Advanced sample injection for serial diffraction at free-electron lasers

X-ray crystallography is a very powerful tool to study the structure of biological molecules such as proteins. Knowledge of the three-dimensional structure of such molecules aids the understanding of biological processes and as such how life works. Traditionally, x-ray crystallography was performed by exposing one or a few cryo-cooled crystals to the x-ray beam and rotating these crystals to obtain the 3D Bragg reflection data necessary for structural reconstruction. With the advent of instruments with highly intense x-ray beams -- namely micro-focus beamlines at 3rd generation synchrotron sources and x-ray free-electron lasers -- the serial approach to x-ray crystallography has expanded on classical crystallography. In serial crystallography (SX), each room temperature crystal typically gives rise to only one diffraction pattern and is discarded after the x-ray exposure. Rather than merging the reflection intensities of many diffraction patterns from the same crystal, data from many crystals is merged into one intensity map. The highly intense x-ray beams enable the measurement of diffraction patterns from crystals with a size of only a few micrometers -- a $10^6$-fold decrease in crystal volume compared to classical crystallography. Thus, SX expands the realm of crystallography to molecules that do not grow large crystals easily such as many membrane proteins, to molecules with metal centers that are highly susceptible to radiation-induced damage and to time-resolved studies of irreversible processes.The aim of this thesis was the development of new instrumentation to improve on three aspects of serial crystallography -- reduction of background scattering intensities, reduction of required sample, and reduction of x-ray time needed. These improvements will be particularly important for experiments that require the accurate measurement of small signal variations such as time-resolved studies. Such studies require the collection of large numbers of diffraction patterns to measure variations in the molecular structure factors during the reaction process. The increase in x-ray intensity for the new x-ray instruments leads to an increase in background photons scattered by imperfections of the x-ray optics at the instrument or by non-sample material in the path of the x-ray beam. Two methods to block such stray x-ray beams are presented in an effort to reduce their impact on the diffraction patterns and thus reduce the number of patterns needed to build up the signal intensities with sufficient accuracy. The x-ray background can be further reduced by optimization of the sample delivery mechanism. Two liquid jet injectors are presented that both reduce the amount of non-sample material that surrounds the crystal in the x-ray interaction region while also reducing the amount of sample needed to collect a full set of reflection intensities. As a final step, the expansion of these injectors to MHz pulse rates available at the most recent generation of x-ray lasers is demonstrated