Labelling quality and chromosome morphology after low temperature FISH analysed by scanning far‐field and near‐field optical microscopy

• Published in: Journal of microscopy (Oxford) 2003-01, Vol.209 (1), p.23-33
• Main Authors: Winkler, R., Perner, B., Rapp, A., Durm, M., Cremer, C., Greulich, K.‐O., Hausmann, M.
• Format: Article
• Language: English
• Subjects: Cell Nucleus - ultrastructure; Cells, Cultured; Centromere labelling of metaphase spreads; Chromosome Structures; Chromosomes - genetics; Chromosomes - ultrastructure; confocal laser scanning microscopy; In Situ Hybridization, Fluorescence - methods; Interphase; low temperature FISH; Lymphocytes; Metaphase; Microscopy, Fluorescence - instrumentation; pUC 1.77 DNA probe; quantitative fluorescence microscopy; scanning near‐field optical microscopy
• ISSN: 0022-2720; 1365-2818
• DOI: 10.1046/j.1365-2818.2003.01101.x

Summary A non‐enzymatic, low temperature fluorescence in situ hybridization (LTFISH) procedure was applied to metaphase spreads and interphase cell nuclei. In this context ‘low temperature’ means that the denaturation procedure of the chromosomal target DNA usually applied by heat treatment and chaotropic agents such as formamide was completely omitted so that the complete hybridization reaction took place at 37 °C. For LTFISH, the DNA probe had to be single‐stranded, which was achieved by means of separate thermal denaturation of the DNA probe only. The DNA probe pUC1.77 was used for all LTFISH experiments. The labelling quality (number of binding sites, relative background intensity, relative intensity of major and minor binding sites) was analysed by confocal laser scanning microscopy (CLSM). An optimum in specificity and signal quality was obtained for 15 h hybridization time. For this hybridization condition of LTFISH, the chromosomal morphology was analysed by scanning near‐field optical microscopy (SNOM). The results were compared with the morphology of chromosomes after (a) labelling of all centromeres using the same chemical treatment in the FISH procedure but with the application of target denaturation, and (b) labelling of all centromeres using a standard FISH protocol including thermal denaturation of the DNA probe and the chromosomal target. Depending on the FISH‐procedure applied, SNOM images show substantial differences in the chromosome morphology. After LTFISH the chromosome morphology appeared to be much better preserved than after standard FISH. In contrast, the application of the LTFISH chemical treatment accompanied by heat denaturation had a very destructive influence on chromosomal morphology. The results indicate that, at least for certain DNA probes, specific chromosome labelling can be obtained without the usually applied heat and chemical denaturation of the DNA target, resulting in an apparently well preserved chromatin morphology as visualized by SNOM. LTFISH may be therefore a useful labelling technique whenever the chromosomal morphology had to be preserved after specific labelling of DNA regions. Binding mechanisms of single‐stranded DNA probes to double‐stranded DNA targets are discussed.