Perturbation and analysis of biological microenvironments

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Allen, Richard William, 1976-

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Understanding microscale biological processes as cells develop into tissues is one of the most important, yet most difficult, problems in modern biology. Cells encounter a dynamic chemical and physical environment and delineating the myriad of variables proves daunting with even the most sophisticated experiments. This dissertation focuses on the development and application of unique enabling technologies designed to sample and control biological microenvironments. By developing two approaches – one aimed at intracellular biochemistry and another for extracellular targets – based on photochemistry and optical force generation, research presented here will allow new areas of subcellular dynamics to be addressed. On the intracellular side, enzyme-immobilized polymeric microspheres or enzyme microstructures are placed into the cell cytosol via optical tweezers for sustained and localized chemical modification of the intracellular environment. This approach is complemented by the use of extracellular guidance barriers formed from photo-induced crosslinking of proteins. Through the use of minimally toxic photosensitizers and femtosecond (fs) near infrared (NIR) light, it is possible to fabricate three-dimensional protein structures in a living cell’s environment. Moreover, this work explores the ability to form protein structures with enzymatic activity as well as with high aspect-ratio features at micron resolution. Finally, the photochemical transformation of serotonin into a highly fluorescent visible photoproduct is investigated as a means to overcome problems associated with sample size in neurotransmitter detection during synaptic chemical signaling. Optimization of this multiphoton process entails understanding the mechanism by which the photoproduct is created and experiments towards this goal are presented here. Ultimately, the precision and flexibility of these technologies will allow access to new areas of the biosciences.



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