Walter, J.; Joffe, B.; Bolzer, A.; Albiez, H.; Benecletti, P. A.; Müller, Stefan; Speicher, Michael R.; Cremer, T.; Cremer, M.; Solovei, Irina
Towards many colors in FISH on 3D-preserved interphase nuclei.
In: Cytogenetic and Genome Research, No. 3-4: pp. 367-378
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 + widefield 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. Copyright (c) 2006 S. Karger AG, Basel.