This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: Currently, microfluidics technology is being applied to develop micromixers for time resolved cryo-electron microscopy (TRCEM) application. TRCEM requires fast and homogeneous premixing of tiny amounts of macromolecules in the time scale of milliseconds or less. Work is also just beginning in which this technology will be applied to the problems of specimen deposition onto EM grids and rapid freezing. Our goal for the remainder of this grant is to design and test a microfluidics-based device that allows TRCEM experiments to be conducted routinely with millisecond time resolution and requires only the small amounts of biomolecules that are often available to researchers. The nano device design and fabrication is conducted in collaboration with Dr. Toh Ming Lu and Dr. Zonghuan Lu at Rensselaer Polytechnic Institute via subcontract from the P41 grant. Micromixers. Microfabrication is a promising technique to achieve the objective of rapid mixing. Last year we described our initial progress in the fabrication of mixers of various designs and their initial testing. For device prototyping and fluid mixing experiments, polydimethylsiloxane (PDMS) material was used with a fast molding and casting replication technique to prepare devices. After intensive computational and experimental testing using fluorescent molecules and light microscopy we have arrived at a basic design for mixers that is compatible with the kinetic and volume requirements of TRCEM. The final configurations comprise two T-shaped premixers that feed into a single channel containing an array of 2-6 pillars that have a butterfly shape in cross-section. The devices typically have a total volume of less than one microliter, perform optimally at flow rates of 200-360 microliters/sec, and allow complete mixing in less than one millisecond. These results have been presented in two meeting abstracts and a full manuscript has been submitted for publication. Micromixer/microsprayer combined into one "monolithic" device. Substantial progress has been achieved in integrating the opitimized micromixer designs with pneumatic micro-sprayer designs (see above). Five devices have been fabricated from silicon and bonded to a glass cover. Photographs of one of the devices, the one that has been most extensively characterized, are shown below (left panels show photographs of the entire device as viewed from the top and bottom; right panels show magnified views obtained by scanning electron microscopy of the mixer and sprayer regions of the device). As currently implemented, the two solution inlets are connected to a dual syringe pump that delivers reactants to the device. The device has a simple spray nozzle consisting of a single liquid channel surrounded laterally by two air channels. Two other types of sprayer nozzles have been fabricated in which the liquid channel bifurcates into two subchannels. Droplet size distributions have been determined for two of the sprayer designs, but they do not show large differences. All of the devices tested produce rather large distributions of droplet sizes and the average droplet diameter is severalfold larger (~ 20 microns in diameter) than we believe to be optimal (based upon results with the macrosprayer described in aim 1). Nevertheless, when grids containing suitably hydrophilic carbon films are used, even the large microdroplets spread to form a film that is sufficiently thin to allow imaging following freezing. Cryo-EM data has been collected for intact bacterial ribosomes and a preliminary 3D reconstruction (23 ¿ resolution) determined from the micrographs. Encouragingly, we find no evidence that the monolithic mixer/sprayer causes any structural damage to the ribosomes. Also, we find no loss in activity when an enzyme, malate dehydrogenase, is passed through the devices. At the time of writing this report, we are engaged in experiments to characterize the association of ribosomal subunits to form intact ribosomes; the objective here is to search for intermediates in ribosomal assembly that are expected to occur based upon results of biochemistry experiments (e.g. Hennelly et al. (2006) J. Mol. Biol. 346:1243). In the coming months we expect to complete these initial studies on ribosomal subunit assembly and to establish the utility of the microdevices in TRCEM. Also, efforts will continue to optimize the design of the devices, in particular, the microsprayer component.