# Imaging of Cell Structures Using Optimized Soft X-ray Contact Microscopy
## 3 Results and Discussion
### SXCM Imaging of Various Cell Lines  
Cell morphology is essential for cell imaging with SXCM because differences in cell thicknesses and internal complexity influence X-rays' adsorption through the sample. Therefore, in our studies, three human cell lines with significantly different morphology were chosen to show the effect of the soft X-ray radiation absorbed energy on the SXCM based imprint preparation. HCC38 breast cancer cells and hMSC present various shapes and sizes compared to DU145 prostate carcinoma cells under the inverted optical microscope (Figure 7). HCC38 and hMSC cells are of elongated shape with several thin and long protrusions. hMSCs are much bigger, extending up to 400 \(\upmu\)m in length. DU145 prostate carcinoma cells, instead, are smaller and of a rhombic shape. The thickness of the attached DU145 cells is approximately 6 \(\upmu\)m [34], which is even three times more than the thickness of hMSC cells, which is 2.5-3.5 \(\upmu\)m (depending on the number of passages [35]).  
Figure 6: AFM images of cellular imprints of fixed HCC38 breast cancer cells saved in PMMA photoresists by soft X-ray contact microscopy. Images were obtained with SXR exposure of (**a**) 400, (**b**) 600, and (**c**) 800 pulses. Imprints were recorded by AFM working in contact mode (Xe120, Park Systems) with OTR4 cantilevers (0.02 N/m, Bruker). Scan size of \(80\times 80\)\(\upmu\)m\({}^{2}\). Two types of signal are presented: deflection (**top** panel) and height (**bottom** panel).  
Figure 7: Cell morphology: (**a**) DU145 prostate cancer cells, (**b**) HCC38 breast cancer cells, (**c**) hMSC (human mesenchymal stem cells). Images were taken using an inverse optical microscope (Primo Vert, Zeiss) with a 20\(\times\) objective. The scale bar is 200 \(\upmu\)m.  
The images of morphologically different DU145, hMSC, and HCC38 cells obtained by AFM after using SXCM are presented in Figure 8. Each cell line was prepared; PMMA imprinted, and visualized using the same protocol. PMMA photoresist was irradiated with 800 SXR pulses and developed with the optimized MIBK:IPA ratio of 1:2.  
Depending on the cell type, different details were preserved by the SXCM, as revealed by images recorded by AFM working in tapping mode. It turned out that in small and rhombic DU145 cells, the intracellular organelles were densely packed, and it was difficult to visualize their internal structures (Figure 8a). The HCC38 cells are larger and more flattened with clearly visible cell nuclei accompanied by nucleoli (Figure 8b). hMSCs were the largest adherent cells among the cell lines studied. In these cells, the nucleus was less visible. However, it was quite easy to visualize the cytoskeleton elements inside the cell, the sharp edges of the cell membrane, and its projections (Figure 8c). The cell-cell interactions could also be observed (Figure S1).  
In conclusion, our results demonstrate that it is impossible to use the same SXR absorbed energy (expressed as the number of photons or pulses) for imaging the different types of cell. The experimental condition for imprint preparation must be optimized for each cell line separately. However, one can conclude that high SXR absorbed energy can be essential for visualization of the intracellular structures like the nucleus. At low exposures, on the other hand, the membrane protrusions could be visible.