# Imaging of Cell Structures Using Optimized Soft X-ray Contact Microscopy
## 3 Results and Discussion
### Optimization of SXCM Technique for Cell Imaging
#### 3.1.1 Optimization of the PMMA Development  
The development of light-sensitive materials is a critical step in their usage for SXCM because it can affect the contrast of the image, the image resolution, and the shape and threshold parameters of the photoresist's response curve (Figure 3). The PMMA is a positive photoresist (a fraction of the photoresist exposed to radiation is soluble in the photoresist developer) with high spatial resolution. MIBK is a strong solvent for PMMA, that is why MIBK is usually diluted with IPA. IPA is a very weak solvent for PMMA and cannot be used efficiently to dissolve PMMA [29]. In our study, the samples of PMMA were irradiated with 600 SXR pulses, and then chemically developed in different MIBK to IPA ratios for four minutes. This duration of the development phase was confirmed in the independent experiments (data not shown). The thickness of degraded PMMA was \(290\pm 52\), \(223\pm 35\), and \(86\pm 25\) nm for the MIBK:IPA ratio 1:1, 1:2, and 1:3, respectively (Figure 3). The results showed a proportional decrease in the thickness of the dissolved PMMA in relation to the increased concentration of IPA in the mixture. Figure 3: Dissolved polymethyl methacrylate (PMMA) thickness versus MIBK:IPA ratio (600 soft X-ray (SXR) pulses, the development time of 4 min). PMMA thickness was obtained from atomic force microscopy images (AFM, di CPII Veeco) as described in Material and Methods. Data are expressed as a mean \(\pm\) standard deviation from 10 images. It has already been shown that sensitivity, contrast, roughness, and resolution depend on the photoresist and its development conditions [30; 31]. Thus, it is necessary to select the photoresist development conditions required to obtain a high spatial resolution while preserving the cellular structures. The developer concentration must be finely tuned to dissolve the degraded PMMA and leave unaffected the regions unexposed to soft X-ray radiation [32; 33]. [PERSON] and coworkers [31] investigated the influence of an unconventional developer comprising of IPA and water on the photoresist characteristics. Three developers, i.e., water/IPA, MIBK/IPA, and water/MIBK were studied. The water/MIBK mixture was rejected due to the low contrast of the final images. Improvements in sensitivity (~40%), contrast (~20%), and roughness (nearly an order of magnitude) were observed when the 3:7 water/IPA ratio was used when compared to the 1:3 MIBK/IPA developer. In our study, cell imprints were visualized after the development with three different ratios of MIBK to IPA, i.e., 1:1, 1:2, and 1:3 (Figure 4). The MIBK of high concentration in a mixture (pure MIBK or MIBK:IPA--1:1) seems to be too aggressive as the edges of the cells appeared to be smoothed by the developer; also, no cellular protrusions were observed (Figure 4a). In the case of the 1:2 developer, the spreading of cells was preserved (Figure 4b). The central part of the cells is smooth, but the cell nucleus could be identified. The use of MIBK solution that was too diluted (MIBK:IPA 1:3) failed to reveal small and thick details of the imaged sample (Figure 4c). Here, no cell details were observed in the images indicating that such development conditions did not enable to obtain the images with high contrast. Ultrasonically assisted development has been used to improve the resolution and line-edge roughness [31]. However, in our case, PMMA deposited on a silicon wafer was damaged due to the ultrasonic agitation. Therefore, in further steps, the adequate concentration of MIBK was chosen to be 1:2 MIBK:IPA. It can be concluded that in this particular condition where the photoresist of specific molecular weight, thickness, this biological sample, and exposure wavelength were used in the experiments, the optimal concentration of MIBK/IPA for sensitivity, linear response region, and developing time was 1:2.