Highly Sensitive Quantitative Microscopy for Cellular and Subcellular Analysis

Abstract

The purpose of this research has been to implement optical methods to investigate subcellular biological function. The diffraction limit of light for conventional optical microscopy is roughly lamda/2, or 200 nanometers for visible light; significantly larger than the size of most macromolecules. One way to overcome this limitation is through the careful use of polarization optics and choices in fluorescent probes. Like an antenna, fluorescent molecules have an orientation at which they preferentially absorb polarized electromagnetic radiation. By adjusting the excitation polarization and observing the anisotropic polarization of light emitted from the probe, information about the local environment of the fluorescent probe or macromolecules that they are bound to can be gleaned on a scale much smaller than the diffraction limit of light. I use this anisotropic polarization to probe the interior of synaptic vesicles which were genetically modified to lack the SV2 glycoprotein. I also made fluorescent polymer nanoparticles that exhibited high fluorescence anisotropy and demonstrated their value as observers of molecular motor function. This was done by observing the rotation of microtubules as they precessed in a microtubule gliding assay. In chapter 4 I discuss the importance of laser choice on cell viability in single-cell nanosurgery. Finally, chapter 5 is a "how-to" guide for building and using optical tweezers that was written for a book on methods in molecular biology.