Congratulations to Matthew Valles on the successful defense of his Master’s Thesis on February 9.
CROSS-BEAM ILLUMINATION GEOMETRY
FOR FLUORESCENCE PHOTOACTIVATION LOCALIZATION MICROSCOPY
By Matthew M. Valles
CANDIDATE FOR THE MASTER OF SCIENCE IN PHYSICS DEGREE
Thesis Advisor: Dr. Samuel T. Hess
Fluorescence microscopy is popular for its flexibility, its relative noninvasiveness, its ability to image multiple species simultaneously, and its single-molecule sensitivity. Furthermore, superresolution fluorescence localization microscopy (SRFLM) utilizes photoactivatable fluorescent proteins (PA-FPs) to improve the lateral resolution of conventional fluorescence microscopy by an order of magnitude. There has been relatively little consideration of the effect of excitation laser polarization on the number of localizations and the brightness of emitting molecules. This study examines the effect of excitation wavelength polarization on the number of localizations and the brightness of molecules by comparing results using two lasers to simultaneously illuminate the sample with different polarizations. The first type of geometry uses two collinear excitation beams perpendicular to the sample stage (widefield). The second geometry uses two nearly orthogonal excitation beams which overlap in the area imaged (cross-beam).
Dendra2-HA is the influenza virus protein hemagglutinin (HA) tagged with the fluorescent protein Dendra2. This fluorescent protein chimera is commonly used in SRFLM studies related to the spread of influenza virus in mammalian cells, such as NIH3T3 cells. With equal excitation rates resulting from the two beam orientations (i.e. the cross-beam), with 73 degrees between the beams, SRFLM yielded more localizations and a narrower brightness histogram in comparison to results using the widefield geometry. Simulation results show a similar trend, but are not analytically in agreement with those of the experiment. Maximization of the number of localizations combined with minimization of the brightness histogram widths can be achieved with ninety degrees of separation between the beams and intensities resulting in equal excitation rates for all molecular orientations. The results obtained from experiment and simulation suggest that the cross-beam orientation has the potential to improve the capabilities of SRFLM for super-resolution imaging of multiple fluorescent species.