# Nanometer-Resolution Imaging of Living Cells Using Soft X-ray Contact Microscopy
## 3 Cell Imaging with SXCM
In 2016, a desktop laboratory system for SXCM with a compact laser-plasma source based on a gas puff target was developed by our group [44]. Since that time, the morphological features of fixed normal human cells (such as bladder HCV29 cells and keratinocytes) and cancer cells (colorectal carcinoma CT26 and urinary bladder carcinoma T24) were revealed [42; 54; 67]. The SXCM technique was further employed to resolve cancer-metastasis-related issues. First, the optimization of SXCM parameters for imaging cancer-cell structures was performed [44]. It revealed that the relationship between the exposure conditions and the quality of cell imaging with the SXCM was crucial in the efficient visualization of cell structures. A spatial resolution was also influenced by cell thickness and internal complexity. The experimental condition for imprint preparation should be optimized for various biological specimens. A high SXR absorbed energy is essential for visualizing intracellular structures, such as the nucleus. However, the membrane protrusions are visible only at low exposures (Figure 3). The selection of appropriate exposure parameters allowed the dynamic tracking of adhesion structures, such as focal adhesions (FA), within human triple-negative breast cancer (TNBC) cells [9]. Found in 15-20% of breast cancer patients, TNBC is an aggressive cancer subtype with a poor prognosis because of the lack of targeted therapies. Detailed clinical studies on the adhesion and migration of cancer cells are crucial. These processes involve the actin cytoskeleton and its interactions with the cell membrane and the extracellular matrix (ECM). The actin cytoskeleton is linked to integrin heterodimers and other adhesion proteins that comprise FAs structures. Based on AFM images of FAs, the sizes of both the agglomerates and the native single focal adhesion were calculated (Figure 4). The length of the FA aggregates was 3.11 \(\upmu\)m, the size of a single FA's \"dot head\" diameter was 0.84 \(\upmu\)m, and the length of the \"tail\" was 1.94 \(\upmu\)m. The distribution and size of the adhesive complexes in the SXCM images corresponded to the confocal scanning laser fluorescence (CLSM) microscopy images. The SXCM technique provides a higher-resolution and more detailed view of adhesive complexes' internal substructures than CLSM. However, the iden  
Figure 3: 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 exposures of (**A**) 400, (**B**) 600, and (**C**) 800 pulses. The nucleoli of the nuclei are indicated by arrows and the endoplasmic reticulum, vacuoles, and mitochondria are outlined with a frame. Imprints were recorded by AFM working in contact mode (Xe120, Park Systems Suwon, South Korea) with OTR4 cantilevers (0.02 N/m, Bruker, Camarillo, CA, USA). Scan size of 80 \(\times\) 80 \(\upmu\)m\({}^{2}\). A deflection signal is presented. Reproduced with permission from [PERSON] et al. [44]. tification of proteins within adhesion complexes can be achieved only via immunostaining and fluorescent microscopy imaging. Currently developed super-resolution methods, such as stimulated emission depletion (STED) or photoactivated localization microscopy (PALM), which allow the observation of structures such as FAs in living cells, require genetically encoded fluorophores to express the desired proteins. These super-resolution techniques use the GFP-tagging method or modified cell line, obtaining a stably integrated transgene that leads to protein-translation disorders. The inclusion of such a large tag as GFP could potentially affect its stability and ability to bind other proteins by disrupting the native folding pattern of the protein of interest, causing protein destabilization [68; 69]. However, to select transferred cells or to induce expression, substances (including antibiotics) are used in the culture of labeled cell lines that may also affect the condition of the cells.