BACKGROUND: The recently developed technique of high-resolution cytometry (HRCM) enables automated acquisition and analysis of fluorescent in situ hybridization (FISH)-stained cell nuclei using conventional wide-field fluorescence microscopy. The method has now been extended to confocal imaging and offers the opportunity to combine the advantages of confocal and wide-field modes. METHODS: We have automated image acquisition and analysis from a standard inverted fluorescence microscope equipped with a confocal module with Nipkow disk and a cooled digital CCD camera. The system is fully controlled by a high-performance computer that performs both acquisition and related on-line image analysis. The system can be used either for an automatic two (2D) and three-dimensional (3D) analysis of FISH- stained interphase nuclei or for a semiautomatic 3D analysis of FISH-stained cells in tissues. The user can select which fluorochromes are acquired using wide-field mode and which using confocal mode. The wide-field and confocal images are overlaid automatically in computer memory. The developed software compensates automatically for both chromatic color shifts and spatial shifts caused by switching to a different imaging mode. RESULTS: Using the combined confocal and wide-field HRCM technique, it is possible to take advantage of both imaging modes. Images of some dyes (such as small hybridization dots or counterstain images of individual interphase nuclei) do not require confocal quality and can be acquired quickly in wide-field mode. On the contrary, images of other dyes (such as chromosome territories or counterstain images of cells in tissues) do require improved quality and are acquired in confocal mode. The dual-mode approach is two to three times faster compared with the single-mode confocal approach and the spectrum of its applications is much broader compared with both single-mode confocal and single-mode wide-field systems. CONCLUSIONS: The combination of high speed specific to the wide-field mode and high quality specific to the confocal mode gives optimal system performance. Copyright 2001 Wiley-Liss, Inc.
BACKGROUND: The recently developed technique of high-resolution cytometry (HRCM) enables automated acquisition and analysis of fluorescent in situ hybridization (FISH)-stained cell nuclei using conventional wide-field fluorescence microscopy. The method has now been extended to confocal imaging and offers the opportunity to combine the advantages of confocal and wide-field modes. METHODS: We have automated image acquisition and analysis from a standard inverted fluorescence microscope equipped with a confocal module with Nipkow disk and a cooled digital CCD camera. The system is fully controlled by a high-performance computer that performs both acquisition and related on-line image analysis. The system can be used either for an automatic two (2D) and three-dimensional (3D) analysis of FISH- stained interphase nuclei or for a semiautomatic 3D analysis of FISH-stained cells in tissues. The user can select which fluorochromes are acquired using wide-field mode and which using confocal mode. The wide-field and confocal images are overlaid automatically in computer memory. The developed software compensates automatically for both chromatic color shifts and spatial shifts caused by switching to a different imaging mode. RESULTS: Using the combined confocal and wide-field HRCM technique, it is possible to take advantage of both imaging modes. Images of some dyes (such as small hybridization dots or counterstain images of individual interphase nuclei) do not require confocal quality and can be acquired quickly in wide-field mode. On the contrary, images of other dyes (such as chromosome territories or counterstain images of cells in tissues) do require improved quality and are acquired in confocal mode. The dual-mode approach is two to three times faster compared with the single-mode confocal approach and the spectrum of its applications is much broader compared with both single-mode confocal and single-mode wide-field systems. CONCLUSIONS: The combination of high speed specific to the wide-field mode and high quality specific to the confocal mode gives optimal system performance. Copyright 2001 Wiley-Liss, Inc.
Authors: Emilie Lukásová; Stanislav Kozubek; Martin Falk; Michal Kozubek; Jan Zaloudík; Václav Vagunda; Zdenek Pavlovský Journal: Chromosoma Date: 2004-01-13 Impact factor: 4.316
Authors: Miroslav Varecha; Michal Zimmermann; Jana Amrichová; Vladimír Ulman; Pavel Matula; Michal Kozubek Journal: J Biomed Sci Date: 2009-07-06 Impact factor: 8.410
Authors: Martin Falk; Iva Falková; Olga Kopečná; Alena Bačíková; Eva Pagáčová; Daniel Šimek; Martin Golan; Stanislav Kozubek; Michaela Pekarová; Shelby E Follett; Bořivoj Klejdus; K Wade Elliott; Krisztina Varga; Olga Teplá; Irena Kratochvílová Journal: Sci Rep Date: 2018-10-02 Impact factor: 4.379
Authors: Eva Pagáčová; Lenka Štefančíková; Franz Schmidt-Kaler; Georg Hildenbrand; Tomáš Vičar; Daniel Depeš; Jin-Ho Lee; Felix Bestvater; Sandrine Lacombe; Erika Porcel; Stéphane Roux; Frederik Wenz; Olga Kopečná; Iva Falková; Michael Hausmann; Martin Falk Journal: Int J Mol Sci Date: 2019-01-30 Impact factor: 5.923