We present a crystallography chip enabling in situ room temperature crystallography at microfocus synchrotron beamlines and X-ray free-electron laser (X-FEL) sources. Compared to other in situ approaches, we observe extremely low background and high diffraction data quality. The chip design is robust and allows fast and efficient loading of thousands of small crystals. The ability to load a large number of protein crystals, at room temperature and with high efficiency, into prescribed positions enables high throughput automated serial crystallography with microfocus synchrotron beamlines. In addition, we demonstrate the application of this chip for femtosecond time-resolved serial crystallography at the Linac Coherent Light Source (LCLS, Menlo Park, California, USA). The chip concept enables multiple images to be acquired from each crystal, allowing differential detection of changes in diffraction intensities in order to obtain high signal-to-noise and fully exploit the time resolution capabilities of XFELs.

The use of electron probes for in situ imaging of solution phase systems has been a long held objective, largely driven by the prospect of atomic resolution of molecular structural dynamics relevant to chemistry and biology. Here, we present a nanofluidic sample cell with active feedback to maintain stable flow conditions for pathlengths varying from 45 nm to several 100 nm, over a useable viewing area of 50 x 50 μm. Using this concept, we demonstrate nanometer resolution for imaging weakly scattering polymer and highly scattering nanoparticles side by side with a conventional transmission microscope. The ability to flow liquids allows control over sample content and on-the-fly sample exchange, opening up the field of high-throughput electron microscopy. The nanofluidic cell design is distinguished by straightforward, reliable, operation with external liquid specimen control for imaging in (scanning) transmission mode and holds great promise for reciprocal space imaging in femtosecond electron diffraction studies of solution phase reaction dynamics

In this paper, we show the design and theoretical calculation of our new femtosecond electron source based on rf-accelerator generating 2-5 MeV electron bunches with high electron density and high coherence length

In situ imaging using (scanning) transmission electron microscopes has proven to be an extremely important and powerful cross-disciplinary scientific technique. In particular nanotechnology and materials sciences have special interest in assembly and disintegration processes, in growth and shapetuning of (nano)-particles, 1,2 and, furthermore in mechanistic studies of chemical reactions underlying these processes. However, limitations for in liquid and in situ imaging utilizing electron microscopy arise from experimental conditions required to minimize disturbing electron scatter. Within current sample preparation methods, these limitations are difficult to achieve: 3 Nanometer thin and vacuum compatible samples, which are additionally easy to use, reliable and provide relatively high-throughput flow are pending.Here, we present a nanofluidic sample cell allowing for exquisite control over the liquid layer thickness, currently >40 nanometer, in order to preserve the highest possible spatial resolution for the in situ study of liquid/solutions using electron microscopy. Preparing such ultra thin liquid layers enables us to use a variety of electron microscopes with different lens configurations and electron energies for our imaging experiments. We provide liquid flow through our nanocell by applying differential pressure with feedback control external to the microscope column and therewith allowing for on-the-fly sample exchange within the imaging area (Figure 1).In this contribution, we show the ability to clearly image gold nanoparticles as small as 5nm, and polymer-based nanoparticles as small as 36nm, over fluid layers as thin as 50nm. Thicker layers, up to 280nm, show clear degradation in spatial resolution but still deliver good quality imaging. We studied the imaging resolution dependent on the liquid layer thickness, obtaining a linear relation between the two (Figure 2). Furthermore, we demonstrate unidirectional flow in the design concept using gold nanorods pumped directionally through the imaging area upon an applied external force, while tumbling in Brownian motion once the external pressure is released. Having shown the capabilities of our nanofluidic design, we further show preliminary results of in situ imaging, studying gold nanorods to illustrate applications in the study of materials and amyloid fibrils as example of biological applications. Taken together, these systems highlight the variety of applications our nanofluidic cell is able to address. This design is distinguished by straightforward, reliable operation in different, commonly used electron microscopes.In future experiments, we will expand the use of our unique ability to control the path thickness and flow directionality. The latter will be key to enable time resolved electron diffraction in the liquid phase, allowing studies of a wide range of chemical reaction mechanisms on an atomic or molecular level. 404

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.