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1. Fundamental limits of measuring single-molecule rotational mobility

   https://doi.org/10.1117/12.2506712  

   Zhang, Oumeng; Lew, Matthew D. (February 2019, Proc. SPIE 10884, Single Molecule Spectroscopy and Superresolution Imaging XII)

   Various methods exist for measuring molecular orientation, thereby providing insight into biochemical activities at nanoscale. Since fluorescence intensity and not electric field is detected, these methods are limited to measuring even-order moments of molecular orientation. However, any measurement noise, for example photon shot noise, will result in nonzero measurements of any of these even-order moments, thereby causing rotationally-free molecules to appear to be partially constrained. Here, we build a model to quantify measurement errors in rotational mobility. Our theoretical framework enables scientists to choose the optimal single-molecule orientation measurement technique for any desired measurement accuracy and photon budget. 

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2. Six-dimensional single-molecule imaging with isotropic resolution using a multi-view reflector microscope

   https://doi.org/10.1038/s41566-022-01116-6  

   Zhang, Oumeng; Guo, Zijian; He, Yuanyuan; Wu, Tingting; Vahey, Michael D.; Lew, Matthew D. (December 2022, Nature Photonics)

   Imaging of both the positions and orientations of single fluorophores, termed single-molecule orientation-localization microscopy, is a powerful tool for the study of biochemical processes. However, the limited photon budget associated with single-molecule fluorescence makes high-dimensional imaging with isotropic, nanoscale spatial resolution a formidable challenge. Here we realize a radially and azimuthally polarized multi-view reflector (raMVR) microscope for the imaging of the three-dimensional (3D) positions and 3D orientations of single molecules, with precisions of 10.9 nm and 2.0° over a 1.5-μm depth range. The raMVR microscope achieves 6D super-resolution imaging of Nile red molecules transiently bound to lipid-coated spheres, accurately resolving their spherical morphology, despite refractive-index mismatch. By observing the rotational dynamics of Nile red, raMVR images also resolve the infiltration of lipid membranes by amyloid-beta oligomers without covalent labelling. Finally, we demonstrate 6D imaging of cell membranes, where the orientations of specific fluorophores reveal heterogeneity in membrane fluidity. With its nearly isotropic 3D spatial resolution and orientation measurement precision, we expect the raMVR microscope to enable 6D imaging of molecular dynamics within biological and chemical systems with exceptional detail. 

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3. Single-molecule orientation localization microscopy I: fundamental limits

   https://doi.org/10.1364/JOSAA.411981  

   Zhang, Oumeng; Lew, Matthew_D (February 2021, Journal of the Optical Society of America A)

   Precisely measuring the three-dimensional position and orientation of individual fluorophores is challenging due to the substantial photon shot noise in single-molecule experiments. Facing this limited photon budget, numerous techniques have been developed to encode 2D and 3D position and 2D and 3D orientation information into fluorescence images. In this work, we adapt classical and quantum estimation theory and propose a mathematical framework to derive the best possible precision for measuring the position and orientation of dipole-like emitters for any fixed imaging system. We find that it is impossible to design an instrument that achieves the maximum sensitivity limit for measuring all possible rotational motions. Further, our vectorial dipole imaging model shows that the best quantum-limited localization precision is 4%–8% worse than that suggested by a scalar monopole model. Overall, we conclude that no single instrument can be optimized for maximum precision across all possible 2D and 3D localization and orientation measurement tasks. 

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4. SpotClean adjusts for spot swapping in spatial transcriptomics data

   https://doi.org/10.1038/s41467-022-30587-y  

   Ni, Zijian; Prasad, Aman; Chen, Shuyang; Halberg, Richard B.; Arkin, Lisa M.; Drolet, Beth A.; Newton, Michael A.; Kendziorski, Christina (December 2022, Nature Communications)

   Abstract Spatial transcriptomics is a powerful and widely used approach for profiling the gene expression landscape across a tissue with emerging applications in molecular medicine and tumor diagnostics. Recent spatial transcriptomics experiments utilize slides containing thousands of spots with spot-specific barcodes that bind RNA. Ideally, unique molecular identifiers (UMIs) at a spot measure spot-specific expression, but this is often not the case in practice due to bleed from nearby spots, an artifact we refer to as spot swapping. To improve the power and precision of downstream analyses in spatial transcriptomics experiments, we propose SpotClean, a probabilistic model that adjusts for spot swapping to provide more accurate estimates of gene-specific UMI counts. SpotClean provides substantial improvements in marker gene analyses and in clustering, especially when tissue regions are not easily separated. As demonstrated in multiple studies of cancer, SpotClean improves tumor versus normal tissue delineation and improves tumor burden estimation thus increasing the potential for clinical and diagnostic applications of spatial transcriptomics technologies. 

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5. Single-molecule orientation localization microscopy for resolving structural heterogeneities between amyloid fibrils

   https://doi.org/10.1364/OPTICA.388157  

   Ding, Tianben; Wu, Tingting; Mazidi, Hesam; Zhang, Oumeng; Lew, Matthew_D (June 2020, Optica)

   Simultaneous measurements of single-molecule positions and orientations provide critical insight into a variety of biological and chemical processes. Various engineered point spread functions (PSFs) have been introduced for measuring the orientation and rotational diffusion of dipole-like emitters, but the widely used Cramér-Rao bound (CRB) only evaluates performance for one specific orientation at a time. Here, we report a performance metric, termed variance upper bound (VUB), that yields a global maximum CRB for all possible molecular orientations, thereby enabling the measurement performance of any PSF to be computed efficiently ( $\sim <\#comment/>1000\times <\#comment/>$faster than calculating average CRB). Our VUB reveals that the simple polarized standard PSF provides robust and precise orientation measurements if emitters are near a refractive index interface. Using this PSF, we measure the orientations and positions of Nile red (NR) molecules transiently bound to amyloid aggregates. Our super-resolved images reveal the main binding mode of NR on amyloid fiber surfaces, as well as structural heterogeneities along amyloid fibrillar networks, that cannot be resolved by single-molecule localization alone. 

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