Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as it is a well-characterized model system, useful for proof-of-principle experiments, system optimization and algorithm development. RDV, an icosahedral virus of about 70 nm in diameter, was aerosolized and injected into the approximately 0.1 μm diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 Ångström were recorded. The diffraction data are available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development, the contents of which are described here.

Background: Numerous studies have shown that nitrogen (N) deposition decreases biodiversity in terrestrial ecosystems. To explain the N-induced species loss, three functionally based hypotheses have been proposed: the aboveground competition hypothesis, the belowground competition hypothesis, and the total competition hypothesis. However, none of them is supported sufficiently by field experiments. A main challenge to testing these hypotheses is to ascertain the role of shoot and root competition in controlling plant responses to N enrichment. Simultaneously examining both aboveground and belowground responses in natural ecosystems is logistically complex, and has rarely been done.

Methodology/Principal Findings: In a two-year N addition experiment conducted in a natural grassland ecosystem, we investigated both above- and belowground responses of plants at the individual, species, and community levels. Plants differed significantly in their responses to N addition across the different organizational levels. The community-level species loss was mainly due to the loss of perennial grasses and forbs, while the relative abundance of plant species was dependent mainly on individual-level responses. Plasticity in biomass allocation was much smaller within a species than between species, providing a biological basis for explaining the functionally based species loss. All species increased biomass allocation to aboveground parts, but species with high belowground allocations were replaced by those with high aboveground allocations, indicating that the increased aboveground competition was the key process responsible for the observed diversity loss after N addition in this grassland ecosystem.

Conclusions/Significance: Our findings shed new light on the validity of the three competing hypotheses concerning species loss in response to N enrichment. They also have important implications for predicting the future impacts of N deposition on the structure and functioning of terrestrial ecosystems. In addition, we have developed a new technique for ascertaining the roles of aboveground and belowground competition in determining plant responses to N fertilization.

This study explores the capabilities of the Coherent X-ray Imaging Instrument at the Linac Coherent Light Source to image small biological samples. The weak signal from small samples puts a significant demand on the experiment. Aerosolized Omono River virus particles of ∼40 nm in diameter were injected into the submicrometre X-ray focus at a reduced pressure. Diffraction patterns were recorded on two area detectors. The statistical nature of the measurements from many individual particles provided information about the intensity profile of the X-ray beam, phase variations in the wavefront and the size distribution of the injected particles. The results point to a wider than expected size distribution (from ∼35 to ∼300 nm in diameter). This is likely to be owing to nonvolatile contaminants from larger droplets during aerosolization and droplet evaporation. The results suggest that the concentration of nonvolatile contaminants and the ratio between the volumes of the initial droplet and the sample particles is critical in such studies. The maximum beam intensity in the focus was found to be 1.9 × 10¹² photons per µm² per pulse. The full-width of the focus at half-maximum was estimated to be 500 nm (assuming 20% beamline transmission), and this width is larger than expected. Under these conditions, the diffraction signal from a sample-sized particle remained above the average background to a resolution of 4.25 nm. The results suggest that reducing the size of the initial droplets during aerosolization is necessary to bring small particles into the scope of detailed structural studies with X-ray lasers.

Structural studies on living cells by conventional methods are limited to low resolution because radiation damage kills cells long before the necessary dose for high resolution can be delivered. X-ray free-electron lasers circumvent this problem by outrunning key damage processes with an ultra-short and extremely bright coherent X-ray pulse. Diffraction-before-destruction experiments provide high-resolution data from cells that are alive when the femtosecond X-ray pulse traverses the sample. This paper presents two data sets from micron-sized cyanobacteria obtained at the Linac Coherent Light Source, containing a total of 199,000 diffraction patterns. Utilizing this type of diffraction data will require the development of new analysis methods and algorithms for studying structure and structural variability in large populations of cells and to create abstract models. Such studies will allow us to understand living cells and populations of cells in new ways. New X-ray lasers, like the European XFEL, will produce billions of pulses per day, and could open new areas in structural sciences.