Melanosomes, isolated by centrifugal separation from culture broth of B16 melanoma cells derived from mouse, were observed by scanning electron microscopy (SEM), and by transmission electron microscopy (TEM). Some interesting structural features were found inside and outside of the melanosomes. By SEM observation, the melanosomes were ellipsoid shape, their surface was not smooth and was covered with rough substructure, 10 to 20 nm particles. By TEM, uneven structure and micro particles were observed in the melanosomes. Furthermore, three-dimensional analysis was tried by using the ultra-high voltage electron microscopy(UHVEM). Micrographs of the melanosomes were taken at various tilted angles by UHVEM, after preparing 500 nm thickness specimens stained with lead citrate. From the micrographs collected, the three-dimensional structures were reconstructed by using i-mode software. Melanin stained by lead and non stained parts was clearly observed in the reconstructed structure. Non stained parts were round, regular size, and distributed widely in the melanosomes.
Cite this paper
Akazaki, S. , Takahashi, T. , Nakano, Y. , Nishida, T. , Mori, H. , Takaoka, A. , Aoki, H. , Chen, H. , Kunisada, T. and Koike, K. (2014) Three-Dimensional Analysis of Melanosomes Isolated from B16 Melanoma Cells by Using Ultra High Voltage Electron Microscopy. Microscopy Research
, 1-8. doi: 10.4236/mr.2014.21001
 D. N. Peles, L Hong, D. N. Hu, S. Ito, R. J. Nemanich and J. D. Simon “Human Iridal Stroma Melanosomes of Varying Pheomelanin Contents Prossess a Common Eumelanic Outer Surface,” Journal of Physical Chemistry B, Vol. 113, No. 32, 2009, pp.11346-11351.
 J. D. Simon, L. Hong and D. N. Peles “Insights into Melanosomes and Melanin from Some Interesting Spatial and Temporal Properties,” Journal of Physical Chemistry B, Vol. 112, No. 42, 2008, pp. 11201-11217.
 Y. Liu, L. Hong, K. Wakamatsu, S. lto, B. Adhyaru, C. Y. Cheng, C. R. Bowers and J. D. Simon “Comparison of Structural and Chemical Properties of Melanosomes Isolated from Retinal Pigment Epithelium, Iris and Choroid of Newborn and Mature Bovine Eyes,” Photochemistry and Photobiology, Vol. 81, No. 3, 2005, pp. 510-518.
 Y. Liu, L. Hong, K. Wakamatsu, S. lto, B. Adhyaru, C. Y. Cheng, C. R. Bowers and J. D. Simon “Comparison of Structural and Chemical Properties of Black and Red Human Hair Melanosomes,” Photochemistry and Photo- biology, Vol. 81, No. 1, 2005, pp. 135-144.
 Y. Liu, V. R. Kempf, J. B. Nofsinger, E. E. Weinert, M. Rudnicki, K. Wakamatsu, S. lto and J. D. Simon “Comparison of the Structural and Physical Properties of Hu- man Hair Eumelanin Following Enzymatic or Acid/Base Extraction,” Pigment Cell Research, Vol. 16, No. 4, 2003, pp. 355-365.
 Y. Yamaguchi and V. J. Hearing “Physiological Factors That Regulate Skin Pigmentation,” Biofactors, Vol. 35, No. 2, 2009, pp. 193-199.
 K. Koike, A. Mamada, S. Nagase, O. Yoshida and A. Takaoka “Structure Analysis of Human Hair Fiber with High Voltage Electron Microscopy (HVEM),” Microscopy, Vol. 43, No. 4, 2008, pp. 258-262.
 T. Nishida, R. Yoshimura and Y. Endo, “Three- Dimensional Distribution of TrkA Neurotrophin Receptors in Neurite Varicosities of Differentiated PC12 Cells Treated with NGF Determined by Immunoelectron Tomography,” Cell Tissue Research, Vol. 35, No. 1, 2013, pp. 1-13.
 H. Watabe, T. Kushimoto, J. C. Valencia and V. J. Hearing “Isolation of Melanosomes,” Current Protocols in Cell Biology, Vol. 3, 2005, pp. 1-3, 14, 16.
 A. Kawase, T. Kushimoto, Y. Kawa, K. Ohsumi, H. Ni- shikawa, T. Kawakami, M. Mizoguchi and Y. Soma “Proteomic Analysis of Immature Murine Melanocytes at Different Stages of Maturation: A Crucial Role for Calreticulin,” Journal of Dermatological Science, Vol. 49, No. 1, 2008, pp. 43-52.
 A.Takaoka, T. Hasegawa, K. Yoshida and H. Mori “Microscopic Tomography with Ultra-HVEM and Applications,” Ultramicroscopy, Vol. 108, No. 3, 2008, pp. 230- 238. http://dx.doi.org/10.1016/j.ultramic.2007.06.008
 J. A. Brumbaugh “Ultrastructural Differences between Forming Eumelanin and Pheomelanin as Revealed by the Pink-Eye Mutation in the Fowl,” Developmental Biology, Vol. 18, No. 4, 1968, pp. 375-390.
 H. Nakagawa and G. Imokawa “Characterization of Melanogenesis in Normal Human Epidermal Melanocytes by Chemical and Ultrastructural Analysis,” Pigment Cell Research, Vol. 9, 4, 1996, pp. 175-178.
 R. D. Grickman, S. L. Jaques, R.T. Hall and N. Kumar “Revisiting the Internal Absorption Coefficient of the Retinal Pigment Epithelium Melanosome,” Proceedings of SPIE, Vol. 4257, 2001, pp. 131-141.
 S. Ito, Y. Nakanishi, R. K. Valenzuela, M. H. Brilliant, L. Kolbe and K. Wakamatsu, “Usefulness of Alkaline Hydrogen Peroxide Oxidation to Analyze Eumelanine and Pheomelanine in Various Tissue Samples: Application to Chemical Analysis of Human Hair Melanins,” Pigment Cell Melanoma Research, Vol. 24, No. 4, 2011, pp. 605- 613. http://dx.doi.org/10.1111/j.1755-148X.2011.00864.x
 A. Biesemeier, U. Schraermeyer and O. Eibl “Quantitative Chemical Analysis of Ocular Melanosomes in Stained and Non-Stained Tissues,” Micron, Vol. 42, No. 5, 2011, pp. 461-470.