Nonlinear laser microfabrication in biological environments
Nielson, Rex Young, 1969-
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Microscope optics have long been used to observe biological samples, when used in conjunction with a laser light source, they can also be a powerful means to probe and manipulate cells. This dissertation describes the development of methodologies for laser-based microfabrication in biological environments. These techniques use pulsed laser light at a wavelength transparent to the experimental medium except in a region with submicron dimensions defined by the focus of a high numerical aperture objective. At the focal region, high intensity light can modify sample material. The localized nature of this energetic event allows it to be accomplished in the vicinity of living cells, enabling microfabrication strategies that are used to probe and modify extracellular environments with high resolution. The basic principles of this process are explored and its use in several applications for cell culture manipulation are described. In one methodology the focused laser induces physical and chemical events that lead to the formation of a micron-scale solid from a precursor protein solution. By translating the relative position of the beam in the solution, arbitrary three-dimensional structures can be formed. The use of protein microstructures as a platform for probing and manipulating cellular microenvironments is investigated and an advanced method of rapidly patterning elaborate structures with a spatial light modulator is demonstrated. The high intensity laser focus is used in a second strategy to create microfluidic conduits in a device consisting of two stacked flow channels, one containing adherent cells in buffer and the other a reagent solution. With laser ablation of a pore, highly defined reagent plumes are directed into the cell-containing chamber where they can dose multiple specifically targeted regions with subcellular specificity. The unique microfabrication technologies described in this dissertation could prove to be of use for researchers developing diagnostic and therapeutic devices and could lead to more advanced tools for studying the basic biology of cells.