Abstract
For many scientific questions gaining three-dimensional insight into a specimen can provide valuable information. We here present an instrument called "tOMography Nano crYo (OMNY)," dedicated to high resolution 3D scanning x-ray microscopy at cryogenic conditions via hard X-ray ptychography. Ptychography is a lens-less imaging method requiring accurate sample positioning. In OMNY, this in achieved via dedicated laser interferometry and closed-loop position control reaching sub-10 nm positioning accuracy. Cryogenic sample conditions are maintained via conductive cooling. 90 K can be reached when using liquid nitrogen as coolant, and 10 K is possible with liquid helium. A cryogenic sample-change mechanism permits measurements of cryogenically fixed specimens. We compare images obtained with OMNY with older measurements performed using a nitrogen gas cryo-jet of stained, epoxy-embedded retina tissue and of frozen-hydrated Chlamydomonas cells.
Highlights
Ptychographic x-ray computed tomography (PXCT) is one of many methods for gaining access to three-dimensional internal information of a specimen.1 With several keV photon energies, it has the potential to bridge the resolution gap between traditional x-ray imaging methods and electron microscopy, imaging in the spatial resolution range approaching 10 nm in high-contrast samples having a thickness of tens of microns.2,3 Ptychography is a scanning microscopy technique where a sample is illuminated by a spatially confined coherent beam.4,5 The sample is scanned across the beam such that neighboring illuminated areas partially overlap, and far-field diffraction patterns are recorded for each position
The xy position data of Fresnel zone plate (FZP) versus order sorting aperture (OSA) are directly fed to proportional integral differential (PID) control loops which generate the required output signal to keep the relative position between these elements stable in closed loop
We suspect that the observable cell structures in the present measurement were altered by water crystallization: For instance, the shape of cell walls (CW), pyrenoid (P), and starch platelets (SP) deviates significantly from the more rounded shapes they exhibit in high-pressure frozen specimens31 or in plunge frozen specimens immersed in a thin layer of water without a glass capillary
Summary
Ptychographic x-ray computed tomography (PXCT) is one of many methods for gaining access to three-dimensional internal information of a specimen. With several keV photon energies, it has the potential to bridge the resolution gap between traditional x-ray imaging methods and electron microscopy, imaging in the spatial resolution range approaching 10 nm in high-contrast samples having a thickness of tens of microns. Ptychography is a scanning microscopy technique where a sample is illuminated by a spatially confined coherent beam. The sample is scanned across the beam such that neighboring illuminated areas partially overlap, and far-field diffraction patterns are recorded for each position. To have ptychographic projections result in a high-resolution 3D image, they have to provide the required resolution and have to be distortion free, meaning that short-range precision of neighboring scanning positions needs to be accurate and long-range precision between extreme scanning points The latter puts strong requirements on thermal drift during the scan and on position distortions, which may be caused by angular error motions of scanning stages or by a small rotation or skewing between the scanning axes and the image pixels. Exteroceptive laser interferometry is used to measure the relative position between the x-ray beam-defining optics and the sample, which allows accurate position measurements and minimizing thermal drifts Such metrology has to be compatible with the rotational degree of freedom required for tomography and led to the development of a dedicated tracking interferometer.. We describe the OMNY instrument in detail and discuss the performance of the cryogenic and positioning system
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