Date of Award
Doctor of Philosophy (PhD)
Single molecules (SMs) have become important tools for studying nanoscale dynamics in biological studies since they were first optically observed in 1989. Numerous techniques, termed single molecule orientation localization microscopy (SMOLM), have been developed to measure the position and orientation of SMs. Due to the challenging signal-to-background ratio (SBR) in typical SM experiments, it is critical to understand whether an imaging system is optimal for a specific target sample. However, the maximum theoretical limit of measurement performance has yet to be quantified. In Chapter 3. We first develop a mathematical framework to compute the accuracy limit in measuring the rotational mobility (wobble) of SMs, revealing an unavoidable bias in the measurements caused by limited SBR. We then adapt classical and quantum estimation theory to derive the best possible precision for measuring both the position and orientation of SMs for any imaging system. We show that the best quantum-limited localization precision based on a vectorial dipole imaging model is ~4-8% worse than that suggested by an approximate scalar monopole model. Further, we derive conditions that enable the orientation of a rotationally-fixed SM to be measured with quantum-limited precision and propose an interferometric imaging system that achieves the precision limit. Interestingly, we find that there exists an information trade-off between precisely measuring the orientation of a molecule versus its wobble. We compare the performance of multiple state-of-the-art and commonly used methods for SMOLM to these limits under typical SM SBR.
Inspired by the performance limits of SMOLM techniques, we develop new methods for measuring the orientation and position of SMs. In Chapter 4, we report a radially and azimuthally polarized (raPol) microscope with high detection and estimation performance. Imaging Nile red (NR) molecules transiently bound within DPPC supported lipid bilayers (SLBs) reveals the existence of binding pockets that moderately limit NR from freely exploring all orientations. Treating the SLBs with cholesterol-loaded methyl-β-cyclodextrin causes NR’s orientational diffusion be significantly more confined. Strikingly, NR's translational diffusion drastically increase despite the cholesterol-induced condensation of the SLBs. In Chapter 5, we report a multi-view reflector (MVR) microscope for 3D SMOLM. The localization and orientation precision using a radially and azimuthally polarized MVR (raMVR) microscope is ~1.4-2.5 times better compared to other state-of-the-art SMOLM techniques. Imaging lipid-coated silica spheres, we show that the raMVR microscope can resolve the 3D position and orientation of NR molecules transiently bind spheres as small as 150 nm and as large as 1 μm. Further, we experimentally show its robustness against aberration caused by refractive index mismatch, making it suitable for imaging cells and extracellular vesicles with various of sizes. The raMVR SMOLM also allows us to resolve membrane versus amyloid aggregates according to the rotational dynamics of NR molecules. These detailed measurements of SM rotational and translational dynamics are made possible by raPol and raMVR's high measurement performance, and we expect the raPol and raMVR systems to be adapted in many future SMOLM studies.
Matthew D. Lew