Abstract
Water-window x-ray microscopy allows two- and three-dimensional (2D and 3D) imaging of intact unstained cells in their cryofixed near-native state with unique contrast and high resolution. Present operational biological water-window microscopes are based at synchrotron facilities, which limits their accessibility and integration with complementary methods. Laboratory-source microscopes have had difficulty addressing relevant biological tasks with proper resolution and contrast due to long exposure times and limited up-time. Here we report on laboratory cryo x-ray microscopy with the exposure time, contrast, and reliability to allow for routine high-spatial resolution 3D imaging of intact cells and cell-cell interactions. Stabilization of the laser-plasma source combined with new optics and sample preparation provide high-resolution cell imaging, both in 2D with ten-second exposures and in 3D with twenty-minute tomography. Examples include monitoring of the distribution of carbon-dense vesicles in starving HEK293T cells and imaging the interaction between natural killer cells and target cells.
Highlights
Water-window x-ray microscopy allows two- and three-dimensional (2D and 3D) imaging of intact unstained cells in their cryofixed near-native state with unique contrast and high resolution
We report on laboratory cryo x-ray microscopy with the exposure time, contrast, and reliability to allow for routine high-spatial resolution 3D imaging of intact cells and cell-cell interactions
We report on laboratory cryo x-ray microscopy with the exposure time, contrast, and reliability to allow for routine high-spatial resolution 3D imaging of processes in intact cells and cell-cell interactions
Summary
Water-window x-ray microscopy allows two- and three-dimensional (2D and 3D) imaging of intact unstained cells in their cryofixed near-native state with unique contrast and high resolution. We report on laboratory cryo x-ray microscopy with the exposure time, contrast, and reliability to allow for routine high-spatial resolution 3D imaging of intact cells and cell-cell interactions. Lens-based x-ray microscopes show better resolution on cryofixed hydrated cells and has demonstrated many relevant biological results Both methods presently rely on large-scale accelerator-based x-ray facilities, synchrotrons or free-electron lasers. We demonstrate laboratory water-window x-ray microscopy with high resolution and high contrast on cryofixed cells with routine 10 s exposure time in 2D imaging and twenty-minute exposure time for 3D tomography Such exposure times and reliability are prerequisites for enabling the tomographic 3D imaging and to allow investigations on realistic biological samples, which typically are large and often heterogeneous, such as in the examples discussed below. Previous laboratory x-ray microscopes provided imaging with similar resolution but with www.nature.com/scientificreports/
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