X-ray transparent flow cell with integrated dielectrophoretic cell handling for single cell water-window microscopy

Soft X-ray (SXR) microscopy represents a powerful, label-free imaging technique for studying cellular structures and intrinsic biophysical properties at nanometric resolution. Operating within the so-called water-window range (284 – 543 eV), this method offers high-contrast visualization of fully hydrated biological specimens, due to the characteristic penetration depth of SXRs in water in this energy range. Despite these advantages, a key limitation remains: the inability to efficiently manipulate and stably trap individual suspended cells or microparticles in laser focus under vacuum conditions, as typically required for SXR imaging. Conventional immobilization strategies often rely on chemical fixation or artificial adhesion, which limit sample mobility and complicate positioning and sample exchange between acquisition runs. To address this challenge, the present study focuses on the development of a microfluidic device specifically designed to be compatible with SXR microscopy, allowing for stable and flexible manipulation, as well as robust trapping of micro-objects, for high-resolution imaging. The proposed system consists of a flow cell incorporating a microfabricated chip, where gold electrodes are photolithographically patterned onto thin silicon nitride (Si₃N₄) membranes. Through the application of negative dielectrophoresis (nDEP), the device allows for the precise, non-invasive manipulation of individual particles in their native liquid environment. This approach enables controlled positioning, trapping and rotation of target micro-objects, while ensuring accurate alignment within the laser focus, ultimately enabling the first successful soft X-ray image of a live cell suspended in liquid, via dielectrophoretic trapping under highvacuum conditions. Moreover, the device was designed with the aim of enabling easy and rapid sample replacement during sequential exposure, without requiring complete venting of the vacuum chamber. This feature increases experimental throughput while maintaining consistent imaging conditions throughout the acquisition process. Overall, this novel approach provides a robust platform for long-term in vivo SXR analysis of suspended biological samples, opening new possibilities for more versatile applications in single cell studies and high-resolution correlative imaging.