The aims of the study were to write an image analysis (IA) program allowing the stereological quantification of human epidermal melanocyte melanization at the ultrastructural level and to specify the suitable preparative methods, in keeping with IA limits and stereological principles. Micrographs of cultured human melanocytes obtained in transmission electron microscopy were digitized with a scanner. The key step of the designed IA program is a thresholding based on the gray levels. Hence, gray level histograms (pixel frequency as a function of gray level) of melanocyte images exhibit a peak specific to melanin. The gray level thresholding used consists in isolating the melanin pixels that form profiles on a binary image and in storing the numerical data produced for a given melanocyte profile. These primary data are used to calculate numerous parameters via stereology with melanocyte cytoplasm and melanized melanosome as main reference spaces. The most important stereological parameters obtained are v(mi,cy) (melanin volume per average cell), v(mi,m) (melanin volume per average melanized melanosome), and nm (number of melanized melanosomes per average cell), and their validity is discussed. Melanocytes embedded in situ were abandoned for stereological reasons but pelleted melanocytes were found suitable. Using this computerized tool and stereology, we are able to perform quantitative studies producing varied data even from small cell samples. To our knowledge, this is the first stereological approach for quantifying intracellular melanization. A quantitative comparison of spectrophotometrical results (melanin assay) with stereological results obtained in ultraviolet B-irradiated Caucasian epidermal melanocytes will be performed in order to appraise this method.

The aim of the study was to compare two methods quantifying eumelanins and pheomelanins, pigments synthesized by melanocytes. One is based on the high performance liquid chromatography (HPLC) quantitation of specific degradation products of each melanin type. The other requires image analysis, transmission electron microscopy (TEM), and stereology. This study was carried out in cultured human melanoma cells and for each line, melanins were measured by HPLC and cells were fixed and embedded as pellets for TEM. Ultrathin sections were treated or not by the alkali elution method allowing the elimination of pheomelanins. The obtained micrographs were analyzed with our image analysis program permitting the estimation of several primary parameters. Stereology was used for estimating melanosomal maturation, intracellular melanins content, and number of melanized melanosomes per cell, for total melanin, eumelanins, or pheomelanins. Our results show a good correlation between both methods for total melanin, particularly when using the cytoplasmic volume density of melanin (r=0.93). Moreover, we report that the number of melanized melanosomes per cell and not the melanosomal maturation is responsible for the differences in total melanin content observed between the different cell lines. However, none of the stereological melanization parameters was correlated in the case of eumelanins or pheomelanins. In order to demonstrate the utter relevancy of this stereological approach, utilization of more pigmented melanoma cells, comparative study of HPLC and stereology, in normal epidermal melanocytes and a new evaluation of the alkali elution method in appropriate animal models would help us to explain the present results.

The aims of the study were to write an image analysis (IA) program allowing the stereological quantification of human epidermal melanocyte melanization at the ultrastructural level and to specify the suitable preparative methods, in keeping with IA limits and stereological principles. Micrographs of cultured human melanocytes obtained in transmission electron microscopy were digitized with a scanner. The key step of the designed IA program is a thresholding based on the gray levels. Hence, gray level histograms (pixel frequency as a function of gray level) of melanocyte images exhibit a peak specific to melanin. The gray level thresholding used consists in isolating the melanin pixels that form profiles on a binary image and in storing the numerical data produced for a given melanocyte profile. These primary data are used to calculate numerous parameters via stereology with melanocyte cytoplasm and melanized melanosome as main reference spaces. The most important stereological parameters obtained are v (mi,cy) (melanin volume per average cell), v (mi,m) (melanin volume per average melanized melanosome), and n m (number of melanized melanosomes per average cell), and their validity is discussed. Melanocytes embedded in situ were abandoned for stereological reasons but pelleted melanocytes were found suitable. Using this computerized tool and stereology, we are able to perform quantitative studies producing varied data even from small cell samples. To our knowledge, this is the first stereological approach for quantifying intracellular melanization. A quantitative comparison of spectrophotometrical results (melanin assay) with stereological results obtained in ultraviolet B‐irradiated Caucasian epidermal melanocytes will be performed in order to appraise this method. Microsc. Res. Tech. 36:188–200, 1997. © 1997 Wiley‐Liss, Inc.

The aim of the study was to compare two methods of quantitating eumelanins and pheomelanins, pigments synthesized by melanocytes. One is based on the high performance liquid chromatography quantitation of specific degradation products of each melanin type. The other requires image analysis, transmission electron microscopy, and stereology. In a previous study, we showed good correlations between both methods for total melanin but not for eumelanins or pheomelanins. We describe here the same comparison in more pigmented cells (nevus cells and stimulated HBL melanoma cells). Transmission electron microscopy micrographs were image analyzed to generate several primary parameters. Stereology was used for estimating melanosomal maturation, intracellular melanin content, and the number of melanized melanosomes per cell, for total melanin, eumelanins, or pheomelanins. Our results showed a good correlation between both methods for total melanin, eumelanins, and pheomelanins with an r equal to 0.99, 0.91, and 0.93, respectively, when all the points were used in the linear regression analyses. In the melanoma cell group (HBL cells cultured in media of different compositions), the chemical and morphometric estimations were not parallel in the case of eumelanins and pheomelanins. In addition, the stereologic and high performance liquid chromatography pheomelanins to eumelanins ratios were still not correlated. These results demonstrate the relevancy of the stereologic method, but the low level of melanization, the possible lack of specificity of melanogenesis in melanoma cells, and a problem of sensitivity of the stereologic method in this context seem to be obstacles in obtaining better results. The utilization of normal human melanocytes could give some answers to our hypotheses.

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