Currently, it is estimated that no fewer than 1 million cytogenetic and molecular cytogenetic analyses are performed per year representing the standard of care in several fields of medicine and the routine clinical work-up for numerous patients suffering from congenital malformations, mental diseases, cancers, or reproductive problems 1. Molecular cytogenetic techniques have been repeatedly proven effective in diagnostics and have been recognized as a valuable addition or even alternative to chromosomal banding 234. Furthermore, contemporary basic biomedical research widely applies molecular cytogenetic technologies 567. Browsing the most popular scientific resources would undoubtedly return several tens of thousands of articles, which mention at least one molecular cytogenetic technique (for more details refer to 3 and web page about multicolor fluorescence in situ hybridization at http://www.med.uni-jena.de/fish/mFISH/mFISHlit.htm managed by Dr. Thomas Liehr, Jena, Germany). Thus, one can be certain that it is hard to overestimate the role of molecular cytogenetics in current biomedicine.

There are two main advantages that molecular cytogenetics possesses: (i) the ability to provide either an on-chip scan of the whole genome at extremely high resolution or visualization of single peculiar genomic loci 468; (ii) the capability to analyze genome organization, structure and behavior in single cells at the DNA (RNA) sequence level 7910. Both are continuously used for biomedical research and molecular diagnosis of chromosome abnormalities in humans 2345678910111213. The first advantage is appreciable when analyzing mixed DNA isolated from large amount of cells. Therefore, it is unsurprising that such approaches are rarely used for single-cell analysis 1014. The second advantage of molecular cytogenetic techniques is consistently emphasized 35678910111213, but is used more commonly for studying mitotic cells via analyzing metaphase chromosomes 371012. However, cells of eukaryotes are more likely to be in interphase. Therefore, during surveys of genome organization, structure and behavior, essential part of cellular life is usually fallen out of researchers' scope. As to molecular diagnosis of chromosome abnormalities, one can notice that interphase analysis is uncommonly applied, as well. The explanation of leaving interphase cytogenetics aside from diagnostics and research might be a suggested lack of reproducibility and low resolution. A brief look through studies of genome architecture in interphase nuclei 1516171819 and somatic genomic variations 7101220212223242526272829 as well as developments in interphase cytogenetics 303132333435 will reveal such assumptions unsupported and will show that laboratories elaborating such techniques are able to solve different practical and research tasks without major difficulties 37121314151617181920212223242526272829303132333435. It seems thereby that preferences to use interphase molecular cytogenetic techniques suffer rather from "insufficient publicity" than from "technological underdevelopment".

Looking through the voluminous amount of reviews dedicated to molecular cytogenetics, we have found occasional descriptions of both technological and theoretical side of visualizing human chromosomes in interphase. Consequently, we were forced to conclude that undeservedly little attention is paid to interphase molecular cytogenetics in modern biomedical literature. Additionally, technical side of the application is even more rarely addressed. To fill this gap, we have attempted to give an overview of currently applied molecular cytogenetic techniques with a special emphasis on their technological abilities for studying human interphase chromosomes.

The overwhelming majority of molecular cytogenetic techniques are based on hybridization. There are currently two essential platforms available for developments in molecular cytogenetics: fluorescence in situ hybridization (FISH) including comparative genomic hybridization (CGH) 336 and peptide nucleic acid (PNA) probing for analysis of chromosomal DNA 3738. Alternatively, another technique uses primed in situ labelling (PRINS) reaction 3738. The resolution and level of excellence of all these techniques are established against cytogenetic banding analysis, which remains the golden standard in this instance 36.Single-cell molecular cytogenetic analysis can be performed either through analysis of metaphase plates or through analysis of interphase nuclei. Studying metaphase plates has been long described to be successful using several detection technologies (i.e. spectral karyotyping -- SKY or multicolor FISH -- MFISH) and different DNA probe sets (chromosome-enumeration/centromeric, site-specific, whole-painting, microdissected) 2356791011121330363940414243444546. In general, if modified, almost all these techniques can be applied to interphase cells, but this "transfer of technology" requires significant efforts 237101213303132333547. Generally, all molecular cytogenetic assays that provide for visualization of genomic loci in an interphase nucleus are termed interphase FISH or I-FISH 35. Table 1 gives an overview of molecular cytogenetic techniques that are used for metaphase and interphase analysis with special attention to the resolution and to the modifications for studying single cells. The impossibility of listing all known molecular cytogenetic approaches seems to be apparent, but even a short description of such techniques (Table 1) shows molecular cytogenetics able to perform high-resolution analysis of chromosomal structure and behavior at all stages of cell cycle, being, nevertheless, more frequently use to detect metaphase chromosome imbalances and rearrangements or to operate with total DNA for probing in CGH analysis 2345671011121314192021222324252627282930313233343536373839404142434445464748495051525354. Further, we attempt to review each aforementioned approach in context of applications to single-cell chromosomal analysis.

I-FISH as all other FISH-based methods roughly requires three steps to be performed: (i) obtaining cells suspensions or performing another preparations of biopsies for the analysis; (ii) denaturation/hybridization; (iii) microscopic visual/digital analysis of hybridization results 133578. The first stage is not associated with any limitation of I-FISH, because any cell type of a human organism can be processed for such analyses 73578. This is considered the essential advantage of interphase molecular cytogenetic techniques in contrast to classical cytogenetics (analysis of metaphase chromosomes) -- the ability to analyze chromosomes in all the tissue (cell) types 37132021222324252627282930313233343536. Classically, I-FISH was suggested to be limited to analyses of specific genomic loci 23713. However, some modifications such as ICS-MCB allow to get a view of interphase chromosomes in their integrity 2324262728293133343574. As mentioned before, ICS-MCB still have a limitation that is referred to the possibility of studying only one homologous chromosome pair per analysis (metaphase chromosomal analysis allows to visualize all chromosomes of a cell), being, however, the unique way to visualize the whole banded chromosome in a nucleus 2333. Denaturation and hybridization steps of I-FISH are performed identically to metaphase FISH-based approaches 1335. Therefore, no additional drawbacks can be attributed to these procedures during interphase molecular cytogenetic studies. Scoring of I-FISH results is usually performed via conventional visual analysis 3579. However, there are numerous possibilities to apply digital analysis for studying interphase chromosomes. These include, but are not restricted to, QFISH, analysis of signal co-localization (oncocytogenetic studies of gene fusions because of translocations in interphase nuclei), ICS-MCB (visualization of chromosomal structures), increasing of FISH result visibility, automatic signal detection 79. Furthermore, digital analysis is a need for multicolor FISH-based assays (SKY, MFISH, multiprobe interpahse FISH or mFISH), which are usually applied to increase the potential of FISH applications through simultaneous analysis of multiple targets 2312132021222324252627282930353680. Combining several aforementioned techniques mFISH with 2-5 probes (colors) per assay, QFISH and ICS-MCB) has become a basis for an integrated approach proven to be of highest efficiency for molecular diagnosis and genome/chromosome researches at supramolecular level in interphase 7101213202122232425262728293032333541607881. The type of FISH result evaluation (i.e. visual or digital) is determined by the type of assay or, more precisely, by features of DNA probes (amount of probes per reaction and DNA sequence affinity) and detection. Therefore, to get an overview it is to subdivide I-FISH techniques this way. Table 2 gives such overview.

Spatial chromosome organization in interphase has been repeatedly shown to be a driving force for numerous crucial intracellular processes. It is suggested that specific arrangement of interphase chromosomes is likely to associate with genome activity, normal/abnormal cell division, chromosome rearrangements occurring during meiosis and mitosis 715161769197071727593100101. To get an integrated view of genome organization in interphase, numerous approaches should be applied. The leading role in these studies is played by I-FISH 7807293. There could be several applications of I-FISH approaches for interphase chromosome analysis on this occasion: (i) identification of chromosome positioning and its relation to other nuclear compartments (nucleolus, Cajal bodies, nuclear speckles etc.) -- I-FISH with wcp, interphase MFISH or ICS-MCB 1931233335347071727493; (ii) studying correlation between positioning of specific genomic loci in relation to each other (i.e. association of whole chromosomes or their regions) and their behavior (transcriptional/replicative activity) for elucidating functional meaning of nuclear organization and its driving forces -- I-FISH with centromeric, site-specific and wcp, mFISH/QIFSH or ICS-MCB 719312333353234576669727475939495100; (iii) analysis of chromosome behavior in relation to genome, epigenome and proteome changes for delineation of possible consequences of specific interphase chromosome architecture (i.e. occurring of somatic chromosomal mutations in cancers) -- I-FISH with centromeric, site-specific and wcp, mFISH/QIFSH, ICS-MCB and Immuno-FISH 71516171819346971727475939495100101. Additional complication of I-FISH analysis of spatial chromosome organization is associated with structural preservation of nuclei. It is to note, that some researchers report about dependence of fixation type on I-FISH results 7293, whereas others do not 71. Regardless these debates, an alternative for I-FISH spatial genome analysis could be a suspension FISH (S-FISH) technique 102. The advantage of this approach is related to possibility of studying three-dimensional (3D) preserved nuclei from any human tissue, whereas other 3D preservation techniques require specific conditions of cell cultivation. The latter makes I-FISH to lose its main advantage. Together, it is to conclude that comprehensive description of functional significance of nuclear organization requires application of almost all known interphase molecular cytogenetic techniques.

During the last half decade, genomic variations -- a source of human healthy and pathological diversity -- have become a major focus of current biomedical research. Being involved in evolutionary and disease pathways, variations of the human genome are considered the main target of researches aimed to uncover disease mechanisms and species origins 103. Soon after description of high rate of interindividual genomic diversification, it has been hypothesized that related processes-- somatic genomic variations -- lie at the origin of intercellular genomic differences. Moreover, somatic variability of cellular genomes was proposed as a mechanism for complex human diseases 71012. The latter has been partially confirmed by high-resolution interphase molecular cytogenetic (molecular neurocytogenetic) studies of neurological and psychiatric diseases 720212223242526272829. The growing evidence for contribution of somatic genomic variations to the key physiological processes has been used for further hypothesizing about the emerging role of cell-to-cell genome variability in normal/abnormal human intrauterine development (including exogenous effects), cancerization, tissue-specific pathology (i.e. targeted neurodegeneration), sex differences in complex diseases, responses to molecular therapy of debilating neurological disorders 2122242829104105106107108109. Altogether, this forms a basis for forthcoming researches in the field of single-cell biology. All these achievements were the result of numerous developments in interphase molecular cytogenetics. To prove it, we would like to refer to determination of stochastic (sporadic or background) aneuploidy level in human tissues (Table 3) 20212223245928293581109110111112. Looking through these data, it is hard to avoid the conclusion that aneuploidy rates become more reasonable if high-resolution I-FISH approaches are applied. Additionally, interindividual genomic variations can be detected in interphase by a parent-of-origin-determination FISH (pod-FISH) technique 113. Together, I-FISH can be proposed as a required addition for studying genomic variations at microscopic and submicroscopic levels.

Molecular cytogenetic identification of chromosomal aberrations by I-FISH has been already mentioned in this review. Here, we would like to make some additional comments related to more specific problems of medical cytogenetics and to show again that studying chromosomes in interphase nuclei has profound effects on molecular cancer and prenatal diagnosis 114115. It is obvious that it is almost impossible to refer all the studies that used I-FISH. Here, we have preferred to describe several difficulties encountered during I-FISH introduction and usage for diagnostic purposes. Newly introduced interphase techniques (i.e. ICS-MCB) were used for research purposes only and, therefore, have not been tested for diagnostic validity. Despite of limiting practical application of these I-FISH protocols, related drawbacks can be easily eliminated by forthcoming studies. Another problem comes from the diagnosis of chromosomal mosaicism. There do not exist commonly accepted guidelines or criteria for mosaicism definition 71035. Regardless some attempts (for details see 35), there is still no consensus concerning this topic. The solution would be a large-scale study aimed to uncover somatic genomic variations in unaffected human tissues. Hopefully, similar studies have been already launched 20212223245928293581. Finally, there are still no data or recommendations concerning correlation between metaphase and interphase diagnostic analysis of the same individual. In other words, it is still poorly understood what data is more valid. The structural point of view insists that metaphase analysis of chromosomes is more precise. From the other hand, mosaics require large cell populations to be analyzed. It becomes even more difficult to solve this problem when cases of complex, hidden (cryptic) or dynamic mosaicism are attempted to be described. Metaphase analysis in these case is indispensable for thorough definition of all cell lines, because simple I-FISH analyses are unable to precise a percentage of each cell line 116117. Moreover, some studies require additional data to obtain, i.e. parental origin of chromosomes or some epigenetic features for more thorough confirmational or exclusive diagnosis. To get this opportunity, it is to apply QFISH 32 or pod-FISH 112.

It is widely accepted that molecular cytogenetic diagnosis should be performed using a panel of techniques 12345678910. It could be either a combination of molecular cytogenetic techniques that use different platforms (i.e. FISH+CGH) or consecutive metaphase and interphase FISH analyses in cases of complex mosaics or balanced structural chromosome abnormalities. Thus, regardless significant developments in the field of molecular interphase cytogenetics, I-FISH techniques remain an addition to metaphase cytogenetics or whole genome screening approaches based on array CGH. The exception is few targeted assays for identification of known caner-associated translocations in interphase and preimplantation genetic diagnosis. Consequently, I-FISH should be more thoroughly analyzed in terms of the diagnostic potential to take a well-deserved place among genetic testing procedures.

Structural and behavioral properties of human interphase chromosomes in different tissue/cell types in health and disease remain largely unknown. To date, only fragmentary data on distantly related areas of interphase chromosome biology are available without an integral view of chromosome behavior and arrangement along cell cycle. An overview of molecular cytogenetic techniques for visualizing chromosomes in interphase evidences that a strong technological basis does exist for high-resolution analyses of chromosomes of almost all human tissues. Three main directions of I-FISH application has been advanced by developments in interphase molecular cytogenetics which has provided for possibilities to define functional consequences of spatiotemporal chromosome arrangement in the nuclei, to elucidate the role of such immense intercellular genomic diversity (somatic genomic variations), to propose new diagnostic solutions for medical genetics and oncology. I-FISH is the unique way to study variations and behavior of the genome in all the cell types of human organism, at all stages of cell cycle and at molecular and supramolecular resolutions. Thus, developments in interphase molecular cytogenetics open numerous prospects for genetics, cellular and molecular biology, genomic/molecular medicine. Taking into account data on technological aspects of studying human interphase chromosomes, we conclude that this biomedical direction has the potential to provide revolutionary solutions for basic and applied biomedical research in fields of human genetics and cell biology. This would be undoubtedly the result of combination of interphase molecular cytogenetic techniques (i.e. mFISH, QFISH, ICS-MCB, S-FISH, pod-FISH, Immuno-FISH etc), which has already given rise to several discoveries in current biomedicine.

The authors declare that they have no competing interests.

SGV, YBY and IYI wrote the manuscript. All authors read and approved the final manuscript.