In 1974, Evans first combined chromosome banding techniques with in situ hybridization to improve localization accuracy. In the late 1970s, researchers began exploring fluorescence-labeled in situ hybridization, known as FISH. In 1981, Harper successfully localized a single-copy DNA sequence to a G-banded specimen, marking significant progress in chromosome localization techniques. The 1990s saw rapid development and widespread application of FISH due to the need for high-resolution human genome mapping as part of the Human Genome Project.
FISH (fluorescence in situ hybridization) is an important non-radioactive in situ hybridization technique. The basic principle of FISH is that if the target DNA on a chromosome or DNA fiber section is homologous and complementary to the nucleic acid probe used, they can form a hybrid after denaturation, annealing, and renaturation.
By labeling one of the nucleotides in the nucleic acid probe with a reporter molecule such as biotin or digoxigenin, the specific immunochemical reaction between the reporter molecule and a fluorescently labeled avidin can be utilized for qualitative, quantitative, or relative positional analysis of the target DNA under a fluorescence detection system.
In situ hybridization probes can be divided into radioactive and non-radioactive based on the type of labeling molecules used. Radioactive probes have the advantage of being less demanding on sample preparation and can enhance signal strength by extending exposure time, making them relatively sensitive. However, radioactive probes are unstable, have long auto-radiography times, and the scattered radiation results in low spatial resolution, making isotope operation cumbersome.
Fluorescence labeling systems overcome these shortcomings, leading to the development of FISH technology. As a non-radioactive detection system, FISH has several advantages:
Disadvantages:
FISH technology can be used for chromosome localization of known genes or sequences, and also for studying uncloned genes, genetic markers, and chromosome abnormalities. It is particularly advantageous in studies involving gene qualification, quantification, integration, and expression.
After the establishment of basic FISH technology, FISH was used not only for single gene or nucleic acid detection but also expanded to multicolor FISH for simultaneous detection of multiple gene sites. This expanded from gene detection to genome, chromosome, in situ detection of mRNAs in live cells, and nucleic acid detection at the tissue level. Early probes were larger, prepared through vector proliferation, nick translation, in vitro transcription, and random primer DNA synthesis to obtain specific hybridization clones.
However, large fragment probes typically contained repeat sequences, resulting in high fluorescent backgrounds. Using unlabeled nucleic acids for pre-treatment to bind to non-specific sites to inhibit non-specific hybridization can overcome these problems, allowing researchers to expand detection targets and achieve whole chromosome staining.
In cytogenetics, FISH technology has significantly improved chromosome analysis, such as using comparative genomic hybridization (CGH) to detect chromosome region deletions and duplications. Large fragment probes, once non-specifically bound to samples, would form a signal, confusing gene detection on chromosomes, necessitating shearing into smaller fragments (<200 nucleotides).
Advances in detection methods and software have reduced FISH detection requirements and increased sensitivity. Precise computational image processing algorithms have formed high-resolution sub-microscopic probe techniques.
As detection targets become smaller, FISH technology has been used for hidden subtelomeric karyotype gene rearrangements and precise chromosome mapping and single-copy mRNA detection.
The expansion of FISH detection scope led to a rapid increase in FISH technology applications in the 1990s. FISH-related branch technologies enabled simultaneous detection of an increasing number of different types of sites.
Initially, different fluorophores were used to detect multiple sites, such as dual-color fluorescence for detecting specific nucleic acid sequences, where each chromosome, gene, or transcript was represented by a distinguishable fluorescent signal. Subsequent color coding schemes further expanded FISH applications, mainly based on the proportion of each color in the total color to depict multiple sites.
These methods, or their combinations, have enabled detection of up to 12 sites. Using a computer-translated five-color scheme, all human chromosomes can be detected simultaneously, marking a milestone in FISH multi-site detection. Although various methods can detect mRNAs, FISH seems more promising for in situ analysis of entire transcripts. Color coding techniques have achieved whole tissue detection.
In 1986, Pinkel et al. first used quantitative analysis of fluorescent images for basic cytogenetic detection, employing a dual-color excitation block device camera to detect fluorescent signals. Quantitative analysis techniques soon applied to mRNA detection. Key aspects of fluorescence detection include signal reproducibility, randomness, and background autofluorescence. Fluorescence varies not only between different samples but also within the same slide or identical cells.
Various methods are used to eliminate autofluorescence in some tissues, such as sodium borohydride treatment or light irradiation pre-treatment during sample preparation to eliminate non-specific background signals. These methods are not entirely effective, so computational arithmetic is typically used during image analysis to remove autofluorescence signals.
Spectral data from fluorescent images include true signals and noise, which are separately analyzed and removed through individual spectral component analysis. Multicolor FISH has limitations, including varying fluorescent intensities and color overlap. However, computer algorithms balance multicolor images, including intensity changes and automatic signal overlap correction.
The limitations of FISH images have not hindered the development of automated decoding algorithms. Using larger probes for DNA site detection and multicolor fluorescence counting algorithms has aided pathologists in achieving automated analysis. Additionally, probe kit applications and point counting methods have provided a platform for convenient detection results.
Although various methods have been used to analyze or optimize automated cell detection systems, manual cytopathological detection remains a highly reliable tissue analysis method. However, the high efficiency of computerized cell preparation, identification, and sample detection on fixed media in future medical diagnoses should not be overlooked. Rapid detection of molecular signals within cells can only be achieved through computerized methods. Currently, automated detection programs have expanded to multi-gene transcription models to detect specific DNA clusters and transcription sites, determining the functional status of cells.
Considering FISH’s early development focused on probe types and detection sites, future advancements in fluorescent detection technology may include expansion in detection fields. Clinical diagnostic applications of fluorescent images require further improvements in detection systems, such as probe binding, imaging, and automated analysis, to avoid operational errors between different processes. Sample thickness limits the types of samples detectable by fluorescent microscopy. Recent laser confocal microscopy and optical X-ray tomography techniques require sample thicknesses of 1-2 mm. An improved optical projection X-ray microtomography technique can image samples up to 15 mm thick, expanding the detection range for biological and diagnostic samples.
FISH technology for detecting RNAs in live cells has also been reported, using either in vivo released fluorescent groups or post-hybridization probe fluorescence detection. Both new methods reduce high background from non-specific labeled probes (such as in live cells) and can track mRNA synthesis and transfer pathways. These methods are easier to detect different target molecules compared to green fluorescent protein (GFP). FISH’s susceptibility to internal probe synthesis in cells affects live cell in situ hybridization detection more than GFP.
FISH needs further improvements to reduce interference backgrounds when detecting gene expression in living organisms and avoid interference from internal hybridization products. FISH and fluorescent protein techniques can be combined to detect target nucleic acids and proteins simultaneously.
The application of multiphoton microscopy further expands the range of fluorescent image applications. Multiphoton microscopy uses laser blocks to emit photons that focus on the microscope to excite the target fluorophore two or three times. Near-infrared excitation light can penetrate biological samples deeper and is less toxic to living samples than visible light. This new method has applied fluorescent images to detecting living systems, even entire animals.
Since biological marker probes cannot yet be synthesized, current applications of in vivo fluorescent imaging are limited to detecting fluorescent molecules or autofluorescence in living organisms.
Autofluorescence signals produced by biological tissues during normal physiological or pathophysiological processes can serve as important diagnostic signals. Once biological probes become feasible, they will be a powerful auxiliary tool for identifying specific nucleic acid sequences, conducting non-invasive diagnostics, and obtaining diagnostic images.
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