University of Texas at Austin researchers Bryan J. Kaehr and Jason B. Shear have developed a way to rapidly generate microscale patterns in biomaterials using a technique dubbed mask-directed multiphoton lithography. Among other applications, they have used the method to transfer the outline of a housefly into biomaterials and to fabricate protein-based microchambers to trap and incubate a single moving bacterium.Using mask-directed multiphoton lithography, researchers fabricated the word “avidin” from a solution containing the biotin-binding protein of the same name (top). They washed it with biotin-fluorescein, and the fluorophore adhered to the letters, which can be seen in the fluorescence image (bottom). Scale bar = 10 μm. Images courtesy of Bryan J. Kaehr and Jason B. Shear, University of Texas at Austin.Unlike conventional multiphoton-based fabrication, this approach does not require point-by-point programming of a scanning stage. As a result, experimental shapes can be readied quickly.“From the time that Bryan thinks of a change, he can modify the computer drawing, print it on a transparency, insert the transparency into the mask plane and fabricate a new object — all in something like five to 10 minutes,” said Shear, an associate professor of chemistry and biochemistry, of his student.The technique exploits photoactivated cross-linking of proteins. When exposed to light of the right characteristics, proteins in solution cross-link and become solid. The team accomplished this by raster-scanning a beam over a mask. Optics project the result onto a focal plane, which is where a substrate and the solution sit. Because this is a multiphoton process, the cross-linking is confined to a volume of about a femtoliter; therefore, three-dimensional and graduated shapes are possible.The researchers constructed a Texas-shaped microscale container with a single entrance and a sealed top (left). Twelve hours after the introduction of mobile E. coli into nutrient-rich media, the container is full of bacteria (right). Scale bar = 10 μm.In their setup, the researchers used a Zeiss inverted microscope and a Bio-Rad Laboratories confocal scanner. They created the beam using a femtosecond Ti:sapphire laser from Spectra-Physics that operated between 730 and 740 nm. To give structures the third dimension, they adjusted the laser focus up or down, in some cases using multiple masks as they moved between focal planes. They achieved a practical lateral resolution of about 0.5 μm, but the limiting factors were, in part, mask quality and fabrication speed.For the protein, they used bovine serum albumin with either methylene blue or flavin adenine dinucleotide as a photosensitizer. The latter was used for biocompatible fabrication. In a series of experiments, they created various structures using this method, including protein microchambers with complex layouts, sealed by a solid cover.They also manufactured two-story structures with different layouts on each floor and created a microcontainer with an entrance, which they plugged after a bacterium became trapped inside. In this way, they could contain the multiplying bacteria without having any escape.One application of the technology, according to Shear, might be the creation of cell and tissue replicas. “This technology would make it possible to grow cells in reproducible and, hopefully, lifelike environments.”They also are working on automated mask sequencing via, for example, a micromirror device to allow a wider variety of microstructures.Journal of the American Chemical Society, Feb. 21, 2007, pp. 1904-1905.