## 4 Biological Applications of EUV/SXR Microscopes Based on a Double Stream Gas-Puff Target Source
### A 2D Full Field Imaging of Biological Samples
#### 4.1.2 SXR Bioimaging  
High contrast images, employing \"water window\" radiation were obtained due to a significant difference in absorption coefficients of biological sample constituents, i.e., water (oxygen) and carbon, enabling, in some cases, the observations of small features, approaching the half-pitch spatial resolution  
Figure 14: Images of diatoms acquired (**a**) with the optical microscope and (**b**) with the EUV microscope. The zoomed inset in (**b**) shows small features, of the order of 135 nm. Figure 15: (**a**) _Chrysodidymus syuroideus_ algae imaged with the EUV microscope. In (**b**) small features (\(\sim\)50–100 nm) are resolved. Figure modified, based on [76]. of the microscope. Herein some biological samples imaged will be presented and discussed. For SXR image, an exposure from 1 to 600 SXR pulses was required, at a 10 Hz source repetition rate. The CCD camera was usually cooled down to \(-\)20 \({}^{\circ}\)C during the image acquisition, to decrease its intrinsic thermal noise and its readout was generally set to f\({}_{\text{s}}\) = 2.5 MHz. DNA Plasmids pBR322  
A supercoiled, circular double stranded pBR322 plasmid DNA (from Inspiralis, Norwich, UK), was deposited on top of a 50 nm thick Si\({}_{3}\)N\({}_{4}\) membrane, from a 100 ng/mL solution, subsequently dried in an N\({}_{2}\) atmosphere for 20 min [88]. The image shown in Figure 16a was acquired after 500 SXR pulses and an exposure time of 50 s. It is possible to observe DNA aggregations (from a few \(\upmu\)m down to \(\sim\)200 nm, as shown in Figure 16b [80]). CT 26 Fibroblasts Cells from Mus Musculus Colon Carcinoma  
CT 26 fibroblasts were imaged also in the \"water window\" spectral range. The cells were dehydrated by a series of ethanol-distilled water washes and then cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, MO, USA). More details about the sample preparation are reported in [77]. After dehydration by a series of ethanol-distilled water washes, the sample was deposited on top of a 100 nm Si\({}_{3}\)N\({}_{4}\) membrane. In Figure 17a,c it is possible to observe the cells, 20-30 mm in size, imaged using an optical microscope (40\(\times\) objective and NA = 0.7). The SXR images (Figure 17b-e), acquired with 600 SXR pulses, 1 min exposure, and a 50 kHz CCD readout speed, show enhanced spatial resolution due to the shorter employed wavelength, allowing to clearly observe internal and external structures of the cells. Moreover, the possibility to image small features of the order of \(\sim\)60 nm, as shown in Figure 17e, demonstrate that the SXR microscope clearly goes beyond the capabilities obtainable employing classical visible light microscopy. However, it can be noted that, due to the dehydration shrinkage, in Figure 17b,d, it is possible to observe some distortions in the cell structure, as well as some radiation damages [76; 77]. The same cell culture was prepared with gradual dehydration in EtOH series (final concentration 70%), without any fixation procedure, and deposited on top of a 30 nm thick Si\({}_{3}\)N\({}_{4}\) membrane. Moreover, in this case it was possible to distinguish the internal structures of the cells. Figure 18 shows a direct comparison between the image acquired with an optical microscope (a,c) and with the SXR  
Figure 16: (**a**) Supercoiled pBR322 plasmid DNA, deposited on top of a 50 nm thick Si\({}_{3}\)N\({}_{4}\) membrane, from 100 ng/\(\upmu\)L solution. The zoomed inset in (**b**) shows a 200 nm DNA segment. The image was acquired after 500 SXR pulses (50 s exposure). Figure based on [80]. microscope (b,d). The SXR image, acquired with 200 SXR pulses and an exposure time of 20 s, shows small features, of the order of \(\sim\)240 nm [76,89,90]. Diatoms  
A diatom sample preliminarily imaged with the optical microscope (Figure 19a), was then observed with the SXR \"water window\" microscope (Figure 19b) and with the EUV microscope (Figure 19c). The SXR images were obtained with the SXR image (Figure 19c).