Pathogens, Vol. 11, Pages 1450: Application of Fluorescence In Situ Hybridization (FISH) in Oral Microbial Detection

FISH is one of the most routinely used cytochemical techniques for genetic detection, identification, and localization by a fluorescently labeled nucleic acid probe. It is more specifically used in hybridizing processes with nucleic acid sequences of interest [16]. The vitality of this technology is continuously proved by the evolution of probe design, signal amplification, and multiplexing, broadening the application of experimental research and clinical diagnoses in this field of study [17]. Specifically, FISH and its variants have been applied to nucleic acid investigation, cell metabolic research, oncology diagnostics, and microbiological research [18]. 2.1. Development of FISHIn the 1960s before the advent of FISH, cytochemical methods of detecting and localizing specific intracellular molecules were mainly immunocytochemistry based on in situ hybridization with radiolabeled probes and antigen-antibody interaction with fluorescein-labelled immunoglobulins [19,20,21]. However, radioactive RNA or DNA binding to DNA sequences in situ used to be limited to detecting, characterizing, and localizing specific DNA segments. Due to the low resolving power and long exposure time in autoradiography, this methodology is inaccurate in quantification [17,22]. To compensate for this deficiency, indirect immunofluorescence, a type of immunocytochemistry technique developed in 1965, has been successfully applied in quantitatively analyze biological markers via fluorophore-labeled immunoglobulins that target marker-binding antibodies [21]. As a combination of in situ hybridization and indirect immunofluorescence, a method described by Rudkin et al. in 1977 replaced autoradiography with fluorescence microscopy to detect in situ signals from antibodies against DNA-RNA hybrids [23]. Before long, based on the methods above of nucleic acid hybridization and indirect immunofluorescence without intermediated antibodies, Bauman et al. first applied fluorophore-covalent-labeled RNA probes in specific DNA sequence detection in 1980, representing the birth of FISH [22]. Nonetheless, the application of this original FISH still needs further improvement in probe affinity and signal amplification due to the low signal intensity caused by target inaccessibility and low copy numbers [24].The optimization of probe design and synthesis is crucial to the development of FISH. The establishment of genetic databases made it possible to design probe sequences targeting complementary gene sequences of interest [25]. Probe preparation underwent a development from manual multiple sequence alignment for conserved target regions to probe sequence auto-selecting programs and even web-based platforms for theoretical evaluation of the probe performances in FISH [25,26,27,28,29]. The new technology makes it easier to control the length, complementary sequence, thermodynamic property, potential secondary structure, and specificity of the probe, which are directly related to the successful application of FISH [30].

With accurate probe design methods, FISH properties became even more modern, with changes that include increasing signal intensity and stability, range of targets, and sensitivity. Commonly used variants are: catalyzed reporter deposition FISH (CARD-FISH), gene-FISH, recognition of individual genes FISH (RING-FISH), nucleic acid mimics FISH (NAM-FISH), combinatorial labeling, and spectral imaging FISH (CLASI-FISH), double labeling of oligonucleotide probes for FISH (DOPE-FISH), as described below.

The polynucleotide probe also has an advantage in signal intensity with multiple labels and secondary structures mediating probes-connected networks [31]. RING-FISH achieves high detection efficiency to single genes with multiple labeled transcript polynucleotide probes generating halo-like signals [32]. As an alternative to RING-FISH for prokaryotic cellular microorganisms, two-pass TSA-FISH provides higher efficiency and a better signal-to-noise ratio, making it a useful protocol based on the functional gene for single microbial cell detection [33]. By combining rRNA CARD-FISH and polynucleotide probe gene detection, gene-FISH can be an innovative technique to provide a stable signal and high sensitivity [34]. Furthermore, direct gene-FISH for gene signal quantification can be formed by replacing CARD (and its disabilities) with fluorochrome-labeled probes and super-resolution microscopy [35]. The nucleic acid mimics probe has become the most promisingly high-efficient probe with higher affinity, specificity, and better stability based on resistance to enzymatic degradation [36], used to overcome the issues with weak signal caused by low affinity and vulnerability of DNA or RNA probes. The NAM probes currently being utilized are peptide nucleic acid (PNA), 2’-O-Methyl-RNAs, and locked nucleic acid (LNA) [37]. The variants mentioned above show corresponding advantages in detecting microorganisms, allowing direct identification without cultivation and further detection of microbial community structure and individual function [36,38]. As microbiological research kept expanding from individuals to communities, multiplexing identification was developed to simultaneously analyze multi-microbes and microbiota efficiently. A typical example is CLASI-FISH, which introduces combinatorial labeling and spectral imaging (CLASI) into FISH and can be applied to distinguish several microbes at once by linear unmixing the spectra of fluorophores from overlapping spectra [39]. However, CLASI-FISH application may be limited due to internal sensitivity loss and potential probe binding bias caused by binary combinations, limiting the application of this technique for quantification analyses [40]. Compared to CLASI-FISH, DOPE-FISH provides a double signal intensity as well as stable specificity and higher affinity to targets, but the detectable number of microbes is much lower [41].The variants of FISH are improving, as are their applications based on the detection of nucleic acids. Apart from microbiological applications, FISH has been a gold standard technique in absolute gene copy number quantification in cancer, allowing implementation of precise treatment strategies [42]. Based on imaging spatial transcriptomics, FISH performs cell segmentation for cell interactions and the state of complex tissues for analysis of, for example cell organization in the cerebral cortex and cell fate decisions in organogenesis [43,44,45]. Expansion-Assisted Iterative (EASI)-FISH was developed for the 3D organization of cell types in thick tissue, contributing to characterizing the architecture underpinning brain function [46]. It is reported that the combination of FISH and small and ultrabright fluorescent polymeric nanoparticles functionalized with DNA allows a simpler, faster and sensitive single-cell RNA imaging method for transcriptomic analysis [47]. The characteristic chromosomal abnormalities in cancer cells can be obtained by high-resolution karyotyping by FISH, which can help in differential diagnosis [48]. Other uses of FISH karyotype analysis include genetic diagnosis, prenatal screening, and plant and animal genetic studies [48,49,50,51,52]. But karyotyping is highly dependent on frequency of cytogenetically abnormal cells, in need of enrichment methods such as fluorescence-activated cell sorter (FACS) to improve the sensitivity [53]. Thus, the combination of FISH with other suitable techniques can compensate for some inherent drawbacks and break the application limitations of FISH. The coupling of Flow Cytometry and FISH enables high-throughput quantification of complex whole-cell populations, and the association with FACS (FLOW-FISH-FACS) enables sorting of target microorganisms [54]. In drug discovery, FISH was introduced into high-content screening for intracellular imaging of mRNA to screen mRNA-associated drugs and assess their pharmacological activity [55]. Resolution After Single-strand Exonuclease Resection (RASER)-FISH provides a robust generation of single-stranded DNA with excellent preservation of chromatin structure, nuclear integrity and improved hybridization efficiency, which is achieved by exonuclease digestion rather than physical denaturation by heat and exposure to formamide [56]. 2.2. Procedures and Principles of FISH and Its Variants Used for Oral Microbial DetectionThe general protocols of FISH include (a) specimen treatment, (b) probe denaturation, (c) hybridization, (d) elution, (e) hybridization signal amplification (applicable to biotin-labeled probes), (f) re-staining, (g) encapsulation, and (h) fluorescence microscope observation of FISH results [40,57,58]. Probe labeling and specimen processing require different processing methods according to different detection needs, in order to obtain clearer detection results in the subsequent hybridization process [59,60]. For example, to substantially enlarge the number of distinct taxa in one FISH experiment, CLASI-FISH was created, and the established FISH protocols are ordinarily suitable for the hybridization, but still some changes for the protocol were proposed, such as fixing samples with PFA followed by ethylalcohol [39]. Take CARD-FISH as another instance, to enhance sensitivity and whittle background interference, the protocol resembled the typical FISH, but tyramide-fluorophore and HRP was introduced to replace fluorophore on the probe [61]. All the protocols are detailedly elaborated by the inventors of the FISH variants, so that researchers can consult relevant papers according to application needs. Different physical and chemical environments during hybridization will affect the effect of hybridization, and redyeing will significantly change the clarity of the observed objects, which will lead to significantly different results under the optical microscope [62,63].FISH can evaluate various types of test samples, such as tumor tissue, pathological sections, local animal samples, human tissue samples, etc. For tissue samples: 4% paraformaldehyde fixation (paraffin section), or put into liquid nitrogen, −80 °C storage, sample not less than 100 mg; For cell samples: cell slides prepared with 6-well plates, with no less than 2 × 106 cells per well; For environmental bacteria: sludge samples or sludge particles and other sample forms that can be used for smear or sectioning [6,39,64,65]. Though FISH is widely used in microbiological detection and biofilm analysis, some techniques belonging to nucleic acid, immunity, and single-cell techniques also have been applied in different emphases of microbial research due to their different advantages. Ten common techniques (including FISH) in microbial research were selected and compared based on several performance factors, including detectable resolution, applications in oral microbiology, culture reliance, quantification appliance, capability for microbiota analysis, and unsearched species (Table 1). In summary, some features of FISH make it an advantageous technique, e.g., precision, culture-independent, unsearched species detectability for microbiota analysis for extra semi-quantification and intuitive spectral imaging.

Furthermore, most oral microorganisms are unculturable, and the community structure and components are complex and changeable, and these properties make FISH very suitable for oral microbial research. Among many variants of FISH, classical FISH and CLASI-FISH are the most popular in terms of their application in oral microbiology. However, it is noteworthy that a new variant called HiPR-FISH is considered to have a promising future. The procedure and principles of these three currently used FISH techniques in oral microbiology will be elaborated throughout this study.

2.2.1. FISHThe most common procedure followed by researchers who apply FISH involves the following four steps: (1) fixation for dehydration to inhibit the action of enzymes without cell and nucleic acid structures destruction (some special cells need extra permeabilization treatment that degrade the cell walls to increase membrane permeability); (2) hybridization between target nucleic acid sequences and specific complementary probes labeled with fluorescent dyes or reporter molecules to be detected by fluorescent antibodies; (3) washing to remove the unbound or loosely bound (usually mismatched) probes; (4) detection and visualization of the probe-bounded cells by typically fluorescence microscopy and some advanced microscopy systems, then access to information for species detection or microbiota analysis [16,36,87]. Hybridization is one of the most decisive steps, strongly influenced by probe properties on specificity and sensitivity [87]. These four steps are also the basic standpoints of modified FISH variants. 2.2.2. CLASI-FISHRegardless of some minor adaptions to classical FISH protocols for CLASI-FISH application, the main improvement of CLASI-FISH are probe design and image acquisition/analysis. CLASI-FISH probes are particularly effective in synthesizing two probes for one targeted sequence and two different sequence-particular fluorophores; thus, the cells of different species are labeled by specific combinations of two highest-intensity fluorophores [88]. For detection, CLASI-FISH applies confocal laser scanning microscopy (CLSM) in image acquisition, allowing the linear spectral unmixing computational analysis to identify the fluorophores with overlapping spectra and analyze the fluorophore composition in each cell [39]. 2.2.3. HiPR-FISHRegarding the expansion of the number of distinguishable species in a single image as CLASI-FISH, it has been previously verified that High Phylogenetic Resolution FISH (HiPR-FISH) achieves significantly higher taxonomic resolution and multiplexity due to a two-step hybrid approach and a routine for automated image segmentation [89]. Two kinds of probes were used for hybridization: the encoding probe in the first step and the readout probe in the second step. The encoding probes are taxon-specific probes that include targeting sequences modified with different flanking readout sequences. The fluorescently labeled readout probes target readout sequences and stochastically bind to the bound encoding probes, representing equal proportions of fluorophores.

Ten distinct fluorophores encode up to 1023 fluorophore combinations, and one species corresponds to a 10-bit binary barcode derived from designed one or more encoding probes binding with relevant readout probes and presenting different spectral components. After image acquisition, automated image segmentation classifies the spectra of images and assigned cells the corresponding barcodes. The reference spectrum for each barcode is established by the Förster resonance energy transfer (FRET) model, and the k-means clustering-denoised and straightened images of cultured single-cell and biofilm samples are segmented by the watershed algorithm that the seed is defined by developed Local Neighborhood Enhancement (LNE). For spatial analysis, the adjacency segmentation is also generated by the watershed algorithm and then calculates the intuitive spatial association matrix. The super-resolution images and 3D datasets are generated the same way, and the 4D data cube needs rendering in ipyvolume.

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