# Imaging of Cell Structures Using Optimized Soft X-ray Contact Microscopy  
[PERSON]  
1 Biomedical Engineering Centre, Institute of Optoelectronics, Military University of Technology, PL-00908 Warsaw, Poland; [EMAIL_ADDRESS] (P.N.O.); [EMAIL_ADDRESS] (A.N.-S.); [EMAIL_ADDRESS] (J.C.)1  
[PERSON]  
2 Laser Technology Division, Institute of Optoelectronics, Military University of Technology, PL-00908 Warsaw, Poland; [EMAIL_ADDRESS] (P.W.); [EMAIL_ADDRESS] (A.B.); [EMAIL_ADDRESS] (H.F)2  
Footnote 1: email: {[PERSON].N.O.), (P.N.O.), (P.N.O.),  
The system was first developed for dried samples [12; 13], showing its applicability potential and the need to optimise soft X-ray (SXR) conditions to visualize the internal cell structures in the thickness and morphological complexity-dependent manner. Currently, the system can imagine living cells in the He-filled environmental chamber using an additional silicon nitride (Si\({}_{3}\)N\({}_{4}\)) membrane, allowing moisture underneath the membrane during the entire exposure. Exposure of the samples is much easier to perform, without the necessity to encapsulate or sandwich the samples between two Si\({}_{3}\)N\({}_{4}\) membranes, as it is done typically using transmission-based electron microscopy (TEM [14; 15; 16]), synchrotron-based [17], and plasma-based [18] SXR microscopes, or in some kind of encapsulated vacuum-tight holder [7].  
In our case, the evacuation of the sample chamber is not necessary; thus, we save time and avoid potential problems with sample degassing if the capsule is not properly sealed. Finally, the SXCM system is based on the source with a gas puff target; thus, it is debris-free and easy for operation and maintenance. It does not produce ablation products associated with laser interactions with solid targets [19], nor does it require any surface polishing before the operation [20].  
The polymethyl methacrylate (PMMA) photoresist is the most often applied detector in SXCM [21; 22]. The exposure of PMMA to ionizing radiation causes breakage of bonds along the polymer chain. The photoresist's subsequent chemical development in a solvent mixture leads to a change in its surface topography. This change depends on the X-ray energy deposited in the photoresist [23]. The cells adherent to the PMMA sheathe its surface from the radiation. Depending on the cell thickness, the 3D visualization of internal structures within eukaryotic cells becomes possible [24; 25].  
Despite the technological progress of the source design and improvement of its parameters, the SXCM analysis of biological specimens still requires optimization due to different cell morphologies. Three morphologically different cell lines were chosen for this study: DU145 prostate carcinoma cell line, HCC38 breast cancer cell line, and Poieties mesenchymal stem cells. The SXR absorbed energy in the photoresist, determined by the number of soft X-ray pulses, and the photoresist development procedure was optimized to obtain images of internal structures of a cell with a resolution of a few tens of nanometer. The novelty of our work lies in demonstrating that each cell line requires different technological approaches to be observed in detail under SXCM.