Multiplex Fluorescence In Situ Hybridization

Multiplex fluorescence in situ hybridization was first described by Speicher et al.[2] and, like SKY, exploits combinatorial probe labeling to uniquely identify each of the human chromosome pairs. Where the two techniques differ is in the mode of fluorescence detection. For m-FISH, this is achieved using single band-pass filters that are specific for each fluorophore employed. Currently, the majority of commercially available probe sets contain five spectrally distinct fluorophores such that each chromosome is labeled down its whole length with one, two, or three different fluorophores. Consequently, only a proportion of the chromosomes is positively hybridized and therefore visible in any one fluorescence channel. Each channel is digitally captured in turn, with modifications to the excitation/emission filters, focal plane, and exposure times necessary between each fluorophore. Cells are also counterstained with DAPI and an image of this is captured to allow the visualization of the whole metaphase enabling the automated delineation of all the chromosomal material in the cell. Once this raw capture sequence has been completed, m-FISH dedicated software processes all six ''raw'' images by registering each image on top of each other (Fig. 1). It is this that produces the final merged or composite pseudo-color m-FISH image, where the color and therefore origin of chromosome represented is determined by the presence or absence of each of the five distinct fluorophores detected on a pixel-by-pixel basis. A combination of optimal FISH biology, hardware, focus, registration of raw images, and signal-to-noise ratio of each of the individual fluorophores used determined the quality of image produced. Therefore standard of analysis is tightly linked to the quality of each of the six raw images initially captured.

There are problems associated with the direct analysis of the final pseudocolor m-FISH image however. A chromosome is determined to be apparently normal if no color junctions are observed down its entire length, thus excluding the chance of detecting inversions, deletions, or homologous pair interactions, unless of course there are

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook


Post a comment