Soft X-ray tomography (SXT) is a powerful microscopy technique for studying three-dimensional (3D) structures in biological and materials research. Off-line fluorescence microscopy (FM) is often used to identify types of organelles and cell conditions. A correlative combination of FM and SXT provides functional information in the cellular ultrastructure context. However, deformations and displacements often occur during the sample transfer and loading procedure, reducing the precision of the two-modal image registration. These issues can be addressed by integrating a fluorescence microscope into an SXT system, as reported for a synchrotron-based microscope in a paper discussed below, which SiriusXT have also accomplished on a lab-based soft X-ray microscope, the SXT-100.
In the referenced paper, researchers report an inline fluorescence microscope built into a synchrotron-based soft X-ray microscope. The authors combined built-in LF with SXT imaging to identify apoptotic, necrotic and normal cells.
Apoptosis is controlled cell death, accompanied by cell shrinkage, nuclear fragmentation, DNA fragmentation and mRNA decay.
Necrosis is uncontrolled cell death featured by swelling of the cell organelles, plasma membrane rupture, and cell lysis.
Cryo soft X-ray tomography, which employs low-energy X-rays, is used for imaging the 3D ultrastructure of whole cells with a resolution of nearly 50 nm. Cells or tissue blocks up to 10 µm thick are simply flash-frozen. No chemical fixation, labelling, or staining is required because the contrast is formed naturally at the given X-ray energy range. This near-native state structural imaging modality provides volumetric information highly complementary to that obtained with electron and fluorescence microscopes.
In this research, an inline FM was used to measure the fluorescence intensity of cells incubated with an apoptosis dye for caspase–3/7 enzyme activity. Combining structural information from soft X-ray transmission images of cells, the authors could easily distinguish between apoptotic, necrotic, or normal cells avoiding sample transfers between microscopes. The difference in overall morphology, X-ray absorption properties of organelles, and local 3D structure of cells was further investigated by SXT. The authors conclude that this correlative imaging platform and method would provide insight and assistance in cell selection for imaging by quickly assessing cell condition and function as long as appropriate fluorescent markers are applied.
Introducing a built-in light fluorescence microscope into the SXT-100 immediately boosted data collection throughput. As demonstrated by the authors, the inline fluorescence microscope will also improve the correlation between structural and functional imaging of cells.
On-Line Fluorescence Microscopy for Identification and Imaging of Apoptotic Cell with Synchrotron-Based Soft X-ray Tomography
Chao Zhang, Zhao Wu, Zheng Dang, Lijiao Tian, Yong Guan, Gang Liu and YangChao Tian, On-Line Fluorescence Microscopy for Identification and Imaging of Apoptotic Cell with Synchrotron-Based Soft X-ray Tomography. Micromachines. 2023; 14(2):326. https://doi.org/10.3390/mi14020326