Rapid and Quantitative Imaging of Excitation Polarized Fluorescence Reveals Ordered Septin Dynamics in Live Yeast

Abstract

We report an imaging method for fast, sensitive analysis of the orientation of fluorescent molecules by employing a liquid-crystal based universal polarizer in the optical path of a wide-field light microscope. We developed specific acquisition and processing algorithms for measuring the anisotropy and for correcting artifacts caused by fluorescence bleaching, background light, and differential transmission of optical components. We call this approach the Fluorescence LC-PolScope and we used it to analyze the architectural dynamics of septin-green fluorescent protein (septin-GFP) constructs in the neck region of budding yeast. We describe three different states of highly anisotropic septin arrays in which the prevailing orientation of GFP dipoles was either parallel or perpendicular to the mother-bud axis. The transitions between these ordered states were characterized by transient isotropic states. To analyze the patterns of polarized fluorescence, we modeled the alignment of septin-GFP constructs in different stages of septin ring formation. Based on our model, our experimental data are consistent with the formation of paired rather than single filaments and the axis of the α-helical septin terminus linked to a GFP molecule is likely oriented normal to the cell surface. The Fluorescence LC-PolScope combines the molecular specificity of fluorescence tagging with the structural specificity of polarized light analysis.