# Nanometer-Resolution Imaging of Living Cells Using Soft X-ray Contact Microscopy
## 4 SXCM and Correlative Microscopy  
Interpreting the results taken with an X-ray microscope often requires the confirmation and identification of cellular structures or proteins. Based on the progress in engineering and advanced microscopy, correlative techniques could solve these issues. Furthermore, the development of various correlative techniques has reduced the resolution of various techniques by as much as an order of magnitude [71; 72]. The current status of correlative methods based on X-ray microscopy is presented in Table 2. Soft X-ray cryo-microscopy/tomography provides the three-dimensional structural information with nanometer resolution of biological objects up to 12 \(\upmu\)m thick and can be correlated with conventional and super-resolution fluorescence microscopy. So far, (cryo)SXM, (cryo)SXT, and related techniques (such as laser-plasma soft X-ray microscope (LPSXM)) have been correlated with conventional (or cryo-) fluorescence microscopy [73; 74; 75] and super-resolution fluorescence techniques, such as SIM [76; 77]. This has enabled the study of the role of specific proteins in thick cellular objects, such as the nucleus, without serial sectioning. The proof of principle was presented on mammalian cells and insects [73; 74; 76; 77]. Moreover, X-ray ptychography with XRF and X-ray holography was combined with X-ray scanning diffraction with STED [78; 79]. The possibility of imaging _Chlamydomonas reinhardtii algae_ in its native hydrated state by rapid and continuous X-ray scanning was presented in the correlative approach of ptychographic transmission imaging with sub-20-nanometer resolution and X-ray fluorescence imaging with sub-100-nanometer resolution of diffusible and bound ions in cryo-preserved, hydrated cells, without the need to add specific labels [78]. The correlation of holographic X-ray imaging, X-ray scanning diffraction, and STED microscopy for studying labeled and unlabeled structures in a complementary \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multicolumn{2}{c}{**Techniques**} & & & & & \\ \hline \hline
**X-ray Technique** & **Correlated** & **X-ray Source** & **Sample** & **Object of Interest** & **Spatial Resolution** & **References** \\ \hline Cryo-SXM/SXT & Cryo-FL microscopy & Synchrotron & Cryo-preserved; fluorescently tagged & & & & \\ \hline Cryo-SXM/SXT & Cryo-FL microscopy & Synchrotron & Cryo-preserved; fluorescently tagged & & & & \\ \hline Cryo-SXM/SXT & Cryo-FL microscopy & Synchrotron & Cryo-preserved; fluorescently tagged & & & & \\ \hline Cryo-SXM/SXT & Cryo-FL microscopy & Synchrotron & Cryo-preserved; fluorescently tagged & & & & \\ \hline LPSXM & FL microscopy & Laser plasma & Hydrated; fluorescently tagged & & & & \\ \hline X-ray ptychography & XRF & Synchrotron & Cryo-preserved & _Chlamydomonas reinhardtii_ & 85 & & [201] [78] \\ \hline X-ray holography, & & & Dry/fixed; cryo-preserved; fluorescently tagged & & & & \\ \hline Cryo-SXT & Cryo-SIM & Synchrotron & Cryo-preserved; fluorescently tagged & & & & \\ \hline STXM & XRF & Synchrotron & Paraffin-embedded & & & & \\ \hline Cryo-SXT & Cryo-SIM & Synchrotron & Cryo-preserved; fluorescently tagged & & & & \\ \hline \hline \end{tabular}  
* NS—not specified; LPSXM: laser-plasma soft X-ray microscopy; FL: fluorescence, SIM: super-resolution structured illumination microscopy, SXM: soft X-ray microscopy; SXT: soft X-ray tomography, STED: stimulated emission depletion, XRF: X-ray fluorescence.  
\end{table} 
Table 2: Selected current biological and medical applications of X-ray-related correlative microscopy. manner was demonstrated [79]. A compressive sensing method combining STXM with XRF for investigating complex biological systems solved the problem of examining large areas at high resolutions, which was very time-consuming and often restricted to pre-selected representative regions [80]. The combination of SXT with SIM gave an excellent resolution of 25-50 nm [77].