# Nanometer-Resolution Imaging of Living Cells Using Soft X-ray Contact Microscopy
## 1 Introduction  
Observing live intact cells is an indispensable requisite for progress in biomedical sciences. The visualization of internal cellular structures requires imaging techniques of nanometric spatial resolutions. Only such these resolutions can solve many biological problems and explain the mechanisms biologists encounter in cell biology [1; 2]. Many well-established imaging techniques in the major areas of optical microscopy, electron-based microscopy, and scanning-probe microscopy fulfill this requirement [3; 4; 5; 6; 7]. One of them is soft X-ray microscopy, which provides a unique set of capabilities between visible light and electron microscopy [8]. Soft X-ray microscopes for cell imaging are typically operated using radiation in the \"water window\", which is the spectral region between the K-shell absorption edges of carbon (284 eV, \(\lambda\) = 4.4 nm) and oxygen (543 eV, \(\lambda\) = 2.3 nm). X-ray radiation in this range can penetrate relatively thick hydrated specimens. The high natural contrast of the images is due to the difference in radiation absorption within the organic material containing carbon and water. It allows the imaging of intact cells with nanometric resolution in their native state without freezing, chemical fixation, chemical staining, orsectioning. Thus, soft X-ray microscopy can analyze the structures of live cells in a hydrated condition and study dynamics in cell morphology [5; 9; 10; 11].  
Soft X-ray microscopes, which are currently used for cell imaging, fall into two classes: full-field imaging transmission X-ray microscopes (TXMs) and scanning transmission X-ray microscopes (STXMs). Both microscopes are based on diffraction optics in the form of a Fresnel-zone plate, a diffractive optical element consisting of radially symmetric alternating opaque and transparent rings, called zones [12]. TXMs typically use a Fresnel-zone plate as a diffractive lens to form a magnified X-ray image of a sample on a 2D detector, usually a CCD camera. It is possible to obtain 3D images of biological micro objects using computed tomography methods. However, the biological specimens must be preserved in cryogenic temperatures due to the damaging effect of the absorbed X-rays during multi-projection exposition and image acquisition [13]. In the STXM techniques, a Fresnel-zone plate is used to demagnify an X-ray source to a small, focused spot, through which the sample is scanned. The images are obtained computationally from the transmitted X-ray intensity recorded as a function of the sample position. The spatial resolutions of soft X-ray microscopes, determined by the width of the outermost zone of a Fresnel zone plate, approached 10 nm in some demonstrations [14; 15] and are typically in the range of 30-50 nm [16].  
In recent years, advances in the technology used to manufacture Fresnel-zone plates and CCD detectors and the growing interest in the use of synchrotron radiation in biological research have resulted in the development of several systems for soft X-ray microscopy and tomography that enable the imaging of cells, which cannot be achieved with other imaging techniques [17; 18; 19; 20].  
However, the use of synchrotron radiation is limited to highly specialized experimental centers. Thus, there is a need for a desk-top laboratory-based and user-friendly soft X-ray microscopy system that biologists can operate. This review presents the soft X-ray contact microscopy (SXCM) technique for imaging living cells with nanometer resolution. This technique is well-established and relatively simple. Moreover, adapting the microscope design to users' requirements makes this technique a helpful instrument in biomedical research, as a complement to standard imaging methods. This study presents the development of this technique, its current applications, troubleshooting, and future development in in comparison other techniques based on the current status of X-ray-based correlative microscopy.