Fluorescent in situ hybridization was initially developed in the late 1980's from radioactive hybridization procedures used for mapping human genes. Eventually, this technology was utilized for the for the characterization of chromosomal rearrangements and marker chromosomes, the detection of microdeletions, and the pre-natal diagnosis of common aneuploidies in clinical cytogenetics laboratories. Today, numerous DNA probes have been commercialized, further promoting the wide-spread clinical applications of molecular cytogenetics.
With current FISH techniques, deletion or rearrangement of a single gene can be detected, cryptic chromosome translocations can be visualized, the copy number of oncogenes amplified in tumor cells can be assessed, and very complex rearrangements can be fully characterized.
Using interphase FISH, genomic alterations can be studied in virtually all types of human tissues at any stage of cell division without the need of cell culture and chromosome preparation.
Fluorescent in situ hybridization can be used to detect and quantify specific microorganisms while maintaining their morphological integrity, without nucleic acid extraction. Sample cells are fixed with chemicals to increase their permeability and allow the probe to enter the cells. Fixatives fall into two general classes: 1) precipitants like ethanol or methanol and 2) cross-linking agents like aldehydes (e.g. paraformaldehyde). After fixation the sample is spotted into wells of Teflon-covered slides, which have been gelatin-coated to aid the attachment of the cells.
The sample is air-dried and then dehydrated by serial immersion of the slide in ethanol series with increasing concentration. After hybridization with probe (Figure 1) in hybridization buffer, the excess probe is washed, and the slides air dried and then visualized using fluorescence microscopy. Whole cell hybridization with fluorescently labelled probes can also be combined with flow cytometry for rapid counting or collection of cells.
Figure 1. Hybridization of the functional and taxon specific probes within the cell.
The term in situ hybridization is restricted to whole cell hybridizations in which the cells are detected in their natural microhabitat. With the method one can not only determine the cell morphology of an uncultured microorganism and its abundance, but also analyze spatial distributions in situ. When combined to confocal scanning laser microscopy, fluorescent in situ hybridization (FISH) can assist the visualization of three dimensional arrangement of cells, such as in biofilms or flocs.
The advantage of in situ hybridization is that it can give quantitative information about the number of specific microorganisms. However, in situ hybridization also has limitations. Frequently encountered problems include no signals or low signal intensity. This can be caused by noncomplementarity of probe and target, ineffective probe labelling or nonoptimal hybrization conditions. Low signal intensity can be caused by small numbers or insufficient accessibility of the target molecules (rRNA). Dormant or metabolically inactive cells may not contain enough rRNA to show fluorescent signals after hybridization. The factors that hinder probe binding include limited diffusion of rRNA-targeted probes due to cell peripheries (probably cell wall) and prevention caused by higher order structures in the ribosomes.
The type of fixative influences the permeability of the cell wall. Although gram negative bacteria usually are readily permeabilized with e.g. paraformaldehyde, gram positive bacteria may need additional enzymatic, heat or ethanol-formalin treatment. With complex environmental samples it is difficult to achieve a good compromise between sufficient cell permeabilization for efficient hybridization and good preservation of morphological detail. The target accessibility can sometimes be improved by addition of formamide to the hybridization buffer. Addition of this denaturing solvent weakens the effects of hydrogen bonds, thereby 'softening' hindrance by higher-order structures. There is some indication that certain regions in rRNA might be inaccessible in certain phylogenetic groups. Sometimes, shifting the target site by just few nucleotides has a major influence on the probe sensitivity.
Finally, the quantification of the number of microorganisms with in situ hybridization can be problematic when cells have irregular shapes or when they form chains or compact micro-colonies. In situ detectability is also influenced by non-specific binding and autofluorescence of cells and surrounding material.
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