3-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 fluoresc ence distribution was performed by means of confocal laser scanning mi croscopy (CLSM). Human IGR melanoma cells grown in monolayer or multic ellular spheroid culture were studied for elucidating mitochondrial me mbrane potential characteristics, and cell and nucleus volume dimensio ns. Microspheres 6 mu m in diameter loaded with rhodamine B were used to calibrate our instruments for performing 3D imaging of optical sect ions as obtained by CLSM. Accurate optical slicing is only possible ta king into consideration the physical characteristics of the objectives used like chromatic and spherical aberrations, depth discrimination o r cover slip correction and the temperature dependence of the immersio n medium. While 3D imaging of optical slices can be carried out showin g the original shape of the object being tested without physical disto rtion, 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 gradie nt 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 mono layer or multicellular spheroid culture, which have been loaded with r hodamine 123, show the location of the dye predominantly within the cy toplasm of the cells with a remarkable heterogeneity of fluorescence i ntensity within and between single cells, indicating differences in th e 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 calibrat ed 3D imaging as a valuable noninvasive tool to visualize the heteroge neity of cell parameters under different cell culture conditions.