Procedures for cryogenic X-ray ptychographic imaging of biological samples

Abstract

Biological sample-preparation procedures have been developed for imaging human chromosomes under cryogenic conditions. A new experimental setup, developed for imaging frozen samples using beamline I13 at Diamond Light Source, is described. This manuscript describes the equipment and experimental procedures as well as the authors' first ptychographic reconstructions using X-rays.

X-ray Ptychography Imaging of Human Chromosomes After Low-dose Irradiation

“…Soft X-ray ptychography has been applied to obtain chemical information from~2-μm-thick freezedried Deinococcus radiodurans, without slicing and staining at a photon energy of 512 eV in the water window (Beckers et al, 2011). X-ray ptychography has been demonstrated to obtained quantitative information of human metaphase chromosome to build a preliminary karyotype on the basis of mass (Shemilt et al, 2015), and recently to image a human nucleus under cryogenic condition (Yusuf et al, 2017).…”

“…3b) across a scan of 32×32 points with 1-μm steps and an Xray exposure time of 0.3 s. These data collection schemes allow adequate overlap of the probe positions to solve the phase problem (Batey, 2014). The samples were imaged at room temperature, although attempts to handle cryopreserved samples have been reported elsewhere (Yusuf et al, 2017). The obtained diffraction patterns were reconstructed using 100 iterations of the ptychographic algorithms.…”

Multiscale X-Ray Analysis of Biological Cells and Tissues by Scanning Diffraction and Coherent Imaging

“…Vitrified samples have to be kept at cryogenic temperatures at all times, either by mounting the sample onto a cold finger and inserting the sample into a vacuum chamber, or by rapid mounting of the sample into a cold nitrogen gas stream. Cryogenic chambers for X-ray experiments on vitrified samples have been described before [155][156][157] but are highly specialized developments with limited access and flexibility, even though the demand for such developments is currently rising [95,158,159].…”

Use of 3D imaging for providing insights into high-order structure of mitotic chromosomes

“…To date, there is no report showing 3D metaphase chromosomes imaged with such techniques but these are under development. 3D Cryo FIB-SEM and Cryo 3D CDI have already been performed on cells (Schertel et al 2013; Rodriguez et al 2015), and Cryo X-ray Ptychographic imaging has been attempted on nuclei (Yusuf et al 2017). Super resolution microscopy is proven promising for investigation chromatin structure (Sydor et al 2015).…”