Laboratory cryo x-ray microscopy for 3D cell imaging

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.

Visualization of internal 3D structure of small live seed on germination by laboratory-based X-ray microscopy with phase contrast computed tomography

“…Further improvement of the spatial resolution is required to expand the applicability to the subcellular level. This subcellular level observation at submicron resolutions may require the more advanced technology to fix the cellular motions during the data collection, like the cryoprotection in the soft X-ray tomography [32,33].…”

Perspective: Towards single shot time-resolved microscopy using short wavelength table-top light sources

“…All mentioned techniques can be combined with a rotating sample, enabling, instead of 2D-, 3D-image reconstruction or tomography. Recent examples for 3D-CDI, 34–36 3D-ptychography, 11,37 and 3D-zone-plate imaging 21,38 demonstrate the exquisite capabilities of these techniques.…”

X-ray tomography of cryopreserved human prostate cancer cells: mitochondrial targeting by an organoiridium photosensitiser

“…Cryo-SXT makes use of samples that have been vitrified, to immobilise and cryo-preserve cell structure, and therefore delivers 3D imaging of hydrated biological samples (cells and thin tissue slices) under near-native conditions at high resolution (in the tens of nanometre range) [27,28]. This provides detailed in situ structural information on the distribution and morphology of cell organelles [29,30]. Hence, this technique has the potential to provide remarkable insights into the changes induced upon the exposure of cells to activated metallodrug candidates [31].…”