Towards many colors in FISH on 3D-preserved interphase nucleiWalter J.b · Joffe B.a · Bolzer A.a · Albiez H.a · Benedetti P.A.d · Müller S.a · Speicher M.R.c · Cremer T.a · Cremer M.a · Solovei I.a
aTILL I.D. GmbH, c\o BioImaging Zentrum, Martinsried; bChair of Anthropology and Human Genetics, Department of Biology II, Ludwig-Maximilians-University, Martinsried (Germany); cIPCF-CNR, Pisa (Italy); dTechnical University and GSF, Institute of Human Genetics, Munich (Germany)
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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.
© 2006 S. Karger AG, Basel
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