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 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.
Kahn
E
E
INSERM U494, CHU Pitié-Salpêtrière, Paris, France. kahn@imed.jussieu.fr
Frouin
F
F
Souchier
C
C
Bernengo
J C
JC
Bruzzoni-Giovanelli
H
H
Clément
O
O
Frija
G
G
Di Paola
R
R
Calvo
F
F
Linares-Cruz
G
G
D005063:Europium; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D007211:Indoles; D007834:Lasers; D018613:Microscopy, Confocal / Q000379:methods*; D008863:Microspheres; D009943:Organophosphorus Compounds; D011419:Propidium; D011799:Quinazolines; D052999:Quinazolinones; D012235:Rhodamines; D020298:U937 Cells; D014466:Ultraviolet Rays
D005456:Fluorescent Dyes; D007211:Indoles; D009943:Organophosphorus Compounds; D011799:Quinazolines; D052999:Quinazolinones; D012235:Rhodamines; C079177:2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone; D011419:Propidium; C009434:tetramethylrhodamine isothiocyanate; D005063:Europium; C007293:DAPI; D016650:Fluorescein-5-isothiocyanate