Three-dimensional imaging of rhodamine 123 fluorescence distribution in human melanoma cells by means of confocal laser scanning microscopy.
Three-dimensional (3D) imaging of intracellular rhodamine 123 fluorescence distribution was performed by means of confocal laser scanning microscopy (CLSM). Human IGR melanoma cells grown in monolayer or multicellular spheroid culture were studied for elucidating mitochondrial membrane potential characteristics, and cell and nucleus volume dimensions. Microspheres 6 microns in diameter loaded with rhodamine B were used to calibrate our instruments for performing 3D imaging of optical sections as obtained by CLSM. Accurate optical slicing is only possible taking into consideration the physical characteristics of the objectives used like chromatic and spherical aberrations, depth discrimination or cover slip correction and the temperature dependence of the immersion medium. While 3D imaging of optical slices can be carried out showing the original shape of the object being tested without physical distortion, 3D images of microspheres show well-reproducible structures of rhodamine B fluorescence. These can be explained by a superposition of two effects, namely scattering of the fluorescence light and a gradient of the electromagnetic field strength of the laser beam due to the shape of the object. 3D imaging of optical slices of IGR cells in monolayer or multicellular spheroid culture, which have been loaded with rhodamine 123, show the location of the dye predominantly within the cytoplasm of the cells with a remarkable heterogeneity of fluorescence intensity within and between single cells, indicating differences in the mitochondrial membrane potential and thus in the metabolic activity. Due to the heterogeneity of the cell shape the cell nucleus occupies between 4 and 14% of the total cell volume. These data reveal calibrated 3D imaging as a valuable noninvasive tool to visualize the heterogeneity of cell parameters under different cell culture conditions.
Porwol
T
T
Max-Planck-Institut für molekulare Physiologie, Dortmund, Germany.
Merten
E
E
Opitz
N
N
Acker
H
H
D005456:Fluorescent Dyes; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008545:Melanoma; D018613:Microscopy, Confocal / Q000379:methods*; D020112:Rhodamine 123; D012235:Rhodamines; D014407:Tumor Cells, Cultured
D005456:Fluorescent Dyes; D012235:Rhodamines; D020112:Rhodamine 123