-chromosome specific (i.e., they hybridize to DNA of a particular chromosome at lower Cot 1 / 2 than with DNA of other chromosomes; C ⁇ t 1 / 2 being the time required for one half of an initial concentration (c 0 ) of probe to hybridize to its complement).
• probes are feature (e.g., telomere, centromere, etc.) specific. Both types of probes may be used if desired. Sources of such probes are available from the American Type Culture
• the oligonucleotide probes used in accordance with the methods of the present invention are of a size sufficient to permit probe penetration and to optimize reannealing hybridization.
• labeled DNA fragments smaller than 500 nucleotides in length, and more preferably of approximately 150-250 nucleotides in length, probes are employed.
• Probes of such length can be made by synthetic or semi-synthetic means, or can be obtained from longer polynucleotides using restriction endonucleases or other techniques suitable for fragmenting DNA molecules. Alternatively, longer probes (such as polynucleotides) may be employed.
• the oligonucleotide probes are synthesized so as to contain biotinylated or otherwise modified nucleotide residues. Methods for accomplishing such biotinylation or modification are described in U.S. Patent Nos. 4,687,732; 4,711,955; 5,328,824; and 5,449,767, each herein incorporated by reference. Biotinylated nucleotides and probes are obtainable from Enzo Biochem, Boehringer Mannheim, Amersham and other companies.
• biotinylated or otherwise modified nucleotides are produced by reacting a nucleoside or nucleotide with a mercuric salt under conditions sufficient to form a mercurated nucleoside or nucleotide derivative.
• the mercurated product is then reacted in the presence of a palladium catalyst with a moiety (e.g., a biotin group) having a reactive terminal group and comprising three or more carbon atoms.
• a palladium catalyst with a moiety having a reactive terminal group and comprising three or more carbon atoms.
• This reaction adds the moiety to the purine or pyrimidine ring of the nucleoside or nucleotide.
• such modified probes are used in conjunction with competitor DNA in the manner described by Ward et al.
• Competitor DNA is DNA that acts to suppress hybridization signals from ubiquitous repeated sequences present in human and other mammalian DNAs.
• alu or kpn fragments can be employed, as described by
• probe DNA bearing a detectable label and competitor DNA are combined under conditions sufficient to permit hybridization to occur between molecules having complementary sequences.
• two sequences are said to be able to hybridize to one another if they are complementary and are thus capable of forming a stable anti-parallel double-stranded nucleic acid structure.
• Conditions of nucleic acid hybridization suitable for forming such double stranded structures are described by Maniatis, T., et al. (In: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1982)), by Haymes, B.D., et al.
• sequences need not exhibit precise complementarity, but need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure. Thus, departures from complete complementarity are permissible, so long as such departures are not sufficient to completely preclude hybridization and formation of a double- stranded structure.
• chromosome 1 contains approximately 5.3 times as much DNA as is present in chromosome 21. Thus, a proportionally higher probe concentration would be employed when using chromosome 1 specific probes.
• the resulting hybridization mixture is then treated (e.g., by heating) to denature the DNA present and is incubated at approximately 37 °C for a time sufficient to promote partial reannealing.
• the sample containing chromosomal DNA to be identified is also heated to render it susceptible to being hybridized to the probe.
• the hybridization mixture and the sample are then combined, under conditions sufficient to permit hybridization to occur. Thereafter, the detection and analysis of the hybridized product is conducted by detecting the fluorophore label of the probe in any of the methods described below.
• probes are labeled with biotinylated nucleotides, and permitted to hybridize to chromosomal DNA. After hybridization, the hybridized complexes are incubated in the presence of streptavidin, that had been conjugated to one or more fluors. The streptavidin binds to the biotinylated probe of the hybridized complex thereby permitting detection of the complex, as described below.
• streptavidin binds to the biotinylated probe of the hybridized complex thereby permitting detection of the complex, as described below.
• One aspect of the present invention concerns the identification of a set of seven fluors that are be well resolvable by the excitation-emission contrast (EEC) method.
• EEC excitation-emission contrast
• multi-fluor combinatorial labeling depends in general on acquiring and analyzing the spectral signature of each object i.e., obtaining the relative weighting coefficients of the component fluors.
• Contrast ratio plots were first computed for each of the fluors vs. its two neighbors. These plots indicate regions where pairwise contrast is high enough to be useful. A constraint on the practically attainable contrast is that regions of high contrast generally lie far down the flanks of at least one of the spectra i.e., where excitation and /or emission are strongly sub-optimum. Further, to attain the required degree of selectivity it is necessary to use filters of bandwidths in the range 5-15 nm (cf. approx. 50 nm for 'standard' filter sets). Together, these impose a severe sensitivity penalty. The goal of 10% maximum crosstalk represents an acceptable, practical compromise between sensitivity and selectivity.
• Heat filters routinely used in microscopy are completely inadequate to alleviate this problem. Thus, extensive additional blocking was required.
• available commercial interference filters for infra-red blocking filters also transmit poorly in the near UV, and thus cannot be inserted in the excitation path. Instead, it was found necessary to put the IR blocking filters into the emission path. To minimize loss of image quality by insertion of these filters in the image path, they are placed inside the CCD camera, immediately in front of the window. In practice, two interchangeable filters were chosen, one for use with Cy5, Cy5.5 (Oriel #58893; 740 nm cutoff) and one for use with Cy7 (Oriel 58895; 790 nm cutoff).
• the first member of the set of fluors is the counterstain DAPI, which gives a weak G-like banding pattern. Five of the remaining six fluors may be used combinatorially to paint the entire human chromosome set. All are available as avidin conjugates (for secondary detection of biotinylated probe libraries) or directly linked to dUTP (for direct labeling).
• fluors comprise the preferred fluors of the present invention and are: 4'-6-diamidino 2-phenyl indole (DAPI), fluorescein (FITC), and the new generation cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
• DAPI 4'-6-diamidino 2-phenyl indole
• FITC fluorescein
• new generation cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
• the absorption and emission maxima for the respective fluors are: DAPI (Absorption maximum: 350 nm; Emission maximum: 456 nm), FITC (Absorption maximum: 490 nm; Emission maximum: 520 nm), Cy3 (Absorption maximum: 554 nm; Emission maximum: 568 nm), Cy3.5 (Absorption maximum: 581 nm; Emission maximum: 588 nm), Cy5 (Absorption maximum: 652 nm; Emission maximum: 672 nm), Cy7 (Absorption maximum: 755 nm; Emission maximum: 778 nm).
• DAPI Absorption maximum: 350 nm; Emission maximum: 456 nm
• FITC Absorption maximum: 490 nm; Emission maximum: 520 nm
• Cy3 Absorption maximum: 554 nm; Emission maximum: 568 nm
• Cy3.5 Absorption maximum: 581 nm; Emission maximum: 588 nm
• Cy5 Absorption maximum
• fluorescence is arguably the most powerful, because of its high absolute sensitivity and multiparameter discrimination capability.
• Modem electronic cameras used in combination with high numerical aperture microscope objectives and state of the art optical filters are capable of imaging structures labeled with as little as a 10-100 fluor molecules per pixel.
• fluor-tagged single-copy DNA sequences as small as a few hundred bases in size are detectable under favorable conditions.
• the availability of families of spectrally distinguishable fluors makes simultaneous imaging of several different targets in the same specimen possible, either directly or through combinatorial or analog multiplex methods.
• multi-fluor discrimination may be based on differential excitation of the fluors, differential emission, fluorescence lifetime differences, or on more complex but still analyzable observables such as fluorescence anisotropy.
• This discussion assumes an epi-imaging geometry. Table 1 describes the symbols and operators relevant to the theoretical considerations of fluorescence.
• J j ⁇ 5( ⁇ ,p). d ⁇ can be measured with a bolometric detector such as a calibrated micro-thermopile placed at or near the focal plane of the objective lens.
• S/N of each pixel increases indefinitely as (photons detected) 1 / 2 . How rapidly S/N increases depends on excitation strength, but the relationship between S/N and dose does not.
• the effect of non-zero bleaching constant is to change the t 1 / 2 function to an asymptotic function, the form of which depends on the bleaching mechanism.
• the asymptote once again does not depend at all on excitation rate, although the speed of approach to the asymptote does. If finite camera noise is added to photob leaching, it is found that the S/N climbs to a maximum value, then falls as the fluor is exhausted.
• the microscope objective compresses the excitation beam, in a nonconfocal microscope it does not focus it to a point and so has no bearing on the fluorescence image resolving power (incoherent emitter).
• the commonest excitation source for fluorescence microscopy is the high pressure short mercury arc, whose spectrum consists of pressure- broadened lines from the UV to the middle region of the visible spectrum (principal wavelengths are 334.1 nm, 365.6 nm, 404.7 mm, 435.8 nm, 546.1 nm. 577.9 nm), superimposed on a weaker thermal continuum. Many fluorophores have excitation spectra that overlap one or other of the mercury lines to an acceptable extent. Others (of which the best known is FITC) do not, but may be adequately excited by the continuum if a wide enough excitation bandwidth is employed. Another common source is the high pressure xenon short arc, which produces an almost uniform continuum from ca.
• a high CW power or pulsed quartz halogen lamp outperforms the xenon beyond about 450 nm.
• Certain fluors are well matched to laser excitation (e.g., Ar+ @488 nm for FITC, He-Ne @ 632.8 nm for Cy5, semiconductor diode - pumped YAG @ 680 nm for Cy5.5). In single-fluor imaging, use of the available spectral bandwidth is rarely stringent.
• the excitation filter Fl and dichroic beamsplitter DBl can usually be chosen to give adequate overlap between the source spectrum and the fluor excitation spectrum. If an arc line is available, the Fl bandpass need be no wider than the line. If not, and part of the thermal continuum must be used, the wider the Fl bandpass the greater will be the available excitation flux. However, with low noise integrating detectors the goal of high excitation efficiency is generally secondary to the need to exclude excitation light from the emission path. This limits how close the excitation and emission bandpasses can be placed to one another, and hence constrains the excitation bandwidth. For fluors with small Stokes' shifts, high quality filters with very steep skirts are required. The excitation filter must be rigorously 'blocked' on the long wavelength side, and have no pinholes, scratches, or light leaks around the edge.
• Dichroic beamsplitters are currently much less 'evolved' than bandpass interference filters, meaning that the slopes of their transmit ⁇ -> reflect transitions are far less than the skirt slopes of premium notch filters, and there may be large spectral intervals where they oscillate between intermediate states of partial reflectance and transmittance.
• the main purpose of a dichroic beamsplitter is to improve the combined efficiency of excitation and emission, rather than to define the wavelength response of the instrument.
• the resolvability of overlapping fluors in imaging microscopy may depend critically on the degree of excitation contrast that can be achieved (see C, below).
• the variation with wavelength of the ratio of the extinction coefficient of two fluors is the excitation contrast spectrum. It can readily be calculated from the digitized absorption spectra. Depending on the overlap of the absorptions, their ratio spectrum may either show a distinct peak or may grow indefinitely large. In either case, it is usually possible to choose an excitation wavelength that favors one fluor over another to a useful extent (from a factor of 3-4 fold up to a hundredfold or more).
• G represents the efficiency with which the optics gather the fluorescence and transmit it to the detector; it may be assumed to be wavelength independent to first order.
• NA numerical aperture
• NA additionally determines the spatial resolving power of the microscope, because it scales the dimensions of the Eisenhofer diffraction pattern produced in the image plane by a point source in the specimen plane.
• Several 'rules' are in use for specifying the resolving power of a lens, depending on how much overlap of the Airy discs of two adjacent objects is deemed to constitute the threshold of resolution.
• image 'noise' at p is determined by the statistical variance in the number S(p) of fluorescent photons detected in - 25 -
• S(p) is an integral of the form:
• f(t) is the photobleaching decay function.
• S/N rises along a more or less complex path to an asymptotic value that corresponds to total exhaustion of the fluor.
• N(p,t) N ⁇ (l-exp-
• a nonideal detector contributes noise of many kinds, detailed analysis of which may be intractable.
• the simplest noise component is fluctuation in the so-called 'dark current,' i.e., the flow of thermally excited carriers within the detector. If this noise is assumed to be random, it adds to the photon shot noise in RMS fashion.
• the S/N after time ⁇ t is F.( ⁇ t) 1 / 2 / (F + D) 1 / 2 ; S/N still increases as ( ⁇ t) 1 / 2 , but more slowly than for a noiseless detector.
• S/N rises initially as ( ⁇ t) 1 / 2 , but at some point reaches a shallow maximum and then begins to fall again, as the fluorescent signal declines but the thermal noise power remains constant. In this case, it is in principle desirable to continuously monitor the S/N of the image, and terminate the exposure when the peak is reached.
• the dichroic beamsplitter transition wavelength is specified at for example, 20 mn to the red of the excitation passband. This ensures a high level of rejection of exciting light reflected and /or scattered from the specimen and/or microscope optics.
• the emission filter cut-on is usually considerably steeper than the dichroic edge, and so can be placed practically coincident with it.
• the most efficient emission filter is a long- pass element.
• the preferred filter of this type is Schott glass, which transmits upwards of 90% of all fluorescence to the red of its cut-on, while rejecting other light (especially any excitation light that gets through the dichroic beamsplitter) to very high order - typically >l ⁇ 5.
• the multiparametric imaging of the present invention not only increases the throughput of information about the system under observation and makes more efficient use of the biological material, but also can reveal spatial and temporal correlations that might otherwise be difficult to establish reliably.
• two or more labels can be used combinatorially, which permits discrimination between many more object types than there are spectrally distinguishable labels.
• Some examples of multi-fluor imaging are: a. The co-distribution of proteins in structures such as microtubule networks may readily be visualized using immunolabels linked to different fluors. b. Multiple genes may be simultaneously mapped by fluorescence in situ hybridization (FISH) to a single metaphase chromosome spread.
• FISH fluorescence in situ hybridization
• Such signals cannot usually be discriminated reliably on the basis of intensity alone, and are usually morphologically identical (diffraction-limited points). However, they are readily discriminated by discrete or combinatorial multi-fluor labeling, c. Identification of small chromosomal translocations is most readily done by painting with chromosome specific DNA probe libraries linked to separable fluors, used either singly or combinatorially. d. Analysis of mixed populations of morphologically identical bacteria can also be achieved using species-specific DNA or ribosomal RNA probes coupled to separable fluors.
• the primary design goal of a multi-fluor imager (in addition to those for of a single fluor imager) is to spectrally resolve the fluorescence at any pixel location into components corresponding to each fluor.
• Methods for spectrally resolving complex signals in fluorescent microscope images are outlined below.
• a solution is to extract the light corresponding to each object with a probe (e.g., a fiber optic) and disperse it with an imaging spectrograph onto a 1 -dimensional array detector.
• a probe e.g., a fiber optic
• an imaging spectrograph onto a 1 -dimensional array detector.
• the most reasonable method for full spectral analysis in microscopy is to image through a variable narrow band filter. An image is recorded at each wavelength; intensity values at a given pixel location through the series represent a weighted emission spectrum that can be fit to a linear combination of the known spectra of the component fluors.
• the coefficients are products of the relative molar amounts of the fluors with their extinction coefficients at the exciting wavelength and their fluorescence quantum yields. If the last two are known, the first is obtainable from the fit. In general, it is necessary to take several such image sets, at several excitation wavelengths, to get a unique fit. With enough iterations, this process generates a 3D surface of intensity values as a function of both excitation and emission wavelength.
• This comprises a complete spectral signature of the pixel, giving a very highly constrained solution for the relative amounts of its component fluors.
• the mole ratios could be mapped back onto the x,y coordinates of the image, with appropriate pseudocolor coding, to give a 'composition map'.
• This general (and quantitative) method has a number of technical difficulties, although none of them is insurmountable. The first is that a large number of images are required to evaluate a single microscope field. Imaging time is long, and extensive differential photobleaching of the fluors would make it impossible to achieve a self consistent "fit" to the spectral data. Instrument stability is also an issue, particularly with arc sources, the output spectra of which change throughout their life. Finally, the amount of computation required to generate a composition map would realistically limit the analysis to small image regions only.
• excitation-emission contrast (EEC) approach is in principle applicable to analysis of images involving multiple fluors with fine-grained distributions of mole fraction (e.g., fluorescence ratio imaging), subject to image S/N and the limitations of differential bleaching rates and source instability.
• EEC excitation-emission contrast
• excitation and emission optics that have no wavelength selectivity cannot be used, because the excitation light scattered into the detector would overwhelm the fluorescence by several orders of magnitude.
• One solution is to use multiple-bandpass filters designed for the specific set of fluors to be used.
• the excitation filter defines narrow passbands that overlap the fluor excitation spectra.
• the emission filter defines similar passbands that interdigitate between the excitation bands and overlap the fluor emission spectra (the reddest fluor could use a long- pass filter).
• the dichroic beamsplitter alternates between reflect (overlapping the excitation passbands) and transmit (overlapping the emission passbands).
• a second theoretical solution would be to excite at a wavelength where all the fluors absorb. This is often possible because many fluors are excitable to states higher than SI using photons in the middle UV, but because of internal relaxation processes give 'normal' fluorescence. For example, many laser dyes can be excited at the nitrogen laser wavelength, 337 nm, far to the blue of their visible absorbances. It would be straightforward to block such exciting light from the emission path, using a long-pass filter (e.g., 380 nm), while allowing all fluorescences to simultaneously reach the detector.
• Drawbacks to the use of UV excitation include increased rates of photochemical decomposition of the fluor, and the expense of suitable UV optics. Thus, the method has not found widespread use.
• the multi-bandpass method has the limitation that construction of multiple bandpass elements giving adequate contrast between more than 3 fluors is extremely difficult.
• a generally more powerful approach is to construct optimized filter sets for each fluor, and switch them as needed.
• the primary goal on the excitation side is high excitation flux, to give a bright image.
• fluors may be imaged by sequentially switching filters that are designed using the same criteria as single-fluor sets (except that long-pass emission filters are proscribed for all but the longest-wavelength fluor). Crosstalk of a few percent is usually allowable, and can be compensated numerically if necessary.
• Two displacement components can be identified: i. a reproducible offset that is unique to each filter set. This component is a fixed vector, and arises mainly from imperfect parallelism (i.e., wedging) between the top and bottom faces of the emission bandpass filter. There is also a small component due to wedging in the dichroic beamsplitter, but since this element is very thin the effect is minor. Since the wedging vector is a constant for each filter set, it can be automatically removed in the computer. The size of the offset can also be reduced to very small values ( ⁇ 0.1 ⁇ ) by selecting emission filters for a high degree of parallelism e.g., by measurement in a laser autocollimator. ii.
• the detection of fluor is accomplished using optical filters, in a modification of the method of Ried, T. et al (Proc. Natl. Acad. Sci. (U.S.A.) 89:1388-1392 (1992), herein incorporated by reference).
• DAPI 4',6-diamidino-2-phenylindole
• the DAPI excitation maximum (347 nm) is to the blue of Cascade Blue (CB)
• CB Cascade Blue
• the fluorescence of DAPI peaks to the red of Cascade Blue, and actually overlaps quite strongly with FITC.
• the usual excitation wavelength for DAPI Hg 366 nm line
• the peak of the DAPI/CB excitation contrast spectrum is at 320 nm, which is too far into the UV to pass through the microscope optics.
• the Ealing 35-2989 interference filter has an appropriate bandpass.
• the excitation contrast ratio for DAPI/CB at 334.1 nm has an absolute value of 4.0. d.
• emission contrast must be used in addition to excitation contrast. Note that DAPI emits the bulk of its fluorescence to the red of CB.
• the wavelength of maximum emission contrast for DAPI vs. both CB and FITC is 490 nm.
• a suitable imaging-grade filter is the Omega 485DF22. There are no mercury lines within its bandpass, so good DAPI images with low flare are expected.
• the calculated emission contrast between DAPI and both Cascade Blue and FITC is 6.8.
• the overall contrast achievable for DAPI vs. Cascade Blue is approximately 27-fold.
• the overall contrast of DAPI vs. FITC is very much higher than this, because at the DAPI excitation wavelength the excitation of FITC is close to zero.
• the Omega 450DRLP02 dichroic beamsplitter is very well matched to the proposed excitation and emission filters.
• Imaging Cascade Blue Cascade Blue has a broad, two-peak excitation spectrum that overlaps DAPI extensively, though not its neighbor on the red side (FITC).
• the Stokes shift for CB is very small, i.e., there is very extensive overlap between its excitation and emission spectra. These factors combine to make imaging CB in the presence of DAPI problematical.
• a. The peak of the CB/DAPI excitation contrast spectrum is at 396-404 nm. However, because of the very small Stokes' shift of CB this is very close to the emission contrast peak (408 nm). Since it is more important for image S/N to collect the emission with high efficiency than to excite with high efficiency, the 400-410 nm region is reserved for the CB fluorescence.
• the best compromise for CB excitation is the Omega 380HT15, which overlaps with the Hg 366 nm line enough to provide good excitation strength.
• the emission filter must be narrow and very carefully placed. A suitable filter is the
• the emission spectrum of FITC overlaps that of Cy3 (on the red side) and both DAPI and CB (on the blue side) to an appreciable extent.
• FITC/Cy3 emission contrast ratio goes to very high values below
• the Omega 530DF30 filter gives very high emission contrast, which compounds the high excitation contrast ratio for FITC vs. Cy3 (8.8) given by the 455DF70. It therefore appears possible to image FITC very cleanly.
• the absorbance peak of Cy3 is at 551 nm, at which wavelength the excitation of FITC is essentially zero (contrast parameter Rb jumps to extremely high values to the red of 525 nm).
• the Cy3 extinction peak overlaps strongly with the Hg 546.1 nm line.
• the excitation contrast ratio for Cy3/Cy3.5 is everywhere small, and varies weakly with wavelength. At 551 nm, the absolute value of the excitation contrast for Cy3 vs. Cy3.5 is less than 2, and it only rises significantly far to the blue where the Cy3 absorbance is very low and FITC absorbance is high.
• the excitation contrast ratio for Cy3.5 relative to Cy5 is also quite large (absolute value approximately 8.0).
• the emission contrast parameter for Cy3.5 vs. Cy3 is small at all wavelengths where the Cy3.5 emission is usefully strong, i.e., isolation of Cy3.5 from Cy3 must rely mainly on excitation contrast.
• the emission contrast for Cy3.5 vs. Cy5 is also large over a considerable spectral interval (and rises to very high values below
• the Omega 590DRLP02 is a suitable dichroic for this channel.
• Cy5/Cy5.5 is poor.
• c There is no Hg line available for exciting Cy5.
• the "official" filter for exciting Cy5 in this way is the Omega 640DF20, which will give an excitation contrast with Cy5 of about 1.8.
• a much brighter source for exciting Cy5 is the He-Ne laser (632.8 nm). It does not, however, improve excitation contrast vs. Cy5.5.
• e The emission contrast for Cy5 vs. Cy3.5 peaks at 673 nm, just to the red of the fluorescence intensity peak.
• the closest available filter is the Omega 660DF32 where the emission contrast ratio is approximately 3.1.
• CY5.5 is the penultimate dye of the set, and is very well separated from Cy7. Thus, only its contrast relative to Cy5 need be considered in detail: a.
• the contrast parameter R y for the Cy5.5/Cy5 pair rises to large values to the red of 670 nm. Thus, it is possible to achieve very high excitation contrast for this pair of fluors (analogously to Cy3.5/Cy3).
• the Hg arc is a poor source for exciting Cy5.5.
• the best available source is a 680 nm diode-pumped frequency doubled YAG microlaser (Amoco), which coincides with the peak of the Cy5.5 absorbance. At 680 nm, the excitation contrast ratio for Cy5.5/Cy5 is 5. 1.
• a suitable excitation filter is the Ealing 35-4068.
• the emission contrast ratio between Cy5.5 and Cy5 peaks at 705 nm, approximately 3 nm to the red of the Cy5.5 intensity curve. The numerical value for S D at this point is 4. If the Omega 700EFLP longpass emission filter is used, a contrast ratio of approximately 3 (averaged out to 800 nm) is expected. This, combined with the high excitation contrast, makes imaging Cy5.5 very clean.
• a bandpass filter such as the Ealing 35-6345 could be substituted for the Omega 700EFLP. The potential advantage would be reduction of the infra-red background . i.e., overall improved image contrast.
• Cy7 is the reddest dye of the set.
• the excitation and emission spectra are well separated from Cy5.5, and are well matched to the Omega 740DF25/770DRLPO2/780EFLP triplet.
• the Oriel 58895 is an appropriate IR blocker for Cy7.
• Filters selected for imaging the DAPI, FITC, Cy3, Cy3.5, Cy5, Cy5.5, Cy7 fluor set are summarized in the Table 2 below. None of these filter sets correspond to the filter sets supplied by manufacturers of conventional fluorescence microscopes as narrow band excitation and fluorescence detection is mandatory to achieve sufficient contrast.
• Cy7 Omega Omega Omega Oriel 58895 740DF25 770DRLP02 780EFLP Characteristics of the microscope system are described in detail by Ballard S.G. et al. (J. Histochem. Cytochem. 42:1755-1759 (1993), herein incorporated by reference).
• a high pressure 75W DC xenon arc (XBO) was used as an excitation source because of its approximately constant spectral power distribution.
• a Zeiss Axioskop microscope workstation equipped with a cooled CCD camera (Photometries CH250) was employed.
• the objective lens was a 63x 1.25NA Plan Neofluar which should be "plan” and apochromatic with a high numerical aperture.
• the filter sets selected were able to discriminate between the six fluors with a maximum contrast (Table 2). To minimize the crosstalk between fluorophores, the filter sets were selected on the basis of maximum spectral discrimination rather than maximum photon throughput. Image exposure times were varied to adjust for photon flux differences and flux excitation cross-sections. Narrow band excitation and fluorescence detection is mandatory to achieve sufficient contrast. Appropriate excitation and emission filter sets were used to optically discriminated these fluors (Table 2).
• the combinatorial labeling strategy relies on accurate measurements of intensity values for each fluorophore. Critical features are accurate alignment of the different images, correction of chromatic aberrations, and specific quantitation of each fluorophore. Because simple manual image manipulation could not realize these demands new software was developed in our lab.
• This program comprises the following steps in sequential order: (1) correction of the geometric image shift; (2) calculation of a DAPI segmentation mask; (3) for each combinatorial fluor, calculation and subtraction of background intensity values, calculation of a threshold value and creation of a segmentation mask; (4) use of this segmentation mask of each fluor to establish a "Boolean" signature of each probe; (5) for each chromosome, display of the chromosome material next to the DAPI image; (6) create a composite gray value image, where each labeled object is encoded with a unique gray value; (7) final presentation of the results using a look-up-table (LUT) that assigns each gray value a particular color
• BDS-Image image analysis package
• Waggoner, A. et al. (Methods Cell Biol 30:449-478 (1989)) and modified (du Manoir, S. et al, Cytometry 9:4-9 (1995); du Manoir, S. et al, Cytometry 9:21- 49 (1995), all herein incorporated by reference).
• the DAPI image was used to define the morphological boundary of each chromosome. Accurate chromosome segmentation was achieved by pre-filtering the images through a top-hat filter (Smith, T.G., et al, J. Neurosci Methods 26:75-81 (1988); modified in du Manoir, S. et al, Cytometry 9:4-9 (1995); du Manoir, S.
• selective excitation of multiple fluors and analysis of fluorescence spectral signatures can be carried out using dispersion optics rather than wavelength-selective transmission filters.
• Such optics may be used to create filters of any passband characteristic, including short-pass, long-pass, single bandpass and multiple bandpass functions.
• a dispersion element (prism or grating) is used in conjunction with a wavelength-selective spatial filter to create the desired spectral response.
• the combination is referred to herein as a "comb filter.”
• a comb filter the spectral distribution of the exciting light may be tailored for optimum simultaneous excitation of multiple fluors.
• the inverse comb filter may also be used to selectively block from the CCD camera only the wavelengths used for excitation: the remaining wavelength intervals (corresponding to the gaps between teeth of the comb) are available for spectral analysis of the fluorescence emitted by the fluors. This analysis constitutes the spectral signature.
• an interferometer may be used in conjunction with an epi-fluorescent microscope.
• a light source for excitation of fluorescence that is either coherent (e.g. an Argon laser) or incoherent (e.g. a Mercury arc lamp) may be used.
• a Mercury- Xenon mixed gas arc lamp is preferred due to its intense Mercury lines and broad Xenon visible and near-infrared continuum.
• the Sagnac interferometer has a larger acceptance angle, greater claritye, and is less sensitive to alignment, vibration, and temperature variations than a similar Michelson interferometer.
• the Sagnac interferometer is a common path interferometer.
• An interferometer consists of two or more interfering beams of light. In a common path interferometer there are two beams each traveling the same path but in opposite directions. The optical paths are produced by reflecting light through beamsplitters, for example.
• Multiple beam interferometers operate by dividing the optical energy from a light source into two substantially equal beams of light. The two beams of light are combined after one is permitted to pass through a sample and the interference pattern (the changes in intensity of the combined light caused by the interference of two beams) is detected.
• the light source is also divided into two substantially equal parts.
• Changing the angle of incidence of light on the beamsplitter, causes the optical path length to be changed along one optical axis of the interferometer.
• the other axis of the detector can sample gray scale.
• the fringe pattern produces an interferogram at each pixel.
• the Fourier Transform of this interferogram yields the spectrum of light falling on that pixel of the CCD.
• FIG. 1 A simple example of a Sagnac interferometer is shown in Figure 1. Disturbances, such as a small shift of one of the optical elements, effect both beams in the same way, and hence have no effect on the measurement. This mechanical stability also makes the interferometer relatively insensitive to temperature changes as well. Thus, another advantage of the common path interferometer is its intrinsic stability. Sagnac interferometers and their use are well known (see, for example,
• the acceptance angle of the interferometer is determined to be:
• w/a tan 30°
• w is the aperture width of the interferometer
• a is the length of each leg
• n is the index of refraction of the interferometer glass. Referring to Figure 1, this yields a full acceptance angle of approximately 8 degrees. In other designs the acceptance angle may be different but in any case the input beam to the interferometer need not be collimated.
• the interference pattern or interferogram is most preferably detected with a CCD camera (such as a Princeton Instruments frame transfer CCD camera) capable of 512 X 512 pixels or larger. Since the interferogram in a Sagnac interferometer has an angular dependence, each pixel of the CCD detector measures a small interval of the interferogram. The fringe spacing of the interferogram is set such that a pixel on the CCD detector can adequately sample the interferogram.
• OPD Optical Path Difference
• OPDpixel ⁇ min/4
• ⁇ m j n is the shortest wavelength in the spectrum to be measured by the interferometer.
• This OPDpj xe ⁇ determines the theoretical limit of the resolving power of the interferometer.
• the interferogram is being moved across the CCD detector, such that the maximum optical path difference is then given by the relation
• OPD max N(OPD pixel )
• N is the linear dimension of the CCD detector in pixels.
• Each angular displacement of the light incident on the interferometer beamsplitter may then correspond to one or several OPDpj xe i' s . And, in the case of a CCD detector, one frame of CCD data is required to sample this angular displacement.
• each pixel comprises an interferogram which contains within it information about the spectrum of light falling on the pixel, the intensity of light falling on that pixel, and the x and y coordinates of the pixel.
• the spectrum of light may be recovered from the interferogram by the use of a computational Fourier Transform algorithm.
• UV light is used to excite the fluorescent probes.
• the UV light may be easily blocked with a long pass interference filter allowing the visible and near-infrared colors to pass through to the interferometer.
• This embodiment has the advantage that UV will excite many of the fluorescent dyes currently in use.
• This embodiment also has the advantage that it will allow better than 90% transmittance of the visible fluorescence to the interferometer.
• the disadvantage of UV is that it photobleaches the dyes faster than visible light.
• Both the input and the output lens of the interferometer are preferably very high efficiency camera lenses, and do not significantly effect the efficiency of imaging.
• the focus of the image within the interferometer is most preferably adjusted so as to be constant for the variable powers of the zoom eyepiece, and thus a microscope having the characteristic of infinite image distance (such as Olympus AX70 microscope) are preferred.
• the above-described interferometer possesses certain advantages over optical filters.
• One key advantage is that all the light emitted by fluorescence is theoretically available for detection, whereas the transmittance of an interference filter is limited.
• Another advantage is that since the filters do not have to be changed, there is no image shift due to the non-parallelism of filters.
• FISH Fluorescent In Situ Hybridization
• cytogenetic diagnosis of genetic disease such as the pre- or post-natal diagnosis of disease, complex tumor karyotyping, the analysis of cryptic translocations. It provides a novel method for automated chromosome identification and analysis.
• diseases prenatal disease, cancers (especially BRCA1 or BRCA2 associated breast cancer), leukemias, Down's Syndrome, etc.) are characterized by rearrangements and other chromosomal abnormalities that can be discerned using the methods of the invention.
• Chromosome karyotyping by conventional cytogenetic banding methods is both time consuming, expensive and not easily automated.
• FISH karyotype Advantages of the FISH karyotype are the instant identification of the chromosomal origin of marker chromosomes, double- minutes and homolgy staining regions ("HSRs"). Even “poor quality" chromosome spreads can be evaluated. If desired, one could design a probe set for particular applications or for particular clinical applications, e.g. hematologic diseases, pre- or postnatal diagnosis. The development of specific probe sets that stain particular regions of chromosomes (e.g. telomeric regions) for the identification of cryptic translocations would overcome limitations of the whole chromosome painting probes.
• probes could be used to generate multicolor "barcodes" on individual chromosomes thereby facilitating the automated analysis of karyotype.
• Probes can also be designed that would be specific for a particular arm of a chromosome, thereby permitting a molecular characterization of translocation breakpoints, hot spots of recombination, etc.
• Other applications would include rapid evolutionary studies, provided that the protocols for multicolor FISH on human chromosomes can be adjusted, as expected, for applications on other species.
• the methods of the invention may also be used to assess the presence or absence of infectious agents (treponema pallidum, rickettsia, borrelia, hepatitis virus, HIV, influenza virus, herpes, Group B streptococcus, diarrhea-causing agents, pathogens causing acute meningitis, etc.) in tissue, or in blood or blood products.
• infectious agents such as treateptema pallidum, rickettsia, borrelia, hepatitis virus, HIV, influenza virus, herpes, Group B streptococcus, diarrhea-causing agents, pathogens causing acute meningitis, etc.
• infectious agents such as treponema pallidum, rickettsia, borrelia, hepatitis virus, HIV, influenza virus, herpes, Group B streptococcus, diarrhea-causing agents, pathogens causing acute meningitis, etc.
• the methods of the present invention permit the rapid serotyping of such agents,
• the methods of the present invention may be used to quantitate microorganisms that are difficult to propagate (such as anaerobic microorganisms involved in periodontal disease).
• the methods of the present invention provide a means for the rapid diagnosis of acute bacterial meningitis.
• serotype-specific probes to perform serological analysis
• probes that are specific to particular drug resistance determinants and thereby rapidly determine not only the presence and identity of an infectious agent, but also its susceptibility or resistance to particular antibiotics.
• the methods of the present invention further permit simultaneous mapping of a large number of different DNA probes.
• the analysis of chromosomal number and architecture in individual intact cells becomes accessible.
• Interphase cytogenetics is already possible with small region specific probes, e.g. YAC-clones.
• the accuracy of such analysis could be increased by a three dimensional analysis using a laser scanning microscope.
• the use of a laser scanning microscope would ultimately allow to visualize all whole chromosome painting probes in interphase nuclei and questions relating to intranuclear chromosomal organization as a function of developmental status, cell cycle or disease state could be addressed.
• Different models about the chromosomal organization in interphase nuclei could finally be explored.
• conventional laser scanning microscopes currently do not allow the excitation of some of the fluorophores used, other, more appropriate fluors or devices may be employed.
• the methods of the present invention enable one to examine chromosome architecture or quantitate the chromosome contents of nuclei in single hybridization experiments.
• Questions relating to intranuclear chromosomal organization as a function of developmental status, cell cycle or disease state can accordingly be addressed.
• the ability to quantitatively assess the levels of multiple mRNAs or proteins in a single cell or to determine if they exhibit different intracellular distributions could prove extremely useful in addressing a myriad of interesting biological questions.
• the multiparametric imaging of the present invention does not merely increases the throughput of information, it also makes more efficient use of the biological material. Thus, it can reveal spatial and temporal correlations as well as mosaicisms that might otherwise be difficult to establish reliably.
• the intracellular distribution of oncoproteins or tumor suppressor proteins can be determined within the same cell simultaneously .
• the chromosomes of a particular karyotype are pseudo-colored to thereby facilitate the assignment of the chromosomes, or the recognition of translocations, deletions, etc.
• the digitized images of the chromosomes may be stored in computer-readable storage device (such as a magnetic or optical disk) to facilitate their comparison with other chromosomal images or their transmission and study.
• probes may be employed that are translocation specific or specific to sub-chromosomal elements or regions, such that the pseudocoloration process displays banded or striped chromosomal images.
• the position and sizes of individual bands is preferably digitized and stored so that an image of the chromosome may be -stored on a computer. Similarly, the precise position of any translocation or other karyotypic abnormality can be discerned and stored.
• the methods of the present invention thus permit karyotypic analyses to be conducted more widely and more accurately than was previously feasible.
• the present invention may thus be used to systematically correlate karyotypic abnormalities with disease or conditions. For example, karyotypes of asymptomatic individuals can be obtained and evaluated in light of any subsequent illness (e.g., cancer,
• Alzheimer's disease, etc. or condition (e.g., hypertension, atherosclerosis, etc.) in order to permit a correlation to be made between a patient's karyotype and his or her predisposition to different diseases and conditions.
• karyotypes of individuals having diagnosed diseases or conditions can be obtained and evaluated in light of the extent of any subsequent progression or remission of the disease or condition so as to permit a correlation to be made between a particular karyotype and the future course of a disease or condition.
• a computer or other digital signal analyzer may be employed to orient and arrange the chromosomal images as well as assigning and identifying the chromosomes of the karyotype.
• a computer or other data processor will, upon assigning a particular chromosome to a particular designation (for example, upon assigning that a particular chromosomal image is the image of the chromosome 7 of the karyotype being evaluated), group the assigned chromosome with its homologue (e.g., the second chromosome 7 of the patient's karyotype) and generate, via a printer, monitor, or other output means, an ordered array of chromosomal images in which each autosomal chromosome is paired with its homologue, and in which the sex chromosomes X and Y are paired together.
• the chromosomal images of such arrays will be the pseudocolorized images discussed above.
• such psudocoloring may be internal to the process of assigning chromosomal identity, and not displayed in the output of the computer or digital signal analyzer. Rather, in this sub-embodiment, the output generated will be the light-microscope visible banding pattern of the metaphase chromosomes of the patient whose karyotype is being evaluated.
• a scale in Morgans or other suitable units
• chromosome painting probes representing the 22 autosomes and the two sex chromosomes were used.
• the DNA probes used were generated by microdissection. Microdissected probes (National Center for Human Genome
• Telenius et al. Telenius, H. et al, Genes, Chromosomes & Cancer 4:257-263 (1992); Telenius, H. et al, Genomics 23:718-725 (1992); Meltzer, P.S. et al, Nature Genetics 2:24-28 (1992); Guan, X.Y. et al, Hum Mol Genet 2:1117-
• cyanines are 1-200,000 and 0.3 (Waggoner, A., Methods in Enzymology 246:362-373 (1995)).
• the probes were subjected to a PCR amplification and labeled by nick translation.
• Fluorescein (Wiegant, J. et al, Nuc Acids Res 29:3237-3241 (1991)), Cy3, and Cy5 were directly linked to DUTP for direct labeling.
• Cy3.5 and Cy7 were available as avidin or anti- digoxin conjugates for secondary detection of biotinylated or digoxinigated probes. They were synthesized using conventional N-succinamide ester coupling chemistry. For each probe one to three separate nick translation reactions were necessary, each with a single labeled fluor-labeled triphosphate or biotin or digoxigenin (Table 3).
• probe concentrations for the hybridization mix had to be established carefully in a large number of control experiments. Hybridization conditions were optimized for these multiplex probes. Thus, probes were denatured and hybridized for two to three nights at 37 °C to metaphase chromosome spreads in a conventional 50% formamide hybridization cocktail. The slides were washed at 45 °C in 50% formamide/ 2 x SSC three times followed by three washes at 60 °C in 0. 1 x SSC to remove excess probe. After a blocking step in 4 x SSC/3% bovine serum albumin for 30 min at 37 °C the biotinylated probes were detected with avidin Cy3.5 and the dig-labeled probes with anti-dig-Cy7.
• Fluorescein-dUTP, Cy3-dUT'P, and Cy5-dUTP did not require any immunological detection step. After final washes at 45 °C with 4 x SSC/0.1% Tween 20 three times, mounting medium and a coverslip were applied and the hybridization signals from each fluor imaged using the filters sets listed in Table 3.
• Figure 1 provides a schematic illustration of the CCD camera and microscope employed in accordance with the present methods.
• Figure 2 shows the raw data from a karyotypic analysis of chromosomes from a bone marrow patient (BM2486). Adjacent to each source image is a chromosome "mask" generated by the software program.
• panels A and B are the DAPI image and mask
• panels C and D are FITC image and mask
• panels E and F are Cy3 image and mask
• panels G and H are Cy3.5 image and mask
• panels I and J are Cy5 image and mask
• panels K and L are Cy7 image and mask.
• Figures 3A and 3B show the identification of individual chromosomes by spectral signature.
• Figure 3A is the same photograph as Figure 2, except that it is gray scale pseudocolored.
• Figure 3B displays the karyotypic array of the chromosomes.
• the exceptional power of the methods of the present invention are illustrated by the ease with which the translocation of chromosomes 5 and 8 are identified in Figures 3A and 3B, relative to conventional non-chromosome specific karyotype analysis.

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