Quantitative conversion of biomass in giant DNA virus infection.

Bioconversion of organic materials is the foundation of many applications in chemical engineering, microbiology and biochemistry. Herein, we introduce a new methodology to quantitatively determine conversion of biomass in viral infections while simultaneously imaging morphological changes of the host cell. As proof of concept, the viral replication of an unidentified giant DNA virus and the cellular response of an amoebal host are studied using soft X-ray microscopy, titration dilution measurements and thermal gravimetric analysis. We find that virions produced inside the cell are visible from 18 h post infection and their numbers increase gradually to a burst size of 280-660 virions. Due to the large size of the virion and its strong X-ray absorption contrast, we estimate that the burst size corresponds to a conversion of 6-12% of carbonaceous biomass from amoebal host to virus. The occurrence of virion production correlates with the appearance of a possible viral factory and morphological changes in the phagosomes and contractile vacuole complex of the amoeba, whereas the nucleus and nucleolus appear unaffected throughout most of the replication cycle.

Laboratory cryo x-ray microscopy for 3D cell imaging.

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.

Tomographic reconstruction in soft x-ray microscopy using focus-stack back-projection.

Tomographic reconstruction in soft x-ray microscopy is a powerful technique for obtaining high-resolution 3D images of biological samples. However, the depth of focus of such zone-plate-based microscopes is typically shorter than the thickness of many relevant biological objects, challenging the validity of the projection assumption used in conventional reconstruction algorithms. In order to make full use of the soft x-ray microscopes' high resolution, the tomographic reconstruction needs to take the depth of focus into account. Here we present a method to achieve high resolution in the full sample when the depth of focus is short compared to the sample thickness. The method relies on the back-projection of focus-stacked image data from x-ray microscopy. We demonstrate the method on theoretical and experimental data.

3D simulation of the image formation in soft x-ray microscopes.

In water-window soft x-ray microscopy the studied object is typically larger than the depth of focus and the sample illumination is often partially coherent. This blurs out-of-focus features and may introduce considerable fringing. Understanding the influence of these phenomena on the image formation is therefore important when interpreting experimental data. Here we present a wave-propagation model operating in 3D for simulating the image formation of thick objects in partially coherent soft x-ray microscopes. The model is compared with present simulation methods as well as with experiments. The results show that our model predicts the image formation of transmission soft x-ray microscopes more accurately than previous models.