Synchotron radiation soft X-ray imaging combined with fluorescence microscopy for single-cell imaging applications

Cells are the fundamental structural and functional units of living organisms, and understanding their structure and molecular composition is essential for elucidating the mechanisms of life. Among the various imaging technologies available for visualising cellular structures, electron microscopy is widely regarded as a ground-breaking tool, offering ultrahigh resolution down to 1 angstrom (Å) and enabling detailed analysis of cellular and subcellular architecture. However, due to the limited penetration capacity of electrons, the imaging depth of electron microscopy is confined to a few hundred nanometres, making it unsuitable for visualising the complete ultrastructure of intact single cells.With significant advances in the brightness and coherence of synchrotron radiation sources and continuous improvements in zone plate fabrication techniques, soft X-ray imaging has made substantial progress in biology, materials science and medical research. In the water window region (280–543 eV), synchrotron-based soft X‐ray imaging leverages the differential absorption properties of organic matter and water within cells to achieve intrinsic contrast without the need for staining. Soft X‐rays in this spectral region are particularly well‐suited for imaging mesoscopic cellular structures, spanning from whole single cells (approximately 10 μm in diameter) to structures approaching the scale of molecular machines (around 50 nm). This technique enables non‐destructive acquisition of three‐dimensional fine structures of cells and their subcellular components and is increasingly becoming an indispensable tool in cellular imaging. Yet, soft X‐ray imaging has limitations in directly detecting and characterising specific intracellular molecules that perform different functions but share similar elemental compositions. In contrast, fluorescence microscopy—a functional imaging technique—can precisely localise functional molecules through molecular labelling. This technique, though powerful, cannot provide comprehensive 3D structural information of entire cells and their subcellular architecture beyond the fluorescent markers. Therefore, integrating soft X‐ray imaging with fluorescence microscopy allows for simultaneous mapping of specific molecular distributions and comprehensive 3D cellular architecture at the single‐cell level. This correlative approach offers valuable insights into the relationship between cellular structure and function, playing a notable role in elucidating the mechanisms underlying disease progression and the actions of therapeutic agents at the cellular level.However, conventional wide-field fluorescence microscopy is constrained by the optical diffraction limit, offering a spatial resolution of approximately 200 nm, significantly lower than the ~30-nm resolution achievable with soft X-ray imaging. The advent of super-resolution fluorescence imaging technologies has successfully overcome this limitation, thereby narrowing the resolution gap between fluorescence and soft X-ray imaging. This advancement facilitates improved alignment and integration of fluorescence and soft X-ray images, substantially enhancing the ability to resolve complex intracellular structures and molecular distributions. This study first introduces the fundamental principles, advantages and challenges of synchrotron soft X‐ray imaging systems, both in China and globally. It then summarises the workflow and registration methods used in correlative soft X‐ray and fluorescence microscopy. The article also offers a systematic review of the status, technical characteristics and current challenges of integrated systems combining synchrotron soft X‐ray imaging with wide‐field, confocal and super‐resolution fluorescence microscopy worldwide. Furthermore, it provides an in‐depth analysis of recent advances in applying this technology to cellular imaging and discusses future development directions for correlative imaging techniques, aiming to provide new perspectives and references for X-ray imaging–based cell biology research.