Date of Award

Spring 5-13-2017

Level of Access

Open-Access Thesis

Degree Name

Master of Science (MS)




Samuel Hess

Second Committee Member

Raymond Astumian

Third Committee Member

John Thompson


Fluorescence microscopy is popular for its noninvasive properties and its use in imaging multiple species, simultaneously. Furthermore, superresolution fluorescence microscopy (SRFLM) utilizes photoswitchable proteins to improve the lateral resolution of conventional fluorescence microscopy by an order of magnitude. There is little work conducted on the study of excitation laser polarizations and their effect on the number of localizations as well as the brightness of molecules. This thesis attempts to study the effect of excitation wavelength polarization on the number of localizations and the brightness of molecules by comparing two orientations of circularly-polarized, excitation lasers. The first type of orientation involves collinear excitation beams perpendicular to the sample stage (widefield). The second involves two, crossed excitation beams so that they overlap only at the area imaged in a sample (cross-beam).

Dendra 2-HA is the fluorescent protein Dendra2 tagged to the influenza virus protein hemagglutinin. The fluorescent protein is commonly used in studies related to the spread of the influenza virus in mammalian cells, such as NIH3T3 cells. With equal excitation rates between the two beam orientations, the cross-beam, with 73 degrees between the beams, yielded more localizations and more narrow brightness histograms than that of the widefield variety. Simulation results show a similar trend, but are not analytically in agreement with those of the experimental variety. Maximization of the number of localizations combined with minimization of the brightness histogram widths can be achieved with ninety degrees between the beams and equal excitation rates, per the simulation. This result suggests that the cross-beam orientation has the potential to be of use in the improvement of multiple fluorescent species superresolution studies.