Although there has been extensive work at many institutions to develop microfabrication and microfluidic technology for biomedical applications, the technology has yet to move broadly into biomedical research laboratories. Part of the issue is a lack of familiarity on the part of many biomedical researchers, but in addition many potential research projects require capabilities or customization absent from the limited number of commercially available products. In an effort to lower these barriers, we have developed a basic in-house microfabrication facility. Although the resolution, device yield, and complexity are somewhat lower than those achievable with a dedicated cleanroom, they are nonetheless sufficient for many experiments on cells. Furthermore, the instrumentation complexity, fabrication cost, and turnaround time are greatly reduced, enabling rapid cycling through design parameters as needed. We are able to reliably pattern single- and multi-layer template features with lateral dimensions of less than 2 microns, and with heights ranging from a few microns to a few hundred microns. We have developed protocols for using these templates to generate microstructured PDMS, agarose, and PEGDA hydrogels, including techniques for making and manipulating thin (<200 micrometer) PDMS layers for use in multilayer devices and bottomless structures. This year, we have continued to refine techniques for incorporating track-etched polymer membranes into multilayer structures for co-culture of different cell types, and worked with SPIS, DCB, CIT to incorporate them into perfusion bioreactors for 3D tissue models. We can also perform surface modification of PDMS and other polymers, including the irreversible bonding of PDMS to glass, and have developed techniques for connecting devices to flow-control instruments such as syringe pumps and pressure controllers. In addition, we have the ability to deposit and pattern metal layers, as well as a heated hydraulic press for hot-embossing thermoplastics, including PMMA, polycarbonate, and COC. We have also used a programmable razor cutter and pressure-sensitive adhesive to directly make thin film structures with heights ranging from 25 to a few hundred microns, and sub-millimeter lateral dimensions. This is a low-cost and convenient method for several applications, including the ready fabrication of flow cells with two glass walls, or for fluidic confinement over already-functionalized surfaces. Finally, we continue to work on finite element modeling of transport in microfabricated structures, developing models for oxygen delivery in a bioreactor with micropillars and for chemokine concentration in a microfluidic hydrogel device. These capabilities have found application in a number of projects, representing a broad variety of interests and institutes. In addition to the representative projects discussed below and others still in the early stages, we have also trained researchers in basic microfabrication techniques, including personnel from other laboratories in NEI, NIAID, NHLBI, NCI, NICHD, and NIBIB. 1) A collaborative effort with LSB, NIAID, to study chemotaxis of primary immune cells in 3-D collagen matrices, for which we have been developing and refining a microfluidic agarose device compatible with high-resolution fluorescence and two-photon imaging. A mixing tee on a separate platform, together with programmable syringe pump flow control, enables the formation of reproducible time-varying spatial gradients. We have also developed finite element models of the original device and used these in combination with quantitative image analysis to assist in characterization and modeling of the 3-D device for different temporal inputs. 2) The fabrication and characterization of thin hybrid polymer films made by spin coating for use in operator-independent, high-resolution, light-activated microdissection. This work was begun as part of an inter-institute Director's Challenge project with researchers in NIMH, NCI, and NICHD, and more recent efforts with NICHD and CIT have focused on refining the instrumentation and microdissection protocols for a variety of targets and stain localizations, from whole-cell and region-of-interest based captures, to subcellular targets, such as axon terminals in brain slices (in collaboration with NIDA). 3) The continuing development, in collaboration with LCE, NHLBI, of gratings for phase-contrast x-ray imaging, which involves the use of the NIST nanofabrication facilities as well as our own equipment. This year our efforts have focused on publication of imaging results from the current generation of gratings. Preliminary work on the next generation of gratings is underway. 4) In collaboration with MDP, NIDDK, the use of coverslip-mounted, bottomless PDMS microwells to confine eggs in order to study fertilization with fast, high-resolution microscopy. For this application, the wells need to be compatible with existing fluorescence imaging and culture systems, and also to enable capture of the eggs with minimal handling. The addition of microchannels connecting the wells has provided space for the cumulus cells as they separate, enabling the eggs to be gently captured and imaged at the coverslip surface. 5) The development and implementation of a PDMS microfluidic gradient generator for the deposition of chondroitin sulfate proteoglycans on substrates for neural cell culture that we are using, in collaboration with DN, CBPC, NHLBI, to gain better understanding of the role these molecules play in axon growth and guidance. We have successfully used these removable devices for deposition of CSPG gradients over an extended area (1.3 mm x 20 mm) of PLL-coated glass, and have used these patterned substrates in the culture of mouse cerebellar granule neurons. Recent efforts have focused on the development of customized image analysis software to extract relevant neurite growth parameters from statistically meaningful numbers of cultured cells (a few thousand cells per substrate). 6) The design, fabrication, modeling, and use of an oxygen-transmissive membrane, patterned with a micropillar array to deliver oxygen to three-dimensional cell culture volumes with in vivo-like spatial distribution, in collaboration with LCB, CCR, NCI and SPIS, CIT. Because the pillar spacing is approximately equal to typical intercapillary distances (200 microns), cells in a Matrigel layer surrounding the pillars can be maintained under hypoxic conditions in an extended 3D volume. Current efforts are focused on the design, fabrication, validation and implementation of a higher throughput system that incorporates the membranes into a multiwell plate format. 7) The use of a microfluidic channel together with custom instrumentation developed in LCMB, NICHD in order to separate the effects of pressure and rapid (msec) shear transients on cultured neural cells, in order to gain a better understanding of the cellular mechanisms of traumatic brain injury. Our group has fabricated the microchannels, performed modelling aimed at understanding the shear transients experienced by the cells as a function of the bulk flow, and consulted extensively on how best to interface the microchannel with the existing instrumentation. 8) At the instigation of LCIMB, NIBIB, the collaborative design and fabrication of an aluminum-patterned window for use in calibrating the measured settling distances in analytical ultracentrifuges. A first generation prototype has been made and tested, and we have begun discussions with researchers at NIST about developing a low-volume, commercially available, certified standard.