Towards many colors in FISH on 3D-preserved interphase nuclei |
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| Authors: | Walter J Joffe B Bolzer A Albiez H Benedetti P A Müller S Speicher M R Cremer T Cremer M Solovei I |
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| Institution: | Chair of Anthropology and Human Genetics, Department of Biology II, Ludwig-Maximilians-University, Martinsried, Germany. |
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| Abstract: | The article reviews the existing methods of multicolor FISH on nuclear targets, first of all, interphase chromosomes. FISH proper and image acquisition are considered as two related components of a single process. We discuss (1) M-FISH (combinatorial labeling + deconvolution + wide-field microscopy); (2) multicolor labeling + SIM (structured illumination microscopy); (3) the standard approach to multicolor FISH + CLSM (confocal laser scanning microscopy; one fluorochrome - one color channel); (4) combinatorial labeling + CLSM; (5) non-combinatorial labeling + CLSM + linear unmixing. Two related issues, deconvolution of images acquired with CLSM and correction of data for chromatic Z-shift, are also discussed. All methods are illustrated with practical examples. Finally, several rules of thumb helping to choose an optimal labeling + microscopy combination for the planned experiment are suggested. |
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