Hardware and Software for High-Resolution Cytometry of FISH Dots in Interphase Cell Nuclei
Recently, we have introduced a high-resolution cytometry (HRCM) technique (Cytometry 36:279-293) as a reasonable compromise between flow cytometry (FCM) and laser scanning cytometry (LSCM) on one hand and confocal laser scanning microscopy (CLSM) on the other hand. Whereas FCM/LSCM and CLSM enable measuring of either large number of cells with low resolution or small number of cells with high resolution, respectively, HRCM can analyze quantities comparable to FCM/LSCM measurements (using overnight acquisition) with an accuracy comparable to CLSM.
The HRCM system consists of a fully motorized fluorescence microscope and a cooled digital CCD camera fully controlled by a high-performance computer which performs both acquisition and related on-line image analysis. It is capable of analyzing microscope slides with FISH-stained interphase nuclei in 2-D as well as in 3-D. Using HRCM, it is possible to analyze multi-color preparations including UV-excited dyes as well as repeatedly hybridized preparations re-acquiring individual nuclei. The speed of the acquisition and analysis is about 50 nuclei per minute in 2-D and 1 nucleus per minute in 3-D but depends on the density of nuclei on the slide; the precision of the lateral and axial measurements is approximately 100 nm.
Recently, we have built a second HRCM instrument (see http://www.fi.muni.cz/lom) which enables switching between conventional and confocal modes. The confocal mode is realized using a 5% Nipkow disk optimized for fluorescence imaging. It offers a better resolution but at a lower speed. The user can choose which fluorochromes are imaged in confocal mode. Thus, the system can be configured for a variety of imaging modes ranging from quick 2-D analysis up to high-resolution confocal 3-D studies.
Both instruments are fully controlled by our own software developed in C/C++ language. The software performs both image acquisition and image analysis. Some image analysis steps can be performed on-line right after image acquisition. This enables to save only those parts of the images which contain objects of interest. The user can configure the image acquisition and analysis process by setting desired parameter values in dialog boxes. After setting the parameters, the software can automatically scan the slide overnight and/or perform analysis of acquired image stacks.
Michal Kozubek, Stanislav Kozubek1, Emilie Lukasova1, Eva Bartova1, Magdalena Skalniková, Pavel Matula, Petr Matula, Pavla Jirsova1, Alena Cafourkova1, Irena Koutna
Faculty of Informatics, Masaryk University, Botanická 68a,