# Soft X-ray Microscopy Techniques for Medical and Biological Imaging at TwinMic--[PERSON]
## 3 Results
### STXM Coupled with LEXRF
#### 3.1.2 Reproductive Medicine  
The combination of STXM with LEXRF allows for an efficient localization of specific elements in biological specimens. In reproductive medicine, elements such as Na or Mg have been shown to be directly related to the functionality of the biological system. The TwinMic setup offers the capability to localize these elements in oocyte and spermatozoa (Figures 6 and 7). At the moment, we cannot provide a quantitative analysis of the elements present in the biological specimens, which requires suitable reference standards  
Figure 5: Absorption (Abs) and differential phase contrast (DPC) images of mouse fibroblast cells cultured on Si3N4 membranes and exposed to a 500 \(\upmu\)M concentration of CoFe2O4 NPs, depicted with the corresponding XRF maps of C, O, Na, Mg, Fe, Co, and Scattering (E0). The maps were collected at 1.5 keV over an area of 40 \(\upmu\)m \(\times\) 70 \(\upmu\)m with a 400 nm step size, with a 4 s/pixel acquisition time for XRF. Scale bar is 10 \(\upmu\)m.  
with similar composition, hard to find in the soft X-ray regime. However, comparisons between samples can still be made by evaluating the relative variation of a specific element.  
Figure 6: Absorption (Abs) and differential phase contrast in X (X mom) and Y (Y mom) images of primary oocyte in an ovarian tissue, depicted with the corresponding XRF maps of O, Na, and Mg. The maps were collected at 1.5 keV over an area of 70 \(\upmu\)m \(\times\) 70 \(\upmu\)m (STXM images) or 60 \(\upmu\)m \(\times\) 60 \(\upmu\)m (XRF maps) with 300 nm and 1 \(\upmu\)m step sizes, respectively, with a 7 s/pixel acquisition time for XRF. Scale bar is 10 \(\upmu\)m.  
Figure 7: Absorption (Abs) and differential phase contrast in X (X mom) and Y (Y mom) images of spermatozoa deposited on Si\({}_{3}\)N\({}_{4}\) windows, depicted with the corresponding XRF maps of O, Na, and Mg. The maps were collected at 1.5 keV over an area of 80 \(\upmu\)m \(\times\) 80 \(\upmu\)m with a 400 nm and 1 \(\upmu\)m step size for STXM images and XRF, respectively, and a 10 s/pixel acquisition time for XRF. Scale bar is 10 \(\upmu\)m.  
In a previous study related to artificial reproductive techniques, we indeed demonstrated that STXM can be of help in analyzing the quality of follicles inside ovary tissue samples and the kind of damage resulting from a slow freezing protocol of cryopreservation, by monitoring the morphology of the cells and the distribution of several physiologically relevant elements such as Na, Mg, and O [36]. In particular, it was found that among other effects, low-quality follicles, frequent in the freeze-thawed tissues, show a high Na level in the ooplasm [78].  
Figure 6 shows the absorption and differential phase contrast images of an oocyte in a human ovarian tissue slide together with the corresponding distribution of oxygen, sodium, and magnesium, which was collected in the frame of this research.  
Analogously, STXM coupled with LEXRF has been successful in revealing elemental changes in spermatozoa treated with photobiomodulation therapy [85] and in capacitation procedures [32], where elements such as Na and Mg seem to play an important role. Additionally, elemental changes were revealed when sperm cells were exposed to oxidative stress conditions [79]. Figure 7 depicts an example of STXM and LEXRF mapping performed on healthy spermatozoa. The data confirm the capability of our technique in localizing specific elements inside sperm heads and tails.