Integrated soft X-ray/visible fluorescence microscope using divided-pupil reflective optics for correlative cellular imaging.

Observing living cells in real time is crucial to elucidating the dynamic processes underlying cellular function. We developed an integrated correlative full-field microscope that enables soft X-ray and visible fluorescence imaging with a common field of view using divided-pupil reflective optics, without mechanical switching. Cellular imaging was performed using a water-window soft X-ray beam generated as the third harmonic of a soft X-ray free-electron laser (SXFEL). Sequential fluorescence and soft X-ray imaging were carried out as a pilot study to verify the performance of the microscope. The system enabled localization of fluorescent agents and visualization of structural details in chemically fixed cells treated with fluorescent beads, as well as correlative visualization of living cells expressing a cell cycle fluorescence marker. As a mechanically switch-free system, the microscope offers a platform for the development of advanced simultaneous correlative imaging of living cells.