Simple Methods to Correlate


Figure 3: Cell shrinkage and distortion generated during dehydration steps. FM and SE- SEM overlay of cells grown on ITO cover slips. Blue: DAPI, Green: HER2-GFP, Red: AKT2-RFP.


characterize the morphology, number, and localiza- tion of nucleoli in cultured breast cancer cells. Te presence of prominent nucleolar structures in tumor tissues is one of the parameters used by pathologists to define the “nuclear grade of tumors” [23]. More- over, larger nucleolar size is recognized as a contribu- tor in both tumor initiation and cancer progression [24]. In this example, the fiducials used for correla- tion were the grids imprinted in the wells where cells were grown. Cells grown on these IBIDI slides were primary fixed as described above and imaged by FM (Figure 1B). However, as we have described elsewhere [3], live cell imaging can also be performed using this substrate. During FM screening, the absence of DAPI staining within the nuclei of the cells revealed the localization of the nucleoli (Figures 5A and D). Based on the ROI locations and using the IBIDI grid that is visible on the block under any optical microscope as reference, we generated 250 nm thick sections aſter staining and embedding the cells. As shown in the image overlays (Figures 5C and F), the absence of DAPI signal shown in Figures 5A and 5D provides an indication of nucleoli presence in the BSE-SEM


Figure 4: MFC7 breast cancer cells grown on ITO cover slips and labeled for membrane, cellular, and nuclear markers. (A) 20× brightfield with fluorescence signal overlays. (B) 40× GFP and RFP tagged proteins together with DAPI signal overlayed with transmitted light. (C) 40× fluorescence signal overlayed with the SE-SEM. (D) Higher magnification of labeled region of interest showing more cellular details. Scale bars: A: 400 μm, B: 100 μm, C: 80 μm, D: 20 μm. Images show missing cells between FM and SEM due to sample preparation as well as anisotropic shrinkage.


2020 July • www.microscopy-today.com 27


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