Application of 3D Reflectance Confocal Microscopy: Melanocytic Proliferations as 3D Models; JAAD Supplemental Material
Melanoma on chronically sun-damaged skin (MSDS) is challenging to detect. Reflectance confocal microscopy (RCM) increases diagnostic accuracy, but relies upon interpretation of 2D horizontal sections. Our objective was to determine whether existing technology could be used to create 3D models, which would expand on data available for discriminating skin biology. Supplemental Document: Expanded manuscript describing the background and process. Supplemental Figure 1. Object files. 3D Model from confocal image stack of a selected large atypical cell showing multiple dendrites extending from the body of the cell and nuclear abnormalities. The opacity of the cell body is reduced, such that the nucleus can be seen internally. The object can be rotated freely and examined from any angle using a 3D viewer. NOTE: In order to view the 3D object file interactively and with transparency using Windows 10 3D Viewer, please unzip file containing the .mtl and .obj to the DESKTOP (not a network drive or server). Once both files are on the desktop, double clicking on the .obj file will allow the native 3D viewer to open the model. Supplemental Figure 2. 3D reconstruction of fields of melanoma cells sampled from confocal stacks. The left panel reveals marked pleomorphism while the right panel reveals a relatively uniform, although atypical, collection of cells. Scale bar is 50µm. Supplemental Figure 3. Volume rendering of a stack of confocal images of melanoma on sun-damaged skin viewed along the z axis. Most refractile melanocytic cells are located in the basal layer at approx. 38µm from stratum corneum (yellow arrow), while rare pagetoid cells are present higher in the epidermis (green arrow). This image also reveals marked flattening of the DEJ (orange bracket). Scale Bar is 50µm. Supplemental Figure 4. Movie (.AVI) of a 3D reconstruction of a confocal image stack examining the DEJ by showing the dermal papillae, the blood vessels inside the DP and an adjacent hair follicle.
Steps to reproduce
Technology: Confocal images were acquired using the Vivascope 1500. The slices were spaced at 1.52µm in the z direction with 500µm x 500µm size in the x and y directions. Stacks were obtained from the stratum corneum to the level of superficial dermis down to an approximate maximum depth of 200µm. Software: The confocal image stacks were imported into ImageJ, where they were aligned and preprocessed. Entire stacks were selected for rendering, cell bodies and other structures were selected for segmentation and reconstruction. Reconstructions are generated with TrakEm2, using manual segmentation techniques. When atypical nucleated cells were reconstructed, the opacity of the cell body was reduced in the model, making the nuclei visible as it extends through the inside cavity of the cell body. When processing stacks, the non-cubic voxels were resampled to allow for more accurate visualization.