Confocal multilaser focusing and single-laser characterization of ultraviolet excitable stains of cellular preparations

Background: The aims of this study were (1) to realign cellular preparation s when spots and structures are excited by different lasers of a confocal l aser scanning microscope (multilaser studies); (2) to avoid the use of real igment methods by selecting fluorochromes that call be excited by only one laser (single-laser experiments). Methods: In multilaser studies, we used propidium iodide fluorescent beads, as well as tetramethyl rhodamine isothiocyanate (TRITC), fluorescein isoth iocyanate (FITC), and 4'-6 diamidino-2-phenylindole (DAPI)-stained human ca ncer lines. The) were excited using HeNe, argon, and ultraviolet (UV) argon laser lines of a confocal laser scanning microscope. Single-laser experime nts using UV excitation only were performed using europium as a model fur m agnetic resonance paramagnetic contrast agents. Nuclei of human cancer line s and tissue were counterstained by DAPI and cytoplasms were labeled with E LF(TM)-97 substrates. Factor analysis of medical images (FAMIS) and correla tion methods were used to realign shifted images, focus images, and charact erize each fluorochrome when necessary. Results: In multilaser studies, superimposition of factor images corrected Z shifts and correlation methods provided X, Y correction values. in single -laser experiments, each fluorochrome was clearly distinguished in the grou p of fluorochromes. Estimated images in both studies showed colocalizations of structures. Conclusions: It is possible to characterize differences in the focus and al ignment of fluorescent probes and to correct them. It is also possible to s tudy colocalization of UV excitable fluorochromes (DAPI, ELF-97, europium) in cellular and tissular preparations via multilaser or single-laser experi ments. Cytometry 40:42-49, 2000. (C) 2000 Wiley-Liss, Inc.