Fluorescence in situ Hybridization
Hardware and Software Implications in the Research Laboratory

Nearly a quarter-century has passed since the first research articles introducing in situ hybridization as a method of detecting and studying DNA sequences in chromosomes and cells appeared in the literature. Over the past 15 years, however, a revolution in light microscopy has occurred through the development of fluorescence techniques that allow unprecedented ease, precision, and accuracy in locating, identifying, and recording data on the genetic makeup of biomedical samples.

The power of in situ hybridization can be greatly extended by the simultaneous use of multiple fluorescent colors. Multicolor fluorescence in situ hybridization (FISH), in its simplest form, can be used to identify as many labeled features as there are different fluorophores used in the hybridization. By using not only single colors, but also combinations of colors, many more labeled features can be simultaneously detected in individual cells using digital imaging microscopy.

Presented in Figure 1 is a typical multicolor FISH specimen. Normal male lymphocytes were hybridized with FITC biotin-labeled Chr2l and ChrY probes and CY3 digoxigenin-labeled Chrl3 and ChrY probes. At the upper left is an image of DNA nuclei stained with DAPI, taken using a DAPI filter set. At upper right is an image of Chr21 and ChrY stained with FITC, taken using an FITC filter set. At lower left is an image of Chrl3 and ChrY stained with CY3, taken using a CY3 filter set. The image in the lower right is the joined color composite image showing all target chromosomes in color. The specimen was provided by Dr. Tim Houseal, Integrated Genetics, Framingham, Massachusetts.

The techniques of multicolor FISH, combined with digital imaging technology, today offer unparalleled capabilities for nonisotopic detection of multiple nucleic acid sequences for the analysis of cell components, chromosomes, and genes.

Fluorescence, a phenomenon whereby a chemical excited at one light wavelength emits light at a different and usually longer wavelength, is used throughout the life sciences to study a wide variety of structures and intracellular activities. Advances in probe and microscope technology have led to the rapid development of techniques for fluorescence over the past decade.

This review article will cover the basics of the FISH technique; limitations that researchers have faced over the years in using FISH; recent developments in hardware, software, probes, and reagents that have affected the development of the technique; and current findings in the field. New developments in FISH as the technique begins to expand beyond pure research into clinical diagnostic settings will also be reviewed.

Overview of the FISH Technique

The use of FISH is growing rapidly in genomics, cytogenetics, prenatal research, tumor biology, radiation labels, gene mapping, gene amplification, and basic biomedical research. In principle, the technique is quite straightforward.

The hybridization reaction identifies, or labels, target genomic sequences so their location and size can be studied. DNA or RNA sequences from appropriate, chromosome-specific probes are first labeled with reporter molecules, which are later identified through fluorescence microscopy. The labeled DNA or RNA probe is then hybridized to the metaphase chromosomes or interphase nuclei on a slide. After washing and signal amplification, the specimen is screened for the reporter molecules by fluorescence microscopy.

FISH allows very precise spatial resolution of morphological and genomic structures. The technique is rapid, simple to implement, and offers great probe stability. The genome of a particular species, entire chromosomes, chromosomal-specific regions, or single-copy unique sequences can be identified, depending on the probes used.

Previous Limitations

Until recently, FISH was limited by the hardware, software, reagents, probe technology, and cost involved in implementing the technique.

Commercially available microscope hardware optimized for multicolor FISH was not available until the mid-1990s. Prior to that, microscopes had to be customized for FISH applications. Most microscope optics were not designed to detect the low light levels inherent in FISH signals. As the genomic resolution of the technique has increased dramatically, the requirements on microscope optics have further increased. Chromatic aberrations among multiple wavelengths have been a problem. For multicolor analysis in particular, all lenses, including the collector lens, had to be chromatically corrected. In addition, epi-fluorescence light sources were difficult to align for uniform illumination.

Analysis of multicolor FISH images requires isolation of the various signals either with (a) individual filter cubes; or (b), utilization of an excitation filter wheel with multipass dichroic and barrier filters. Recent developments in filter technology corrected some of the previous problems encountered through optical misalignments caused by mechanical switching of individual filter cubes. Excitation filter wheels used with multi-pass dichroic and barrier filters can be used effectively for up to three colors by employing separate excitation filters for each color with no registration shift. But, for more than three colors, single-pass filters still had to be used.

High-speed color film or CCD (charge coupled device) cameras were used to collect data, and had problems with color fidelity. In addition, there were limitations to the process of superimposing images of different colors acquired with different probes within a single specimen.

Imaging software that could quantitatively analyze samples prepared with fluorescent reagents was also limited because the existing image analysis systems were not optimized to work with fluorescent samples. Visual analysis is a labor- intensive and often subjective procedure, and in some cases, analysis of fluorescent samples was difficult and equivocal without the use of advanced fluorescence imaging capabilities. Researchers typically had to have a software development staff create their own in-house image analysis software.

The reagents and probes themselves were not sufficient for all applications. For instance, the efficiency of hybridization site detection decreased with decreasing probe size, creating significant limits to what could be observed via fluorescence microscopy. The number of differently colored fluorescent dyes was limited, and the photostability of the dyes was poor. But new developments in fluorescent dye technology and spin-off technology from the federally funded Human Genome Project are now having an impact. There are probes for all the human chromosomes and a growing number of new gene-specific probes are available. In situ hybridization kits and fluorescently labeled probes are commercially available from several companies.

Cost was another major hurdle. Since no commercially available FISH system was on the market, researchers had to assemble custom systems, including reagents, probes, microscope, imaging hardware, software, and data analysis and reporting capability. To perform simultaneous multicolor FISH with complex image analysis could cost the researcher upwards of $200,000, a sum not easily accessible to most clinical researchers. As a result, many research scientists who wished to use FISH in their laboratories were inhibited from doing so.

FISH Has Become Widely Available

Many hardware and software manufacturers have developed ways to create affordable commercial alternatives to the custom systems. A spirit of cooperation has developed among many of the firms and laboratories involved in FISH, allowing for new breakthroughs to develop. The authors will reference the developments in their own experience as examples of these kinds of systems.

A system in use in the United States, which provides a mid-priced, commercially available FISH system for research, comprises components from a number of manufacturers and relies on recent developments in image analysis software, microscope hardware, and accessories. Clinical research laboratories will find the system useful for a variety of applications. The system offers the integration and automation to eventually handle clinical test volumes.

The software system illustrated here is MultiFluor (TM) multiparameter imaging software (Biological Detection Systems), a Microsoft® Windows (Microsoft Corp., Bellevue, WA)-based system that can detect, analyze, and display the structural and molecular features of multicolor FISH samples. Analysis time and accuracy are improved by correlating multiple features measured at the various wavelengths in each sample. The system facilitates image acquisition, image storage, database management, microscope automation control, and full-feature graphical data analysis.

Illustrated in Figure 2 is MultiFluor software data review screen. Users can review images and corresponding data together, and correlate multiparameter data from multiple colors (wavelengths) using a variety of interactive graphical plotting tools including histograms, scattergrams, etc. Here, multiple data graphs are shown together with a selected multi-color image set of a cell (showing DAPI-nucleus, FITC-ChrX, CY3-ChrY, and CY5-Chr2l), along with raw data measurements shown in table.

FISH researchers can automatically capture images at multiple wavelengths and multiple focal planes, visualize multicolor FISH probes, annotate and print out images, and store and retrieve large volumes of multicolor image data sets. Multicolor FISH metaphase chromosomes can be analyzed, including gene mapping, CGH (comparative genomic hybridization) ratios, and karyotype generation. User-selected areas of samples can be scanned and analyzed. The software automatically focuses, acquires images at multiple wavelengths, records the slide location of cells, and measures multiple features including probe counts, fluorescence intensity, and cell morphometry. Multiple features from a variety of wavelengths can be correlated.

An additional feature of the system is its ability to work with networked personal computers (PCs). In a typical setup, one PC is an on-line analysis station connected to camera and microscope hardware. This PC handles image acquisition and instantaneous analysis. The other PCs serve as secondary review stations to analyze results produced by the first PC, or to perform specialized analysis off-line.

The software allows all image components to be displayed in vivid pseudocolor for multicolor simultaneous imaging. For example, images of a simultaneous four-color experiment using DAPI (blue), FITC (green), CY3 (red), and a combination of FITC-CY3 (yellow) can all be displayed individually, or combined to form a color composite image (as presented in Figure 1). Each image can be interactively enhanced to reveal characteristics of interest. In addition, histograms, scattergrams, spreadsheets, line plots, and other forms of data tabulation and review are easily created through the software (Figure 2). Finally, data are maintained in an easily accessible popular database format, and can be stored as TIFF, JPEG, GIF, or other files.

Epi-Illumination

Automated microscope hardware is also part of the system. The Optiphot and later model microscope systems (Nikon Instruments Inc., Melville, New York) have an automated, computer-driven XYZ stage and focus system, designed to store the coordinates of each object. Thus, important cellular and chromosomal sites can be instantly recalled on the microscope slide simply be clicking the mouse on the relocate cell button, allowing for more extensive microscope visual examination or review.

Among the most important advantages of the microscope system is the development of an illuminator designed for FISH applications. The Quadfluor (TM) and later model epi-fluorescence illuminators (Nikon Instruments Inc.) accept up to four fluorescence filter cubes and dramatically improve brightness and contrast, which are important benefits for researchers working with multiple fluorochromes.

With the illuminator, researchers performing FISH and other advanced fluorescence techniques can work freely with four or more different probes without ever having to stop to replace filter cubes. Exceptional image registration is ensured by the smooth movement of a very precise linear slider that is used to switch among the four filters.

The filter cubes provide increased brightness and very high contrast through the use of exciter, dichroic, and baffler filter technology; internal light baffling; the high-precision oblique mounting angle of the baffler filter; and proprietary antireflective coatings. In addition, the epi-fluorescence illuminator can accommodate up to four cubes at a time for separating different fluorescent signals, or may be used with a multiband filter cube to image several fluorochromes simultaneously.

High-transmission objectives are also essential for imaging the minute areas of interest on chromosomes. The CFI-60 plan fluor objectives (Nikon Instruments Inc.) feature optical cements and high-transmission coatings to permit broader wavelength ranges and brighter images. The objectives are free of color and spherical aberrations, and offer extremely high contrast and low background autofluorescence.

Additional computer-controlled microscope accessories to create advanced motorized systems that will be modular and even more affordable for research and clinical applications are currently under development by several manufacturers. Cameras are another important consideration for the system. Images can be acquired using digital CCD or intensified video cameras. The key factor in the systems is their ability to perform low-light imaging without sacrificing fidelity. With appropriate software, camera settings can be automatically set via the fully automated microscope for accuracy and ease of use.

Cancer, Prenatal, and Biology Research

The FISH systems described here, designed to be accessible to more researchers than the older, customized systems, are moving into wider use in cancer research, pathology, cytogenetics, and developmental biology. Among their applications are the analysis of interphase cells for multicolor probe spot counts, immunophenotyping, cell morphometry, and DNA content.

The system is in use testing for aberrations in chromosome copy number, correlated with total DNA content, associated with urinary bladder tumors. In prenatal research, the system can be used to test for anneuploidies in interphase nuclei associated with prenatal defects including Down syndrome, Turner syndrome, Klinefelter syndrome, and others. In cell and developmental biology research, the system can be used to map the presence and relative distributions of cell surface markers such as receptors, cytoplasmic markers including cytoskeletal proteins, messenger RNAs, and specific genes.

Diagnostic Potential

For the past decade and a half, researchers have known that the FISH technique has extraordinary potential not only as a tool for pure research but for clinical diagnostic use in such areas as prenatal diagnosis, cytogenetics, and tumor evaluation. The lack of a high-level, moderately priced system has not only slowed down the rate at which FISH has become available to most researchers but has also prevented this inevitable expansion of the technique's accessibility in diagnostic health-care settings.

One of the most important ramifications of this system is that it can provide the high-level results that only costly, custom-configured systems could provide in the past. The dream of applying FISH not only to a wider range of biomedical research applications but also to serve the direct needs of patients will probably be a reality in the not-too-distant future.


BACK TO FLUORESCENCE MICROSCOPY

References

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Contributing Authors

Mel Brenner - Nikon Inc., 1300 Walt Whitman Rd., Melville, New York, 11747.

Terry Dunlay - Biological Detection Systems, Inc., Pittsburgh, Pennsylvania, 15238.