Transmission and Scanning Electron Microscopy and Freeze-Fracture Replication of Normal Human Melanocytes and Human Melanoma Cells in Tissue Culture

Ion beam etching and freeze fracture techniques were utilized in conjunction with scanning electron microscopy to study the ultrastructure of normal and diseased human hair. Topographical differences in the cuticular scale of normal and diseased hair were demonstrated in previous scanning electron microscope studies. In the present study, ion beam etching and freeze fracture techniques were utilized to reveal subsurface ultrastructural features of the cuticle and cortex.Samples of normal and diseased hair including monilethrix, pili torti, pili annulati, and hidrotic ectodermal dysplasia were cut from areas near the base of the hair. In preparation for ion beam etching, untreated hairs were mounted on conducting tape on a conducting silicon substrate. The hairs were ion beam etched by an 18 ky argon ion beam (5μA ion current) from an ETEC ion beam etching device. The ion beam was oriented perpendicular to the substrate. The specimen remained stationary in the beam for exposures of 6 to 8 minutes.

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Three-dimensional relationships between tumor cells and microcirculation with double cyanine immunolabeling, laser scanning confocal microscopy, and computer-assisted reconstruction: an alternative to cast corrosion preparations.

Journal of Histochemistry & Cytochemistry ◽

10.1177/42.5.7908912 ◽

1994 ◽

Vol 42 (5) ◽

pp. 681-686 ◽

Cited By ~ 27

Author(s):

V Rummelt ◽

L M Gardner ◽

R Folberg ◽

S Beck ◽

B Knosp ◽

...

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Blood Vessels ◽

Tumor Cells ◽

Laser Scanning ◽

Three Dimensional ◽

Melanoma Cells ◽

Tumor Blood Vessels ◽

The Relationship ◽

Scanning Electron

The morphology of the microcirculation of uveal melanomas is a reliable market of tumor progression. Scanning electron microscopy of cast corrosion preparations can generate three-dimensional views of these vascular patterns, but this technique sacrifices the tumor parenchyma. Formalin-fixed wet tissue sections 100-150 microns thick from uveal melanomas were stained with the lectin Ulex europaeus agglutinin I (UEAI) and proliferating cell nuclear antigen (PCNA) to demonstrate simultaneously the tumor blood vessels and proliferating tumor cells. Indocarbocyanine (Cy3) was used as a fluorophore for UEAI and indodicarbocyanine (Cy5) was used for PCNA. Double labeled sections were examined with a laser scanning confocal microscope. Images of both stains were digitized at the same 5-microns intervals and each of the two images per interval was combined digitally to form one image. These combined images were visualized through voxel processing to study the relationship between melanoma cells expressing PCNA and various microcirculatory patterns. This technique produces images comparable to scanning electron microscopy of cast corrosion preparations while permitting simultaneous localization of melanoma cells expressing PCNA. The microcirculatory tree can be viewed from any perspective and the relationship between tumor cells and the tumor blood vessels can be studied concurrently in three dimensions. This technique is an alternative to cast corrosion preparations.

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