DESCRIPTION: The proposed work will lead to the development of new methods and breakthroughs in determining the structure and dynamics of biomolecules of significant biomedical relevance. Especially in the areas of drug design (e.g., GPCRs), this has been achieved recently using the recently-invented X-ray laser to image protein structures and their dynamics. Further progress in developing this new approach to protein crystallography, from solution scattering, to pump-probe two-color methods, mixing jets and the use of protein nanocrystals, requires the 2-photon polymerization 3D printer, with sub-micron spatial resolution, which is requested in this application. The world's first hard X-ray free-electron lasr (XFEL) started operation in 2009. In 2013, an application (BioXFEL) from a consortium of seven US campuses to the NSF Science and Technology (STC) program was funded to use it for structural biology. These STC awards normally run for a decade. This STC is based on the discovery that the brief femtosecond X-ray pulses produced by an XFEL outrun radiation damage, allowing damage-free crystallography at room temperature, and the formation of very high time-resolution molecular movies, as recently published by the STC, using protein nanocrystals and molecules in solution. But sample delivery is a critical bottleneck in this methodology, since, in our diffract-then-destroy mode, goniometers cannot be used. To date, hand-ground gas-focusing nozzles have been fabricated with micron precision to provide a continuous jet of micron dimensions flowing across the pulsed X-ray beam, carrying in single file, for example protein nanocrystals or single viruses. This allows 120 diffraction patterns to b read out every second. These nozzles are made by the STC Methods Group at ASU. To progress beyond this artisanal cottage industry of nozzle-in-nozzle gas focusing devices made by hand for 33 beamtimes (each about four days, each requiring several nozzles), automated production of nozzles is essential. This can be achieved using the 3D printer requested in this application. Equally important, the printer will allow rapid prototyping of new designs for new imaging modes, and also allow us to optimize the dimensions of many kinds of nozzles. These will include liquid sheet jets for snapshot solution scattering, mixing jets for imaging chemical reactions, viscous jets and switching jets to minimize sample consumption, and pump-probe jets for imaging molecular machines at work using caged-molecule (ligand/drug) methods. The user base for this instrument will be very large. In addition to the 22 faculty funded by the STC at seven campuses and their groups, consistent with its charge from NSF, the STC has assisted in 19 beamtimes led by non-STC investigators over the last year. The automated production of optimized nozzles will reduce protein consumption, provide complete data sets more quickly with less protein, allow more users per week to access the only US XFEL (the LCLS at SLAC), and, most importantly, allow the STC to explore entirely new methods of data collection for both structural dynamics and the static analysis of crystallization-resistant proteins.