Cryo X-ray microscopy with high spatial resolution in amplitude and phase contrast

Area of Science:

• X-ray microscopy
• Biophysics
• Materials science

Background:

• Transmission X-ray microscopes (TXMs) with zone plate optics currently achieve resolutions around 30 nm.
• Imaging radiation-sensitive hydrated biological materials presents challenges due to X-ray damage.
• Cryogenic sample preparation is a potential method to mitigate radiation damage in X-ray microscopy.

Purpose of the Study:

• To investigate the feasibility of achieving high-resolution imaging in hydrated biological samples using TXM.
• To theoretically and experimentally evaluate the impact of X-ray-matter interactions and zone plate optics on image quality and radiation dose.
• To develop and verify optimized phase contrast techniques for imaging cryogenic biological samples.

Main Methods:

• Theoretical modeling of X-ray-matter interaction, image formation with zone plates, and influence of aperture limitations.
• Quantitative evaluation of X-ray-induced damage using radiation-induced kinetics.
• Modification of a TXM to image frozen-hydrated samples at atmospheric pressure.
• Imaging experiments using amplitude and phase contrast modes at 2.4 nm wavelength.

Main Results:

• Theoretical models incorporating limited apertures predict optimal phase contrast for high image quality at low radiation doses.
• Cryogenic samples were found to be structurally stable under X-ray irradiation.
• TXM imaging of frozen-hydrated cells and algae at 2.4 nm wavelength resolved details as small as 35 nm without structural changes.
• Optimized phase contrast imaging enhanced the visibility of internal structures in frozen-hydrated samples.
• Stereo-pair imaging provided 3D visualization of organelles within algae.
• Element analysis and micro-tomography of cryogenic cells were demonstrated.

Conclusions:

• Shock-frozen hydrated biological samples enable high-resolution (35 nm) TXM imaging.
• Optimized phase contrast imaging is crucial for achieving high contrast and low radiation dose in TXM of biological specimens.
• Cryogenic sample preparation preserves structural integrity, allowing for detailed analysis of cellular and subcellular structures.
• TXM of cryogenic samples opens possibilities for 3D imaging, elemental analysis, and micro-tomography of biological systems.