Development of experimental and instrumental systems to study biological systems

Chapters 1-4 of this thesis describes the development of an experimental system to measure diffusion-limited reaction kinetics in a biological environment. About 100 years ago, the relationship between reaction rate and diffusion in homogenous solution, ie water or buffer, was described as a linear relationship by Smoluchowski. Applying this theory naively would suggest that since the diffusion coefficients drop by factors of 4-100 then the rates of reaction would drop by the same amount. However, recent theory and simulations suggest that this does not hold. Even though biological diffusion coefficients drop to 0.1-20% of that in buffer, these recent studies show that the reaction kinetics are much more weakly affected by the biological environment. Due to the lack of experimental evidence for biological diffusion, there is a great need for information in this area. Here, I describe a protein system, exogenous to E. coli¸ that will form a dimer in the presence of a small molecule. ^ I also describe the development of a new type of multivariate hyperspectral Raman instrument (MHI); the instrument is developed for use to study biological tissues and for high speed cell sorting applications. The new instrument design has a large speed advantage over traditional Raman instrumentation for rapid chemical imaging. While the MHI can reproduce the functionality of a traditional Raman spectrometer, its true speed advantage is realized after pre-training on known sample components. The MHI makes use of a spatial light modulator as a programmable optical filter that can be programmed with filters based on multivariate signal processing algorithms, such as PLS, in order to rapidly detect chemical components and create chemical maps. Chapters 5-8 of this thesis describe the development and construction of the MHI, as well as provide proof-of-concept experimental results demonstrating its functionality