Imaging hybridization Fluorescence microscopy

Figure 12. Metaphase cell hybridized with FISH probes, (a) Widefield fluorescence microscopy without deconvolution and (b) Widefield fluorescence microscopy with deconvolution. The chromosomes are seen in blue. The chromosome that is labeled with FISH probes shows green and red spots which are sharper in the deconvolved image (right) compared to the image without deconvolution (left). Image courtesy, Peter Franklin, Applied Precision Inc., Issaquah, WA, USA.

Successful application of this experimental approach depends on several factors synthesis of high-quality hybridization probes, appropriate fixation of the sample, the hybridization procedure, and the fluorescence microscopy approach used to image the specimen. In adapting the technique of three-dimensional in situ hybridization to different organisms and tissue types, the simplest and most invariant aspect of the technology has proved to be the hybridization procedure. Probes must be developed on a custom basis to address the particular questions of the investigator, and equally crucially, fixation conditions need to be adapted with special attention to the physical attributes of the individual specimen. However, once appropriate preparation conditions are established for a particular type of sample, it has been unnecessary to reoptimize the basic hybridization protocol. We discuss each of these experimental issues separately below. 

Figure 3.8 (a) 9-TMR-DNA hybrids immobilized on a glass cover slide and imaged by TIRF microscopy after complexation by Cu(ll), leading to quenching and addition of Na2S204 which recovers the fluorescence emission, (b) Under these conditions, the fluorescence intensity of the molecules... 

Figure 18. Series of time-lapse surface plasmon fluorescence microscopy images taken during hybridization (a) and dissociation (b) of the target T3, to the surface-attached probes PI - P3 (left column of 3 spots PI (MMI to T3), middle column P3 (MMO), right column P2 (MM2 to T3).

Total internal reflection fluorescence (TIRF) microscopy, fluorescence in situ hybridization (FISH), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM). 

The application of in situ hybridization (ISH) has advanced from short lived, non-specific isotopic methods, to very specific, long lived, multiple color fluorescent-ISH probe assays (FISH). Improvements in the optics, filter technology, microscopes, cameras, and data handling by software, have allowed for a cost effective FISH setup to be within reach of most researchers. The application of mFISH (multiplex-FISH), coupled to the advances in digital imaging microscopy, have vastly improved the capabilities for non-isotopic detection and analysis of multiple nucleic acid sequences in chromosomes and genes (1). 

Luminescent RE + chelates have been successfully developed as labels and probes for highly sensitive and selective bioassays in the past two decades. Time-resolved Inmines-cence detection has been widely applied in fluoroimmunoassay, DNA hybridization assay, enzyme assay, cell activity assay and fluorescence imaging microscopy. ... 

Different types of analyses for the detection of micronuclei originating from structural or numerical aberrations are used light microscopy, semiautomated imaging systems, and flow cytometry (FACS analysis) [66, 67], Fluorescent in situ hybridization (FISH) with specific probes (e.g., centromeric probes) can also be used to examine nondisjunctions. Depending on the methodology, specific features of the damage can be characterized and mechanisms of action addressed. 

Ried, T., Baldini, A., Rand, T. C., and Ward, D. C, (1992) Simultaneous visualization of seven different DNA probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy. Proc. Natl. Acad. Sci. USA 89, 1388-1392. 

Fluorescence is the most commonly used imaging mode in confocal microscopy, such as, applications in cell biology [82], [83], which include the use of autofluorescence, specific dyes in combination with antibodies, and in situ hybridization, Comprehensive reviews of available fluor-ochromes are published elsewhere [80], [84]-[87]. UV lasers broaden the spectrum of available fluorescence dyes even the autofluorescence present in reduced pyridine nucleotides [NAD(P)H] can be monitored [88]. 

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