A novel hyperspectral fluorescence microscope for high-resolution 3D optical sectioning of cells and other structures has been designed, constructed, and used to investigate a number of different problems. We have significantly extended new multivariate curve resolution (MCR) data analysis methods to deconvolve the hyperspectral image data and to rapidly extract quantitative 3D concentration distribution maps of all emitting species. The imaging system has many advantages over current confocal imaging systems including simultaneous monitoring of numerous highly overlapped fluorophores, immunity to autofluorescence or impurity fluorescence, enhanced sensitivity, and dramatically improved accuracy, reliability, and dynamic range. Efficient data compression in the spectral dimension has allowed personal computers to perform quantitative analysis of hyperspectral images of large size without loss of image quality. We have also developed and tested software to perform analysis of time resolved hyperspectral images using trilinear multivariate analysis methods. The new imaging system is an enabling technology for numerous applications including (1) 3D composition mapping analysis of multicomponent processes occurring during host-pathogen interactions, (2) monitoring microfluidic processes, (3) imaging of molecular motors and (4) understanding photosynthetic processes in wild type and mutant Synechocystis cyanobacteria.
The formation and functions of living materials and organisms are fundamentally different from those of synthetic materials and devices. Synthetic materials tend to have static structures, and are not capable of adapting to the functional needs of changing environments. In contrast, living systems utilize energy to create, heal, reconfigure, and dismantle materials in a dynamic, non-equilibrium fashion. The overall goal of the project was to organize and reconfigure functional assemblies of nanoparticles using strategies that mimic those found in living systems. Active assembly of nanostructures was studied using active biomolecules to drive the organization and assembly of nanocomposite materials. In this system, kinesin motor proteins and microtubules were used to direct the transport and interactions of nanoparticles at synthetic interfaces. In addition, the kinesin/microtubule transport system was used to actively assemble nanocomposite materials capable of storing significant elastic energy. Novel biophysical measurement tools were also developed for measuring the collective force generated by kinesin motor proteins, which will provide insight on the mechanical constraints of active assembly processes. Responsive reconfiguration of nanostructures was studied in terms of using active biomolecules to mediate the optical properties of quantum dot (QD) arrays through modulation of inter-particle spacing and associated energy transfer interaction. Design rules for kinesin-based transport of a wide range of nanoscale cargo (e.g., nanocrystal quantum dots, micron-sized polymer spheres) were developed. Three-dimensional microtubule organizing centers were assembled in which the polar orientation of the microtubules was controlled by a multi-staged assembly process. Overall, a number of enabling technologies were developed over the course of this project, and will drive the exploitation of energy-driven processes to regulate the assembly, disassembly, and dynamic reorganization of nanomaterials.
The challenge of modeling the organization and function of biological membranes on a solid support has received considerable attention in recent years, primarily driven by potential applications in biosensor design. Affinity-based biosensors show great promise for extremely sensitive detection of BW agents and toxins. Receptor molecules have been successfully incorporated into phospholipid bilayers supported on sensing platforms. However, a collective body of data detailing a mechanistic understanding of membrane processes involved in receptor-substrate interactions and the competition between localized perturbations and delocalized responses resulting in reorganization of transmembrane protein structure, has yet to be produced. This report describes a systematic procedure to develop detailed correlation between (recognition-induced) protein restructuring and function of a ligand gated ion channel by combining single molecule fluorescence spectroscopy and single channel current recordings. This document is divided into three sections: (1) reported are the thermodynamics and diffusion properties of gramicidin using single molecule fluorescence imaging and (2) preliminary work on the 5HT{sub 3} serotonin receptor. Thirdly, we describe the design and fabrication of a miniaturized platform using the concepts of these two technologies (spectroscopic and single channel electrochemical techniques) for single molecule analysis, with a longer term goal of using the physical and electronic changes caused by a specific molecular recognition event as a transduction pathway in affinity based biosensors for biotoxin detection.