# Soft X-ray Microscopy Techniques for Medical and Biological Imaging at TwinMic--[PERSON]
## 2 Materials and Methods
### TwinMic Imaging Modes
#### 2.1.1 Scanning Transmission X-ray Microscopy  
STXM is nowadays a standard X-ray microscopy technique, used especially in the soft or milder X-ray regime. It requires suitable X-ray optics, usually zone plate (ZP) diffractive optics, to focus the X-ray photons on the sample plane, where the specimen is usually scanned across the microprobe. In a few cases, the beam can be scanned across the sample [4]. In scanning techniques, the spatial resolution is determined by the probe size. For each point in the raster scan, the transmitted X-rays are collected by means of suitable detectors (photodiode, ionization chamber, CCD...), allowing one to record the absorption signal. In the case of a two-dimensional detector, as with the TwinMic beamline, in addition to the absorption image, differential phase contrast and darkfield contrast can be acquired simultaneously, providing a complementary imaging mode [48; 49; 50; 51]. According to the incident beam intensity and the detector characteristics, where the transmitted signals are intrinsically acquired in a serial way, STXM images may require from a few minutes to several tens of minutes. However, by means of suitable detectors, the scanning operation mode also allows for the simultaneous acquisition of other signals point-by-point in the raster scan, such as XRF photons [15] or photoelectrons [52] emitted by the sample. Thus, besides morphological images, chemical ones can be obtained. Similarly, by collecting a stack of absorption images across the absorption edge of specific elements, after suitable image alignment, one can extract X-ray absorption near-edge spectroscopy (XANES) signals, which allows for element speciation identification [4; 8; 10]. A schematic view of the TwinMic STXM operation mode is presented in Figure 1. A series of pinholes of different dimensions acts as a secondary source for the microscope. The incoming monochromatic beam is focused on the sample plane through ZP diffractive optics, whose first diffraction order is selected by the order sorting aperture. The size of the focused beam is given by the secondary source size demagnified through the ZP. For the experiments shown here, a 600 \(\upmu\)m diameter Au ZP with a 50 nm outermost zone was used. The transmitted X-rays are collected by an electron multiplying CCD (EMCCD) camera DV860 from Andor Technology (128 \(\times\) 128 pixels, 24 \(\times\) 24 \(\upmu\)m\({}^{2}\) pixel size) by means of an X-ray-visible light converting system constituted by a P43 phosphor screen, a visible light lens, a mirror tilted by 45\({}^{\circ}\), and an objective lens mounted on the CCD camera itself [50]. \begin{table}
\begin{tabular}{c c c c c c} \hline
**Technique** & **Spatial Resolution** & **Typical Acquisition Time**/Pixel [s] & **Overall Acquisition Time** & **Field of View [\(\upmu\)m]** & **Detector Type** \\  & **[\(\upmu\)m]** & **Time/Pixel [s]** & **[h]** & & \\ \hline STXM & 0.08–2 & 0.005–0.05 & 0.1–2 & 10–80 with piezo & \\  & & & & & \\  & & & & & \\  & & & & & \\  & & & & & \\  & & & & & \\  & & & & & \\ \end{tabular} \({}^{1}\) Recently, with compressive sensing (CS) implementation and sample stage upgrade, we have been able to reach fields of view of about 1 mm \(\times\) 1 mm. \({}^{2}\) Electron-multiplying CCD. \({}^{3}\) Here, we report the values for full frame acquisition.  
\end{table} 
Table 1: Summary of TwinMic techniques. Eight silicon drift detectors (SDDs) are mounted circularly and symmetrically, with their axes placed at 20\({}^{\circ}\) from the sample plane and their center 28 mm from the focal spot [15,16]. The output signal of the preamplifier electronics (XGLab srl, Bruker Nano-Analytics), which is placed in the TwinMic vacuum chamber in close proximity to the SDD chip, is then processed through the DANTE Digital Pulse Processor (XGLab srl, Bruker Bruker Nano-Analytics) [53].