Fluorescence microscopy hybridization

A wide variety of measurements can now be made on single molecules, including electrical (e.g. scanning tunnelling microscopy), magnetic (e.g. spin resonance), force (e.g. atomic force microscopy), optical (e.g. near-field and far-field fluorescence microscopies) and hybrid teclmiques. This contribution addresses only Arose teclmiques tliat are at least partially optical. Single-particle electrical and force measurements are discussed in tire sections on scanning probe microscopies (B1.19) and surface forces apparatus (B1.20). 

Liebermann T, Knoll W (2003) Parallel multispot detection of target hybridization to surface-bound probe oligonucleotides of different base mismatch by surface-plasmon field-enhanced fluorescence microscopy. Langmuir 9 1567-1572... 

The RNA molecules, ribosomal RNA (rRNA) and messenger RNA (mRNA) play key roles in the protein synthesis. The amount of RNA in individual cells or in a community may, therefore, be taken as an indicator of protein synthesis and, thus, microbial activity. The number of active cells can be detected by fluorescent in situ hybridization (FISH) (Amann et al. 1995). By this method, individual cells carrying high concentrations of rRNA, situated on ribosomes, are quantified by fluorescence microscopy. The amount of rRNA in a community can also be detected by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), where rRNA extracted from soil is detected by creating a DNA copy and separating by gel electrophoresis (Duineveld et al. 2001). 

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.

The effect of temperature on fluorescence has been studied, as has the effect of salt concentration and water-soluble conjugated polymers. A method for the quantification of ssDNA dsDNA is described, as well as kinetics of mismatch hybridization and the kinetics of collision in short ss-nucleic acids. Fluorescence quenching of Cy-5 labelled oligonucleotides by poly(phenylene ethynylene) particles has been shown to be a more sensitive method than excitation of the Cy-5 fluorophore. An ultrasensitive method for the detection of DNA uses highly fluorescent conjugated nanoparticles, and detection limits below IfM were achieved. DNA transport through a carbon nanotube has also been observed using fluorescence microscopy. " ... 

Direct method in which the probe-target hybrid can be visualized after the hybridization. When fluorochrome-labeled probes are used, the visualization can be achieved by fluorescence microscopy. Various fluorochromes with different emission colors are now available such as AMA, FITC, fluorescein, rhodamine CY3 and Texas red. The use of different probes labeled by different fluorochromes... 

Direct visualization is performed by fluorescence microscopy when fluoro-chrome-labeled probes or florescence-labeled antibodies are used (fluorescence in situ hybridization, FISH). The color of fluorescence depends on the type of used fluorochrome, blue (AMCA), green (fluorescein) and red (rhoda-mine, Texas red, CY3 and TRITC). 

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. 

The embryos can be analyzed by standard in-situ hybridization and immunohis-tochemistry techniques. The site of tissue culture cell implantation can easily be identified using fluorescent microscopy. 

An interesting novelty was reported by a German team in 2009 when they described vesicles composed of amphiphilic nanoparticles. It was demonstrated with TEM and fluorescence microscopy that CdSe/CdS core-shell nanoparticles with a brush-like coating of poly(ethylene oxide) form LUVs and GUVs. The vesicle wall is composed of a single layer of nanoparticles. Nanoparticle vesicles constitute a new class of organic-inorganic hybrid vesicles. 

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