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.
Soft x-ray microscopy in the water window ( ∼ 285 − 535 e V ) is an emerging and unique tool for 2D and 3D imaging of unstained intact cellular samples in their near-native state with few-10-nm detail. However, present microscopes rely on the high x-ray brightness of synchrotron-radiation sources. Having access to water-window microscopy in the home laboratory would increase the impact and extend the applicability of the method. In the present paper, we review three decades of efforts to build laboratory water-window microscopes and conclude that the method is now reaching the maturity to allow biological studies. The instruments as well as their key components are quantitatively and qualitatively compared. We find that the brightness and the reliability of the laboratory source are the most critical parameters, but that the optics as well as the sample preparation also must be optimized to enable high-resolution imaging with adequate exposure times. We then describe the two sister microscopes in Stockholm and Berlin, which allow reliable high-resolution biological imaging with short exposure times of a few tens of seconds in 2D and a few tens of minutes in 3D. They both rely on a liquid-jet laser-plasma source combined with high-reflectivity normal-incidence multilayer condenser optics, high-resolution zone-plate imaging optics, CCD detection, and cryogenic sample handling. Finally, we present several examples of biological imaging demonstrating the unique properties of these instruments.
Microscopic jets of cryogenic substances such as liquid nitrogen are important regenerative high-density targets for high-repetition rate, high-brightness laser-plasma soft x-ray sources. When operated in vacuum such liquid jets exhibit several non-classical instabilities that negatively influence the x-ray source's spatial and temporal stability, yield, and brightness, parameters that all are important for applications such as water-window microscopy. In the present paper, we investigate liquid-nitrogen jets with a flash-illumination imaging system that allows for a quantitative stability analysis with high spatial and temporal resolution. Direct and indirect consequences of evaporation are identified as the key reasons for the observed instabilities. Operating the jets in an approximately 100 mbar ambient atmosphere counteracts the effects of evaporation and produces highly stable liquid nitrogen jets. For operation in vacuum, which is necessary for the laser plasmas, we improve the stability by introducing an external radiative heating element. The method significantly extends the distance from the nozzle that can be used for liquid-jet laser plasmas, which is of importance for high-average-power applications. Finally, we show that laser-plasma operation with the heating-element-stabilized jet shows improved short-term and long-term temporal stability in its water-window x-ray emission.
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.
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