# Nanometer-Resolution Imaging of Living Cells Using Soft X-ray Contact Microscopy
## 3 Cell Imaging with SXCM  
The cell membrane separates the cell's interior from the external environment. The most significant feature of SXCM is the visualization of the cell's interior structures located under the membrane without the requirement of a \"deroofing\" procedure, i.e., a disruption of the plasma membrane. Soft X-ray radiation makes it possible to record and observe the inside of the native cell. The first image of biological samples (diatoms) using SXCM, demonstrating the possibility o investigating biological specimens with a resolution near 10 nm, was obtained by [PERSON] 45 years ago [27]. Further works by this group [55; 57] have shown that SXCM, a relatively new form of ultrastructural imaging, has a resolution that is better than 10 nm and is uniquely suited to the examination of fragile, unstained biological specimens. Therefore, this technique has been fine-tuned over a long period of time, in order to study subtle biological phenomena, as do other microscopic methods that offer similar efficiency. The attempt to image mammalian cells, bacteria, and isolated organelles with SXCM was undertaken several times, as presented in Table 1. The mitotic chromosome structure in human lymphocytes was the first problem to be solved with this technique. The first report by [PERSON] et al. on the observation of isolated, dried single human chromosome fibers at high resolution appeared in 1990, followed by subsequent team publications [36; 58; 59]. The \"beads-on-a-string\" structure with thin filaments (diameters of 7-15 nm) and nucleosomes (diameters of 10-15 nm) in hydrated cells was revealed at a spatial resolution of 10 nm. The visualization of dry or hydrated human macrophages and Leydig cells demonstrated that SXCM allows the morphologies of actual intact cells to be viewed and encourages further technological development for biomedical research [45; 51; 60; 61; 62]. This technique also offered the possibility of monitoring the morphological changes in microorganisms (e.g., upon exposure to antibiotics), as was demonstrated for the Gram-negative bacterium, Pseudomonas aeruginosa, and Candida albicans, a yeast, among others [63; 64]. The development and popularization of the atomic force microscope (AFM) allowed its application in imaging samples with high resolution with SXCM, as described above [65]. It was a cheaper device, with less complex sample preparation and better accessibility for biological laboratories than SEM and TEM. However, it should be noted that the spatial  
Figure 2: Schematic of the desk-top laboratory system for soft X-ray contact microscopy (SXCM) based on a laser plasma soft X-ray source with a double-stream gas puff target. resolution of SXCM is determined not only by the AFM resolution but also by a photoresist and the number of X-ray source pulses. Moreover, only single-shot exposure of X-ray radiation can ensure the elimination of the blurring connected with the thermal effect and Brown motion, enhancing the resolution of an image. As shown in Table 1, the reported spatial resolutions of the imaged samples were in the range of 5-100 nm, but mainly lay within the range of 80-100 nm. PMMA is still the most frequently used photoresist for studying biological objects. EPR was used in 2003 to improve the spatial resolution of submicron structures [38]. The resist could also provide height features as small as 20 nm, and the photoresist response was at least two orders of