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

Abstract

BACKGROUND:

The aims of this study were (1) to realign cellular preparations when spots and structures are excited by different lasers of a confocal laser scanning microscope (multilaser studies); (2) to avoid the use of realigment methods by selecting fluorochromes that can 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 isothiocyanate (FITC), and 4'-6 diamidino-2-phenylindole (DAPI)-stained human cancer lines. They were excited using HeNe, argon, and ultraviolet (UV) argon laser lines of a confocal laser scanning microscope. Single-laser experiments using UV excitation only were performed using europium as a model for magnetic resonance paramagnetic contrast agents. Nuclei of human cancer lines and tissue were counterstained by DAPI and cytoplasms were labeled with ELF-97 substrates. Factor analysis of medical images (FAMIS) and correlation methods were used to realign shifted images, focus images, and characterize 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 group of fluorochromes. Estimated images in both studies showed colocalizations of structures.

CONCLUSIONS:

It is possible to characterize differences in the focus and alignment of fluorescent probes and to correct them. It is also possible to study colocalization of UV excitable fluorochromes (DAPI, ELF-97, europium) in cellular and tissular preparations via multilaser or single-laser experiments.

• Authors: E Kahn, F Frouin, C Souchier, J C Bernengo, H Bruzzoni-Giovanelli, O Clément, G Frija, R Di Paola, F Calvo, G Linares-Cruz
• Journal: Cytometry (Cytometry) Vol. 40 Issue 1 Pg. 42-9 (May 01 2000) ISSN: 0196-4763 [Print] United States
• PMID: 10754516 (Publication Type: Journal Article)
• Copyright: Copyright 2000 Wiley-Liss, Inc.

Chemical References

• Fluorescent Dyes
• Indoles
• Organophosphorus Compounds
• Quinazolines
• Quinazolinones
• Rhodamines
• 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone
• Propidium
• tetramethylrhodamine isothiocyanate
• Europium
• DAPI
• Fluorescein-5-isothiocyanate

Topics

• Europium
• Fluorescein-5-isothiocyanate
• Fluorescent Dyes
• Humans
• Image Processing, Computer-Assisted (methods)
• Indoles
• Lasers
• Microscopy, Confocal (methods)
• Microspheres
• Organophosphorus Compounds
• Propidium
• Quinazolines
• Quinazolinones
• Rhodamines
• U937 Cells
• Ultraviolet Rays