Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
* Author for correspondence (e-mail: rhsinger{at}aecom.yu.edu)
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Summary |
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Key words: FISH, DNA, RNA, Fluorescence, Imaging, Microscopy, Hybridization, Computer image processing
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Early years |
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FISH for visualization of nucleic acids developed as an alternative to
older methods that used radiolabeled probes
(Gall and Pardue, 1969). Early
methods of isotopic detection employed non-specific labeling strategies, such
as the random incorporation of radioactive modified bases into growing cells,
followed by autoradiography. Several drawbacks of isotopic hybridization
inspired the development of new techniques. First, the very nature of
radioactive material requires that the probe is unstable; the isotope decays
over time and so the specific activity of the probe is not constant. Second,
although sensitivity of radiography is generally high, resolution is limited.
Third, long exposure times are often required to produce measurable signals on
radiography film, delaying results of the assay. Fourth, radiolabeled probe is
a relatively costly and hazardous material and it must be transported,
handled, stored and disposed of in accordance with regulations. FISH allowed
significant advances in resolution, speed and safety, and later paved the way
for the development of simultaneous detection of multiple targets,
quantitative analyses and live-cell imaging.
The first application of fluorescent in situ detection came in 1980, when
RNA that was directly labeled on the 3' end with fluorophore was used as
a probe for specific DNA sequences (Bauman
et al., 1980). Enzymatic incorporation of fluorophore-modified
bases throughout the length of the probe has been widely used for the
preparation of fluorescent probes; one color is synthesized at a time
(Wiegant et al., 1991
). The
use of amino-allyl modified bases (Langer
et al., 1981
), which could later be conjugated to any sort of
hapten or fluorophore, was critical for the development of in situ
technologies because it allowed production of an array of low-noise probes by
simple chemistry. Low probe specific activity prevented the assessment of
nucleic acids with low copy number by FISH. Methods of indirect detection
allowed signal output to be increased artificially by the use of secondary
reporters that bind to the hybridization probes. In the early 1980s, assays
featuring nick-translated, biotinylated probes, and secondary detection by
fluorescent streptavidin conjugates were used for detection of DNA
(Manuelidis et al., 1982
) and
mRNA (Singer and Ward, 1982
)
targets. Approximately a decade later, improved labeling of synthetic,
single-stranded DNA probes allowed the chemical preparation of hybridization
probes carrying enough fluorescent molecules to allow direct detection
(Kislauskis et al., 1993
).
Many variations on these themes of indirect and direct labeling have since
been introduced, giving a wide spectrum of detection schemes from which to
choose; the specifications, sensitivity and resolution of these techniques are
well described elsewhere (Raap,
1998
).
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Coming of age |
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One of the problems with larger probes was that they had to be cut into
small pieces of <200 nucleotides
(Lawrence and Singer, 1986).
If a large probe adhered to the sample non-specifically, it would appear to be
a signal, because the great number of fluorochromes would generate an
intensely fluorescent spot. This would, for instance, confound detection of
genes on chromosomes. Double-stranded DNA probes hybridize within tissues and
cells, giving high background. Reduction of probe size led to improved
signal-to-noise ratio and thus to single-copy detection of genes on
chromosomes (Lawrence et al.,
1988
). Enhancements in detection and computer processing
algorithms have subsequently allowed detection of smaller and smaller targets.
Advances in microscope and detector hardware have allowed the low light level
produced by FISH to be recorded and analyzed with increasing sensitivity
(reviewed by Arndt-Jovin et al.,
1985
). Mathematical image-processing algorithms have built on this
progress to yield super-resolution technology to probe at submicroscopic
resolution, using digital image stacks
(Carrington et al., 1995
). In
the trend towards detection of smaller entities, cytogeneticists have focused
on analysis of cryptic sub-telomeric karyotypic rearrangements
(Brown et al., 2000
) and the
precise chromosomal mapping of genes (reviewed by
Palotie et al., 1996
). Those
working on mRNA detection can assay single mRNAs and even parts of RNAs
(Femino et al., 1998
).
New targets led to new applications of the FISH procedure, the popularity
of the assay increasing dramatically in the 1990s
(Fig. 1). The new avenues of
research opened by these applications required that more and more species be
simultaneously detected. At first, this was achieved by simultaneous
visualization of spectrally distinct fluorophores
(Hopman et al., 1986); later,
strategies using two principal encoding schemes augmented the approach. First,
specific nucleic acid identities can be denoted by binary color combinations,
such that each chromosome, gene, or transcript is represented by a unique
combination of distinct fluorescent signatures. A second scheme employs ratio
identity codes that use the same color combination to delineate multiple
targets by varying the relative contribution of each color to the total
signal. Each of these schemes, as well as the concurrent use of both
approaches has raised the number of nucleic acid targets that can be
simultaneously detected to dozens (reviewed by
Fauth and Speicher, 2001
)
(Table 1). A major milestone in
the detection of chromosome targets was the discrimination of all human
chromosomes simultaneously, using computed interpretation of a 5-color scheme
(Schrock et al., 1996
;
Speicher et al., 1996
).
Although mRNAs can also be visualized in a multiplex fashion
(Levsky et al., 2002
), FISH
analysis of the entire transcriptome in situ is a daunting thought
(Fig. 2). One can only
speculate that future technologies will feature increasingly higher-order
multiplexing, until the number of interesting nucleic acid targets is reached.
The technical means for color coding such a large number of entities is
already in place (Han et al.,
2001
), although reduction to practice will be difficult and a
means of deciphering spatially overlapping signals will need to be
developed.
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Quantitation and analysis |
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Difficulties inherent in objective analyses of FISH images have impeded but
not stopped the development of automated algorithms for interpretation.
Detection of DNA targets with large probes and the use of multi-color
fluorescent cytometry algorithms
(Galbraith et al., 1991) have
allowed the production of automated mechanisms for assisting pathologists
(Piper et al., 1994
). In
addition, the use of diagnostic probe sets and dot-counting approaches have
yielded independent platforms capable of making simple diagnostic conclusions
(Piper et al., 1990
). Although
methods have been introduced to analyze and optimize these cell classifiers
(Castleman, 1985
;
Castleman and White, 1980
;
Castleman and White, 1981
),
manual cytopathology remains the gold standard for reliable tissue analysis,
and automated mechanisms that can yield comparable data are still in
development. Nevertheless, the benefits of high-throughput analysis of cell
preparations, namely objective, computerized interpretation of cell samples on
fixed substrates cannot be understated in the future development of diagnostic
medicine. Although morphological analyses remain best suited to human
operators, the ability to assay many molecular signatures rapidly in cells is
only possible through computer-assisted approaches. Automated processing has
recently been extended from the detection of specific DNA loci
(Lawrence et al., 1988
) and
sites of transcription (Lawrence et al.,
1989
) to the determination of functional cell states by multi-gene
transcriptional profiling (Levsky et al.,
2002
). The ability to assess accurately the transcriptional state
of individual cells in situ has begun to influence the way we conceptualize
single-cell versus tissue-level gene expression as well as study transcription
activation, co-expression, and nuclear structure-function
(Levsky and Singer, 2003
).
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Advancing technology |
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FISH techniques for detecting RNAs have been introduced to living cells
(reviewed by Boulon et al.,
2002), using either fluorophores that can be 'un-caged' in vivo
(Politz and Singer, 1999
) or
probes that fluoresce only when hybridized
(Tyagi and Kramer, 1996
). Both
of these innovative approaches circumvent the high background usually found in
scenarios with unbound probe present (such as living cells), to allow the
investigator to follow the creation and travels of mRNAs. These approaches are
more easily applied to different target molecules than non-hybridization based
GFP-fusion protein systems that bind a unique nucleic acid motif
(Bertrand et al., 1998
). One
drawback of live-cell in situ hybridization as opposed to GFP-based assays is
that FISH requires mechanical disturbance of the cell to introduce probes.
These techniques allow deeper study of live gene expression in a minimally
disturbed context, but must be interpreted with consideration of the possible
artifacts that may result as physiological ramifications of hybridization. The
separation drawn between approaches using fluorescent proteins and FISH should
not be considered absolute. In fact, the compatibility of FISH and
technologies employing fluorescent fusion proteins promises to allow
simultaneous monitoring of proteins and nucleic acids of interest.
The use of multi-photon approaches will also expand application of
fluorescence imaging. In multi-photon microscopy, a laser source fires short
bursts of photons that are focused by the microscope to arrive in pairs or
triplets such that they summate to excite the fluorophore of interest.
Near-infrared excitation light is used, which penetrates biological specimens
more deeply and is far less toxic to live samples than visible light. This
scheme has already allowed the application of fluorescence imaging to many
living systems, including whole animals. Owing to limitations of our ability
to introduce synthetic probes into organisms, most current applications of in
vivo fluorescence imaging involve naturally occurring fluorescent molecules or
bioluminescence (reviewed by Contag and
Bachmann, 2002). Native fluorescent signatures that are present in
tissues due to normal physiology or pathophysiological processes can encode
important clinical information (reviewed by
Konig, 2000
). Should a form of
organism-friendly probe become a reality, the power to discern many specific
nucleic acids could be applied to non-invasive diagnostics, providing an
informative adjunct to current methods of medical imaging.
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Diagnostic FISH |
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Surprises are in store, however. The stochastic nature of gene expression
revealed by this kind of approach indicates that perhaps our conception of a
precisely regulated gene expression pattern is too constrained. Higher levels
of tolerance for diversity in cell expression patterns may require a different
model (Levsky and Singer,
2003). Computer-interpreted FISH assays are now sufficiently
advanced to provide enormous amounts of data from a single cell, and even more
from a tissue section. Measurement of expression from 20 genes by scoring for
activity of neither, both, or one of the two alleles as mere binary 'on or
off' signals, yields 320 or greater than three billion bits of
information per cell. If expression information concerning 100 genes were to
be assayed, the information density would increase to 3100 bits (on
the order of 1047). This exponential increase indicates that
high-throughput data processing of gene expression information will have to
evolve with the technology. The mere enormity of data may reveal insights not
dreamt of in our philosophy.
The ability to visualize RNA movement in living cells will provide models for how and where specific sequences are expressed and the steps by which transcripts are processed and exported from the nucleus. Our understanding of infectious disease will benefit from elucidation of how retroviruses direct nuclear import, trafficking and packaging for export into infectious particles. We are just beginning to understand the mechanisms by which specific RNAs are localized to subcellular regions of oocytes and some somatic cells for the purposes of asymmetric translation and how this is used to effect permanent structural changes - for instance, in synaptogenesis. When the tools become available for us to visualize multiple gene expression patterns in living cells, we will finally be able to fulfill the promise of FISH technology by building and testing models of molecular transcriptional dynamics within the true native context of the cell.
The traditional route to diagnosis has been through morphological evaluation of biopsy specimens and the correlation of this analysis with clinical outcomes. The morphological basis of diagnosis has its limitations. It is well known that tumors that look alike under the microscope, and that appear phenotypically similar, may have radically different clinical courses in real patients. The new field of molecular pathology attempts to obviate the ambiguities of morphology by studying the origins of disease through characterization of genetic mutations and gene products. These investigations promise to provide more reliable biomarker information, founded on recent bioinformatics advances made possible by expression studies using micoarrays and serial analysis of gene expression (SAGE). Transcriptional alterations associated with malignant transformation and markers that correlate with cancer progression are being identified. The mechanism by which these data can be incorporated in the pathologist's 'tool box' is currently being developed.
The recently developed tissue microarray technology is an ideal platform for the introduction of high-throughput molecular profiling of tumor specimens at the single cell level. To construct a tissue microarray, small core biopsies are taken from representative areas of paraffin-embedded tumor tissues and assembled in a single block. Microtome sections are taken from the tissue microarray and placed on glass slides for rapid and efficient molecular analyses. In addition to pathological specimens such as tumor tissues, microarrays generally contain corresponding normal tissue and internal controls. The entire group of samples is analyzed simultaneously in one experiment, providing enormous amounts of correlative information about specific biomarkers, in the context of rigorous procedural controls. The next challenge will be to apply multi-gene FISH technology to these samples to correlate putative genes of prognostic value with specific morphological features initially, and then extend studies to samples where the morphology is not sufficiently informative. Certain genes can then be associated with the pre-cancerous state, for instance. Through such developments, one can foresee how molecular pathology could eventually surpass the limitations of morphological pathology. This would allow more judicious use of minimally invasive biopsy techniques that sacrifice retrieved tissue morphology in favor of comfort of the patient. FISH has already colored the way that we visualize and conceptualize genes, chromosomes, transcription and nucleic acid movements. What remains to be seen is how exhaustive molecular analysis of single cells and tissue samples will impact how we identify, diagnose and alter the course of genetic pathology. Over the long term, it is expected that databases correlating gene expression patterns on the single cell level will accumulate as investigators and industries employ the technology of FISH with their favored biomarkers. Ultimately, FISH will be the preferred approach to anticipate the complicated components of gene expression leading to disease.
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Acknowledgments |
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