Principal Investigator: Dr Venera Weinhardt, Centre for Organismal Studies, Heidelberg

Sample Preparation

Samples for cryo-Soft X-ray Microscopy (cryo-SXM) can be prepared on either a flat substrate such as a transmission electron microscopy (TEM) grid, or in a glass capillary. The TEM grid is a suitable substrate for adherent cells, whereby capillaries are ideal for non-adherent cells in suspension. Additionally, a capillary can be rotated through 360° thus avoiding any missing angles. The ideal ‘fixing’ method for a cell (i.e., TEM grid vs capillary) is therefore influenced by whether the cell is adherent or non-adherent and by what organelle or structure within the cell is to be imaged. In the ideal scenario, a soft X-ray microscope should be capable of catering for both cell-fixing techniques. The lab-scale prototype SXM currently supports only the TEM-fixed cells.

CoCID will focus on extending this functionality to support capillary-mounted cells. This will greatly increase the range of biological applications that can be addressed. The imaging performance will be tested on two different viruses, one of which targets the nucleus-based DNA and the other that targets the mRNA in the cell cytoplasm, thus allowing the performance of the different sample mounting techniques to be evaluated.

Cells prepared i) on TEM grid and ii) in glass capillary

BSL safety considerations

Highly contagious emerging viruses such as SARS Cov2 are classified as biosafety level 3(BSL-3), meaning they cannot leave the BSL-3 classified lab environment. Since the lab-scale SXM will be located in a non-BSL lab for this project, viral samples must be first downgraded to BSL-2 status before they can be transported to the SXM for imaging.

Generally, the approach to disabling infectious samples is through aldehyde fixation. This approach is routine in electron microscopy and standard protocols exist for doing this. Chemical fixation has, however, the potential to adversely affect the soft X-ray image contrast. Ideally, cryo-fixed samples would be downgraded to BSL-2 by other methods. One such method is to use a replication of the virus in which the virus is mutated so it cannot exit the infected cell. CoCID will explore soft X-ray imaging of both fixed and replicon bearing samples. For CoCID replicon bearing samples, which are classed as BSL-2, a bio-safe cryo-shuttle will be developed and employed to transport the BSL-2 sample to the SXM for cell imaging.


Soft X-ray Imaging

Soft X-ray microscopy uses X-rays in the ‘water window’ that extends from the K-absorption edge of carbon to the K-edge of oxygen, that is from about 282 eV (λ = 4.4 nm) to 533 eV (λ = 2.3 nm). Water is transparent to these X-rays, but organic molecules are absorbing. Therefore, these X-rays can be used as the basis for microscopy of whole cells in their near-native (frozen) state, without need for any contrast enhancing agents. A 3D tomogram with resolution between 25 nm to 60 nm (full pitch) is produced by rotating the cell over a range of angles, with an image acquired at each tilt angle. The concept is equivalent to a medical CT scan applied at the nanoscale. A soft X-ray tomogram is obtained by rotating the sample through a series of angular increments.

The aim of CoCID WP2 is to compare and benchmark the throughput and image quality of the lab-scale SXM with the established X-ray microscopes at the synchrotron facilities. While sample preparation protocols are the same for laboratory SXM, protocols for image acquisition should be adapted with respect to the new X-ray source, resulting in unique imaging performance in terms of spatial resolution and contrast. To characterize the X-ray imaging performance of the lab-scale SXM, we will use already established protocols to measure the spatial resolution and depth of field of the microscope. This type of analysis will provide specification on cell types and tissue sections which can be imaged with the laboratory SXM as well as detectability of viral particles within cells. The imaging experiments with the laboratory SXM will be performed on the same cell type as used for SXM imaging at the ALBA X-ray microscope, providing a set of possible image acquisition strategies and calibration values for X-ray linear absorption coefficient to simplify analysis of subcellular structures.

Cell Soft X-ray Images showing a) a 2D projection, b) a 2D slice from 3D reconstruction and c) a 3D slice of a correlated 3D reconstruction.

The mobility of single capsids and its correlation with chromatin distribution will be studied by 3D image correlation analyses of LSCM/SDM data. These analyses will be performed to analyse diffusion of capsids in the interchromatin areas. Finally, nuclear mobility of capsids will be modelled by random walk modelling in 3D reconstructions (Aho et. al., 2017, Myllys et. al., 201)of chromatin created from SXM images using automatic segmentation and analysis pipeline created in the project.

Image Correlation:

Correlation of SXM data with fluorescence microscopy (FM) will facilitate the identification of molecular-level interactions within the context of the whole structure of an intact cell, imaged in its natural state. Integration of FM in the prototype SXM, with its objective lens inside the SXM’s sample cryo-vacuum chamber, will result in simplified sample handling, increased imaging speed and higher-fidelity correlated images.

The main purpose of CoCID WP3 is to design and integrate a standard epi-fluorescence microscope in the prototype SXM for correlative SXM-FM studies. The microscope will also serve as a screening device to locate cells of interest for SXM imaging and assess the quality of the ice layer. A long working distance objective lens will be used to avoid excessive radiative heat transfer to the cryo sample. The lens will be mounted on the inside wall of the sample chamber with its optical axis at 90° to the soft X-ray axis, allowing a fluorescent mosaic of the sample to be obtained prior to soft X-ray imaging. Correlation will be done in post processing by overlaying the FM and SXM images using a set of common fiducial markers for alignment. The FM will be a multichannel microscope optimized for the fluorophores used in the CoCID virology studies. Imaging performance will be evaluated by imaging fluorescent nanospheres deposited on a TEM grid and measuring their point spread functions.

Cell image from i) fluorescence microscope and ii) correlated fluorescence and soft x-ray microscope images

Image Analysis

The main purpose of CoCID WP5 is to develop an efficient and user-friendly data pipeline for the lab-scale SXM prototype which will support the virology studies. We will build an integrated workflow that covers the entire tomography data processing pipeline, from automated tilt series alignment to nanometer-scale resolution. To achieve our goals, we will begin by packaging existing software interfaces and rolling these out to CoCID partners. Hence pipeline development will be a user-driven process, building on the initial platform to offer users an optimised pipeline in terms of human bias reduction, enhancement of 3D resolution and increased data throughput.

Data acquisition: With flat specimen holders we will collect data typically over a 110° range (+/- 55°), with a 1° step. For capillaries data will be collected over a 180° range. Division of dose with angle will also be investigated, as will the acquisition of multiple frames per angle, primarily for motion correction.

Automated stack alignment & reconstruction: The principal aim of this task is to consolidate existing interfaces into a single streamlined package, incorporating the aspects of these approaches that are most compatible with the SXM datasets. The data pipeline will be tailored to cater for both TEM grid and capillary sample schemes, as developed in the CoCID project.

CoCID integrated workflow for entire tomography data processing pipeline