Stereological image analysis of cultured human melanocytes observed by transmission electron microscopy

Abstract

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.

Comparison of High Performance Liquid Chromatography and Stereological Image Analysis for the Quantitation of Eumelanins and Pheomelanins in Melanoma Cells*

“…Melanoma cells were fixed in suspension. This technique was specifically elaborated in order to obtain near spherical to spherical melanocytes and to embed them as pellet (Donois et al, 1997~). Melanoma cells were detached, pelleted, washed, and fixed in suspension with glutaraldehyde after leaving the cell suspension for 20 min in cacodylate buffer as previously described (Donois et al, 1997~).…”

“…This technique was specifically elaborated in order to obtain near spherical to spherical melanocytes and to embed them as pellet (Donois et al, 1997~). Melanoma cells were detached, pelleted, washed, and fixed in suspension with glutaraldehyde after leaving the cell suspension for 20 min in cacodylate buffer as previously described (Donois et al, 1997~). After the osmium tetroxide hation (Donois et al, 1997~1, an ethanol dehydration was performed, followed by an infiltration with absolute ethanol:l&epoxypropane (1:l vh, 15 min), 1,2-epoxypropane (15 min), l,2-epoxypropane: complete embedding medium (1:l vh, overnight).…”

“…The Semper 6 Plus image analysis software (Synoptics, Ltd, Cambridge, U.K.) was used to execute our Quantimel program. The latter enables us to extract and quantify melanin profiles and to estimate the following primary data: A,, (nuclear area), Le (cell area), Afi (melanin area), Nm, (number of melanin particles), and Nc,, (number of connections between melanin particles) (Donois et al, 1997~). Melanin was gray level thresholded according to the SEM method by using the SEM2 threshold (Donois et al, 199713).…”

“…After obtaining primary data from image analysis, stereology was used to calculate three-dimensional (3-D) melanization parameters from two-dimensional (2-D) measurements (see Bolender, 1992;Mayhew and White, 1980;Weibel, 1979Weibel, , 1989 for more explanations). The main melanization parameters used permit to estimate the number and the degree of melanization of melanosomes and the volume of melanins per average cell (Donois et al, 1997~); these parameters are described in Table I.…”

Treatment of atrophoderma of Pasini and Pierini‐associated hyperpigmentation with the Q‐switched alexandrite laser: A clinical, histologic, and ultrastructural appraisal

“…Multiple views from a variety of sites within each specimen were obtained. By using a technique of modified stereology [10], melanosome number, size, and volume were investigated. Melanin granules were similarly counted, and their mean size was measured.…”