Open AccessUse of platinum coproporphyrin and delayed luminescence imaging to extend the number of targets FISH karyotyping
Author(s) -
Tanke Hans J.,
De Haas Richard R.,
Sagner Gregor,
Ganser Michael,
van Gijlswijk Rob P. M.
Publication year - 1998
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/(sici)1097-0320(19981201)33:4<453::aid-cyto9>3.0.co;2-s
Subject(s) - luminescence , fluorescence , metaphase , shutter , rhodamine , fluorescence in situ hybridization , fish <actinopterygii> , materials science , optics , biology , optoelectronics , chromosome , physics , genetics , gene , fishery
Combinatorial use of fluorophores in multicolor fluorescence in situ hybridization (FISH) allows for the recognition of all human chromosomes. Here we introduce the concept of the use of delayed luminescence labels such as phosphorescent platinum coproporphyrins (PtCP) to extend the number of simultaneously detectable targets in multicolor FISH karyotyping. PtCP‐conjugated antibodies were used in combination with conventional FISH labels such as cascade blue, fluorescein, lissamine rhodamine, Cy5, and Cy7. Probe sets for all human chromosomes were generated and labeled with these dyes in a combinatorial approach. Delayed luminescence of PtCP was accomplished using a standard fluorescence microscope in which a specially constructed module for visualization of delayed luminescence was incorporated. The module consists of a minichopper incorporated in the standard block that holds the shutter and diaphragm, and a FLC polarizing shutter mounted in a filter holder at the emission side. Multicolor FISH staining was applied to normal metaphase chromosomes and to chromosomes generated from cultured JVM‐2 cells with known translocations. Multicolor FISH images (conventional and delayed) were registered using a slow‐scan CCD camera. Recognition of all 24 chromosomes was feasible, since the delayed PtCP fluorescence (lifetime, 90 μs) could be easily distinguished from the conventional promptly fluorescing dyes. We discuss possibilities for extending the number of targets far beyond the 24 demonstrated so far. Cytometry 33:453–459, 1998. © 1998 Wiley‐Liss, Inc.