0 1992 Wiley-Liss, Inc.

Cytornetry 13:839-845 (1992)

Fluorescence Ratio Measurements of Double-Labeled Probes for Multiple In Situ Hybridization by Digital Imaging Microscopy' P.M. Nederlof,2 S. van der Flier, J. Vrolijk, H.J. Tanke, and A.K. Raap3 Sylvius Laboratory, Department of Cytochemistry and Cytometry, University of Leiden, 2333 A1 Leiden, The Netherlands Received for publication June 26, 1991; accepted March 18, 1992

To expand the multiplicity of the in situ hybridization (ISH) procedure, which is presently limited by the number of fluorochromes spectrally separable in the microscope, a digital fluorescence ratio method is proposed. For this purpose, chromosome-specific repetitive probes were double-labeled with two haptens and hybridized to interphase nuclei of human peripheral blood lymphocytes. The haptens were immunocytochemically detected with specific antibodies conjugated with the fluorochromes FITC or TRITC. The FITC and TRITC fluorescence intensities of spots obtained with different doublehaptenized probes were measured, and the fluorescence ratio was calculated for each ISH spot. Combinations of different haptens, such as biotin, digoxigenin, fluorescein, sulfonate, acetyl amino fluorene (AAF),and mercury (Hg) were used. The fluorescence intensity ratio (FITCI TRITC) of the ISH spots was fairly constant for all combinations used, with coefficients of variation between 10 and 30%.

In situ hybridization (ISH) procedures allow the detection of DNA and RNA sequences in individual cells. Due to improved techniques i t is now feasible to detect unique sequences of only a few kilobases using nonradioactive procedures (4,10,27). Also, the possibility of detecting multiple sequences simultaneously has been actively pursued; this is of particular interest for screening for numerical aberrations in tumors (2,6-8,12,16). Furthermore, structural aberrations such as translocations can be detected in interphase nuclei and in metaphase chromosomes by multiple hybridizations. For that purpose, probes at both sides of the breakpoint are tagged with different

To study the feasibility of a probe identification procedure on the basis of probe hapten ratios, one probe was double-labeled with different ratios, by varying the relative concentrations of the modified nucleotides (biotin-11-dUTP and digoxigenin-11-dUTP)in the nick-translation reaction. Measurement of the FITC and TRITC intensities of the ISH spots showed that the concentration of modified nucleotides used in the labeling procedures was reflected in the mean fluorescence intensity of the ISH spots. Furthermore, the ratio distributions showed little overlap due to the relatively small coefficients of variation. The results indicate that a multiple ISH procedure based on fluorescence ratio imaging of double-labeled probes is feasible. o 1992 Wiley-Liss, Inc. Key terms: Quantification, ratio labeling, ratio imaging, image analysis, CCD camera, multiparameter analysis, microscopy

colors, as was shown for the detection of the Philadelphia chromosome in interphase nuclei by probes for bcr and abl(3,22).For the detection of complex rearrange-

'This research was sponsored in part by the Netherlands Organization for Scientific Research NWO grant 534-060, and NWO grant PGS 90-129.90. 2Present address: Dr. P.M. Nederlof, Department of Laboratory Medicine, Division of Molecular Cytometry, MCB 230, University of California, San Francisco, San Francisco, CA 94143-0808. 3Address reprint requests to Dr. A.K. h a p , Sylvius Laboratory, Department of Cytochemistry and Cytometry, University of Leiden, Wassenaarseweg 72, 2333 A1 Leiden, The Netherlands.

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ments and probe mapping, more than three color hybridizations may be desirable. The multiplicity of the fluorescence ISH is at present limited not by the number of hapten labeling procedures, but by the number of spectrally separable fluorochromes. Although a large number of fluorescent dyes exist (13,26), most are not suitable for multiple ISH, due to their spectral characteristics or their high molecular weight as is the case for most of the phycobiliproteins. In practice only three colors (green, red and blue) can be used, for which the fluorochrome combination fluorescein-isothiocyanate, tetramethyl rhodamine isothiocyanate, and aminomethyl coumarin acetic acid (FITC, TRITC, and AMCA) (14) is available, allowing triple ISH procedures (17). Infra-red dyes such as cyanine dyes (9) may provide one additional color, but require infraredsensitive detectors since the human eye is not sensitive in that part of the spectrum. To overcome the limitation imposed by the restricted number of spectrally separable colors, probes can be double- or triple-labeled with different haptens. The probes are then identified on the basis of their multiple-color composition. Consequently, the number of simultaneously detectable probes can in principle be increased to a maximum of seven (18). To expand the number of simultaneously detectable probes beyond seven, we investigated the feasibility of ratio labeling. Ratio-labeled probes are identified cytometrically, on the basis of their color intensity ratio and not on their color composition alone. For visualization of ISH results, the measured fluorescence ratios may be displayed as pseudo-colors on a monitor. Fluorescence ratio measurements are used in other applications in image cytometry and flow cytometry for identification or quantification of specific molecules or objects. A well-known example is the measurement of intracellular Ca2+ on the basis of indo-1 fluorescence (23). A number of requirements have to be fulfilled to perform ratio imaging for ISH: the hapten ratio of the probe should be preserved during hybrid formation; the immunocytochemical detection of the different haptens should be independent; the ratio of the measured fluorescence intensities obtained with a specific hapten ratio should be reproducible; and, most importantly, the measured fluorescence ratio should show relatively low variation. There are a large number of labeling methods available suitable for fluorescence ISH. For double (ratio) labeling both enzymatic and chemical modification procedures can in principle be used. We performed double labeling with chemical (AAF and Hg), enzymatic (biotin, digoxigenin, and fluorescein), and a combination of both procedures (sulfonate, biotin). For ratio labeling it is important to control the amount of label introduced on the probe. An option is the simultaneous incorporation of two different labels in one reaction by a nick-translation reaction comprising two different haptenized nucleotides. It is also pos-

sible to combine two mono-haptenized probes to acquire a ratio-tagged hybrid. The cytometric requirements for ratio ISH measurements should include a microscope set-up equipped for multiple color detection, and a detector with sensitivity for different wavelengths, high resolution, linear response, large dynamic range, and low noise. The cooled charge coupled device (CCD) cameras fulfill these requirements (1,ll).In this paper we present the first results, which show that the identification of DNA targets by digital fluorescence ratio measurements is feasible.

MATERIALS AND METHODS Preparation of the Slides Human lymphocytes were isolated from peripheral blood by Ficoll gradient. Cells were treated with a hypotonic buffer containing: 50 mM KC1, 10 mM MgCl,, 5 mM Na,HPO,, pH 8.0, and subsequently fixed in several changes of methanolfacetic acid (3:l vh). The nuclei suspension was stored a t -20°C until use. Nuclei were centrifuged on glass and treated with pepsin (Serva, Heidelberg, FRG) 0.01% in 0.01 M HC1 for 15 min at 37"C, after which the slides were post-fixed with 1%formaldehyde in phosphate-buffered saline (PBS): 0.15 mM NaC1, 10 mM Na phosphate, pH 7.2) for 10 min. Double Labeling of Probes A probe specific for the region lq12 of chromosome 1 (pUC1.77) (5) was double-labeled with biotin-11-dUTP (Sigma, Deisenhofen, FRG) and digoxigenin-11-dUTP (Boehringer, Mannheim, FRG) or with biotin and fluorescein-11-dUTP (a generous gift of Boehringer) by nick-translation or with biotin and a sulfon group using the Chemiprobe kit (FMC Bioproducts) according to the manufacturer's instructions (25). Different ratio-labeled probes were obtained by changing the ratios of the modified nucleotides in the nick-translation reaction mixture. The biotin-11-dUTP and digoxigenin-11-dUTP ratios used were: 1:1, l:lO, 1:50, and 1 : l O O . The total amount of modified nucleotides in the nick-translation reaction was kept constant, a t 2.0 nmol per reaction. In addition, 0.5 nmol dTTP was added to increase the incorporation. The amounts of biotin-11-dUTP and digoxigenin-11-dUTP in the reaction mixtures were therefore, respectively: 1:1,0.2:1.8, 0.04:1.96, and 0.02:1.98 (nmo1:nmolj. After labeing, the probes were purified on a Sephadex G-50 fine Pasteur pipet column. Probes were dissolved in the hybridization buffer (60% deionized formamide, 2 x SSC, pH 7) (2 x SSC: 0.3 M NaCl, 30 mM Na citrate, pH 7.2) a t a concentration of 10 ng/p1 with 50 x excess yeast tRNA (Boehringer) and sonicated salmon sperm DNA (Sigma). Hybridization and Detection For each slide a 20 ng probe in 5 ~ 1 6 0 % formamide, 2 x SSC was used. Probe and target were denatured

RATIO FLUORESCENCE IN SITU HYBRIDIZATION

Table 1 Filter Sets Used for Multiple Fluorescence ISH Analysis"

DAPI

FITC

TRITC

Excitation filters LP34OSP380 LP450-SP490 LP53OSP580

Wavelength (nm) Dichroic Emission mirror filters DM400 LP42OSP560 DM510 LP515SP560 DM580 LP580

"ISH, in situ hybridization; DM, dichroic mirror; LP, long wave pass filter; SP, short wave pass filter.

simultaneously under a coverslip for 3 min at 80°C in an incubator. Slides were transferred to a moist chamber and hybridization was performed for 16 h at 37°C. After hybridization of the double-labeled probes, the slides were washed 3 x 5 min with 60% formamide, 2 x SSC pH 7 a t room temperature and 1 x 5 min with 2x ssc. All immunocytochemical detetions were performed in 0.1 M Tris HC1, 0.15 M NaC1, 0.05% Tween-20, pH 7. Slides were washed with this buffer once and subsequently incubated in the same buffer containing 0.5% (wiv) blocking agent (Boehringer) for 20 min a t 37°C. For the detection of the biotiddigoxigenin ratio-labeled probes, the slides were incubated with avidin-FITC (Vector) and a monoclonal mouse antidigoxigenin antibody (Boehringer). A second incubation was done with a TRITC-labeled goat anti-mouse IgG antibody (Sigma). For detection of the fluoresceid biotin-labeled probe the detection consisted of one layer of avidin-TRITC (Vector). For detection of the biotid sulfonate-labeled probe the first incubation consisted of avidin-FITC (Vector) and a monoclonal mouse anti-sulfonate followed by a second layer with a goat antimouse-TRITC. Slides were dehydrated through a n ethanol series and mounted in PBS/glycerol (1:9 v/v) containing diamidinophenylindole (DAPI, 0.5 kg/ml) as a blue fluorescing total DNA counterstain, and 2.3% (w/v) 1,4-di-azobicyclo-(2,2,2)-octane (DABCO) (Sigma, St. Louis, MO) as an anti-fading agent. Since only two fluorochromes were used (FITC and TRITC), a third color could be applied for the overall staining of DNA (DAPI). Microscopy A Leitz Dialux epif luorescence microscope equipped with a 100 W mercury-arc lamp and a CF Fluor 40 x 1.30 0.8 NA oil objective (Nikon) was used for all measurements. The filters used for the selection of the fluorophores are listed in Table 1.

-

I ma ge Collection and Analysis Images were recorded with a cooled CCD camera (Photometrics, Arizona) with a Kodak chip of 1,024 x 1,345elements of 6.8 x 6.8 pm2 each. For each microscope field three images (256 x 256 pixels) were recorded for FITC, TRITC, and DAPI, respectively. The exposure times for the different preparations and

841

wavelengths varied between 2 and 15 s. The different fluorescence intensities were normalized to the intensity corresponding to a n exposure time of 1 s. The programs for the automatic analysis of the images were developed using the TCL-image analysis software package developed at the Technical University Delft (Multihouse, Amsterdam, the Netherlands) running on a SUN workstation interfaced to the camera. The image collection and image analysis was performed as described elsewhere (20). The intensity of a spot was calculated as the integrated intensity of all pixels within the area of the spot corrected for the (local) background. The determination of the spot area was performed automatically without user interaction. The local background was determined by the minimummaximum digital filtering technique (24).The F I T 0 TRITC spot ratio was calculated as the quotient of the integrated FITC and TRITC intensities of the corresponding spots from a pair of images. The image shift caused by the slightly different position of the dichroic mirror in the filter blocks used for the selection of the different fluorophores was reproducible, but could not be adjusted mechanically. The image shift was therefore corrected by pixel-wise shifting of the digitized image according to the shift found with microspheres, which could be excited a t various wavelengths. RESULTS Different double haptenization procedures were investigated. The chromosome 1-specific probe double haptenized with sulfonate and biotin was hybridized to human peripheral blood lymphocyte nuclei, and detected with FITC and TRITC, respectively. In Figure 1A the FITC intensity of each spot is plotted against its corresponding TRITC intensity. The FITC/TRITC intensity ratio distribution showed a variation of only 13%.The variation in both FITC and TRITC intensities was large between different nuclei, with coefficients of variation (CV) of 78% and 81% respectively. The double labeling with sulfonate and biotin was only successful when the sulfonate labeling was performed prior to the biotin labeling, although the nick-translation removed part of the sulfonate label (results not shown). As a result, it was difficult to control the conditions for obtaining defined ratios. A better control of the labeling was obtained by incorporating two haptens in the same labeling reaction, by introducing two modified nucleotides in a nicktranslation reaction. The same probe was double-labeled using fluorescein-11-dUTP and biotin-11-dUTP. In Figure 1B the corresponding FITC and TRITC intensities of each spot are plotted. The FITC/TRITC ratio of each spot was relatively constant, with a CV of 19.7%. The inter-nuclear variation in both FITC and 'TRITC intensities was large between different nuclei (with CVs of 64%and 61%,respectively). In addition to the use of double-haptenized probes, a ratio-labeled target was generated by combining two

842

NEDERLOF ET AL.

/

1000 1000

10000

100000

FTTC intensity (a.u.) l00000U

9

m

fB

FIG.2. Scatter plot of the FITC and TRITC intensity of spots obtained with a mixture of two single-labeled probes, with biotin (FITC) and digoxigenin (TRITC). Mean FITC 14,769 (CV = 40%); mean TRITC 32,249 (CV = 51%);mean FITUTRITC 0.477 (CV = 20%); R = 0.89: N = 161.

100000

v

.-*h 2

3

.-

10000

E

U

E l5

1000

100

1000

10000

innnoo

FITC intensity (a&) FIG.1. Scatter plot of the FITC and TRITC fluorescence spot intensities obtained after ISH of double-labeled probes, specific for chromosome 1,t o human peripheral blood lymphocyte interphase nuclei. A: Double-labeled with biotin (FITC) and sulfonate (TRITC). Mean FITC intensity 8,198 (CV = 78%);mean TRITC intensity 10,997 (CV = 82%);mean FITC/TRITC 0.75 (CV = 9.3%);R = 0.97, N = 61. B Double-labeled with biotin (TRITC) and fluorescein. Mean FITC 7,351 (CV = 64%); mean TRITC 14,221 (CV = 61%); mean FITC! TRITC 0.51 (CV = 21%); R = 0.91; N = 356.

single haptenized probes in a hybridization procedure. A biotin- and digoxigenin-labeled probe were combined in the hybridization mixture; the ISH procedure was kept similar. The fluorescence intensity ratio distribution showed a variation of 20% (CV), and the internuclear variation for the FITC and TRITC intensity was large, with CVs of 39% and 44%, respectively (Fig. 2). To obtain probes with different hapten ratios, the concentration of biotin-11-dUTP and digoxigenin-lldUTP in the nick-translation reaction was varied. Four different probes were obtained in this way, with biotin/ digoxigenin nucleotide ratios of 1:1, l:lO, 1:50, and 1: 100. After hybridization the biotin was developed to FITC and the digoxigenin to TRITC. The effect of variations in the concentration of the

modified nucleotides in the nick-translation reaction on the degree of hapten incorporation was indirectly examined by measurement of the FITC and TRITC intensities after ISH. The mean intensities, standard deviation (SD), and fluorescence ratios are summarized in Table 2. As shown, the mean FITC spot intensities of the different probes tend to increase with increasing bio-11-dUW concentrations in the labeling reaction. The same tendency was found for the mean TRITC intensities and the amount of digoxigenin in the reaction mixture. Also, the ratio of the mean FITC and TRITC intensities corresponds to the biotiddigoxigenin ratio (Table 2). In Figure 3 the FITC intensity of each spot is plotted against the corresponding TRITC intensity of the spots for the 1:1, l:lO, and 1:50 probe. Since the 1:lOO and the 1:50 probes showed considerable overlap, the results obtained with the 1 : l O O probe are not plotted.

DISCUSSION Multiple in situ hybridization procedures are seriously hampered by the fact that the number of simultaneously detectable probes is limited by the number of spectrally separable fluorochromes in the microscope. In practice, so far only three colors can be distinguished, and this number is not expected to increase substantially within the near future. Using these three colors, the number of hybridizations can be expanded by creating combinations of the three colors, resulting in seven permutations. The results presented in this paper show that this number can be increased by the introduction of an additional parameter: the intensity ratio of the colors. Measurement of the FITUTRITC fluorescence ratio of ISH spots obtained by different probes, double-labeled with various combinations of haptens, showed

843

RATIO FLUORESCENCE IN SITU HYBRIDIZATION

Table 2 Quantification of a Ratio-Labeled Probe Specific for Chromosome 1, Labeled With Biotin and Digoxigenin and Visualized With FITC and TRITC, Respectively

TRITC

Bio:dig

Ratio

No. 164 159 163 132

1:l

1:lO 1:50 1:lOO

SD 1164 1836 3414 2765

Mean 2264 3935 6753 4070

(%I

Mean

SD

51 47 57 68

5942 1124 574 388

2915 547 340 262

100000 A

h

. 0

FITUTRITC

FITC

cv

.

that the ratios are constant, with small coefficients of variation. The results obtained with a mixture of two singlehaptenized probes were comparable to those obtained with double-haptenized probes. The advantage of a mixture is the flexibility in varying the ratio, starting from two stock solutions of single-haptenized probes. On the other hand, preferential hybridization of one of the two labels will not occur when the two haptens are on the same molecule. The observation that the internuclear variation of the individual colors was low in this experiment should not be emphasized, since earlier studies (19) show that between experiments the CVs may vary from 35% to as much as 75%. The hapten ratios mentioned throughout the paper refer to the ratio of the labeled nucleotides within the nick-translation reaction. Differently ratio-labeled probes were obtained by varying the amount of labeled nucleotides in the nick-translation reaction. This ratio does not necessarily reflect the ratio of haptens obtained on the probe, since it was noted that polymerase has a preference for biotin above digoxigenin. The effect of changing the concentration of modified nucleotides in the reaction mixture on the degree of labeling of the probe is not known. Measurements showed, how-

cv (%I

Mean

R

49 49 59 68

2.625 0.286 0.085 0.095

0.74 0.93 0.87 0.79

34.6 17.2 27.9 41.5

ever, that the fluorescence intensities increased with increasing concentrations of biotin- or digoxigenin-lldUTP in the reaction mixtures. These results suggested that the amount of label in the nick-translation is reflected in the degree of labeling of the probe. Differences in probe hapten ratios in different experiments are unavoidable, due to variations in nick-translation labeling reactions (for example differences in DNase I activity). This implies that after each ratiolabeling reaction the actual ratio of the obtained probe has to be measured. Another possibility for controling the ratio may be found in the use of a mixture of two singly labeled probes. The desired ratio can easily be adjusted by changing the amount of the singly labeled probes in the hybridization reaction mixture. Variations in detection due to variations in antibody activities will generally affect all ratio-labeled probes to the same extent. These variations can be avoided using the directly fluorochrome-labeled nucleotides for ratio labeling. The fluorescence ratio obtained is a relative value and it depends on a number of parameters, such as the immunocytochemical detection systems, the optical filters, and the exposure times a t the different wavelengths. The mean and standard deviation of the ratio intensity distributions will determine the number of probes (ratios) that can be distinguished. The CVs of the four ratio-labeled probes were not as small as the CV obtained by the biotidsulfonate-labeled probe (9%). Still, three distributions were well separated. It is expected that at least one more ratio is positioned between the distributions of the 1:l and 1:lO hapten ratios. Beyond the 1:l hapten ratio, the inverted ratios can be positioned (up to 50:l; see Fig. 3). This suggests that the number of ratios that might be distinguished is seven for this bi-color approach. This number may be increased by introduction of a third hybridization color. The number of permutations would than be 24, if the accuracy of measurement of the third color is comparable to that of the two colors described. Including a triple-haptenized probe, which was produced successfully using biotin, digoxigenin, and fluorescein, in combination with the double ratioand single-labeled probes, might increase the multiplicity even further. As discussed, a three-color imaging approach could in principle extend the number of

844

NEDERLOF ET AL.

detectable probes; however, without an additional total DNA counterstain for segmentation purposes, this approach is not very practical. With the introduction of new dyes such as the infrared dyes, this approach may be tested. The maximum number of ratios that can be distinguished also depends, besides the CV of the ratio distributions, on the characteristics of the detector, the microscopy, and the cytochemical conditions. The sensitivity of the detector will determine the lower limit to the number of fluorochromes at the target site that can be distinguished. The accuracy of representing the different intensities is determined by the dynamic range and the linear response of the camera. A large dynamic range is required, since ratio labeling implies the introduction of a wide range of different intensities. In general, an 8 bit grey level resolution (256 grey values) is considered to be inadequate. For microscopy, the selectivity of the optical filters should be high; band pass filters with narrow transmission peaks are preferable. High background fluorescence, caused by immunocytochemistry or autofluorescence, will have a negative effect on the signal/noise ratio, and will decrease the effective dynamic range of the detector. In this respect, the use of directly fluorochromized probes is of interest. The experiments presented in this paper were performed on interphase nuclei with chromosome-specific repetitive probes for developmental purposes. For the relative large hybridization spots they provide, a partial image-shift correction was sufficient, since the ratios were determined on basis of the integrated FITC and TRITC spot intensities and not on a pixel by pixel basis. When multiple, smaller targets in close vicinity are to be detected with high spatial resolution and accuracy, it will be diflicult to determine the corresponding FITC and TRITC spots. It may be necessary to determine the fluorescence ratios on a pixel by pixel basis. This requires that the images obtained a t the different wavelengths should exactly coincide. Even a small shift of the image would otherwise perturb the fluorescence ratio measurements. Recent developments in dual band pass filters, which allow excitation and emission for two f luorochromes simultaneously (FITC and Texas Red) (151, will help to solve the image shift problem, although it should be realized that, for the selection of a specific fluorophore by positioning of additional (interference) filters in emission or excitation pathways, image shifts may also be introduced. The dual bandpass filter has been shown to be very useful for the direct visualization of ratiolabeled probes, showing different shades of orange for the greenfred combinations or shades of purple for the redblue combinations. Although this direct visualization of ratio-labeled probes is possible for large targets such as the whole chromosome painting probes, it appeared to be very difficult to distinguish different color shades in smaller targets. To distinguish different colors within a small hybridization spot, intensity mea-

surements and display in pseudo-colors are required (21). Our previous study on quantification of ISH signals showed that the variation of ISH signals is mainly introduced at the cytochemical level. Improvements of ISH quantification procedures, as for the proposed ratio imaging, should therefore be searched for at the cytochemistry level primarily. In conclusion, the experiments described show the feasibility of fluorescence ratio imaging of double-labeled probes for multiple ISH procedures. Presently, we are concerned with the optimization of the procedures for the detection of unique target sequences in both interphase nuclei and metaphase chromosomes. Applications of the fluorescence ratio imaging ISH procedure may be found in tumor cytogenetics for the identification of complex rearrangements by multiple ISH, and for gene mapping purposes. If the ratio and spatial resolution, as well as its accuracy, proves to be sufficient, hybridization-based banding procedures should be feasible in the future.

ACKNOWLEDGMENTS We wish to thank Dr. I.T.Young, Technical University at Delft, the Netherlands, for the use of the CCD camera, and Drs. L. van Vliet and J. Mullikin for software support. LITERATURE CITED 1. Aikens RS,Agard DA, Sedat JW: Solid-state imagers for microscopy. Methods Cell Biol 29:291, 1989. 2. Arnoldus EPJ, Noordermeer IA, Peters, ACB, Voormolen JHC, Bots GTAM, Raap AK, van der Ploeg M Interphase cytogenetics on brain tumors. Genes, Chromosomes Cancer 3:lOl-107, 1991. 3. Arnoldus EPJ, Wiegant J, Noordermeer IA, Wessels JW, Beverstock GC, Grosveld GC, van der Ploeg M, Raap AK: Detection of the Philadelphia chromosome in interphase nuclei. Cytogenet Cell Genet 54:108-111, 1990. 4. Bhatt B, Burns J, Flannery D, McGee JOD: Direct visualization of single copy genes on banded chromosomes by nonisotopic in situ hybridization. Nucleic Acids Res 16:3951-3961, 1988. 5. Cooke HJ, Hindley J: Cloning of human satellite III DNA: Different components are on different chromosomes. Nucleic Acids Res 6:3177-3197, 1979. 6. Cremer T, Landegent J, Brueckner A, Scholl HP, Schardin M, Hager HD, Devilee P, Pearson P, van der Ploeg M . Detection of chromosome aberrations in the human interphase nucleus by visualization of specific target DNAs with radioactive and nonradioactive in situ hybridization techniques: Diagnosis of trisomy 18 with probe L1.84. Hum Genet 74:346-352, 1986. 7. Cremer T, Tessin D, Hopman AHN, Manuelidis L: Rapid interphase and metaphase assessment of specific chromosomal changes in neuroectodermal tumor cells by in situ hybridization with chemically modified DNA probes. Exp Cell Res 176:199220, 1988. 8. Devilee P, Thierry RF, Kievits T, Kolluri R, Hopman AHN, Willard HF, Pearson PL, Cornelisse CJ: Detection of chromosome aneuploidy in interphase nuclei from human primary breast tumors using chromosome specific repetitive DNA probes. Cancer Res 48:5825-5830, 1988. 9. Ernst LA, Gupta RK, Mujumdar RB, Waggoner AS: Cyanine dye labeling reagents for sulfhydryl groups. Cytometry 10:3-10, 1989. 10 Garson FA, van den Berghe JA, Kemshead JT: Novel non-isotopic in situ hybridization technique detects small (1Kb) unique se-

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