La Riviere
Patrick J.
La Riviere
Patrick J.
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ArticleReflective imaging improves spatiotemporal resolution and collection efficiency in light sheet microscopy(Nature Publishing Group, 2017-11-13) Wu, Yicong ; Kumar, Abhishek ; Smith, Corey ; Ardiel, Evan L. ; Chandris, Panagiotis ; Christensen, Ryan ; Rey-Suarez, Ivan ; Guo, Min ; Vishwasrao, Harshad D. ; Chen, Jiji ; Tang, Jianyong ; Upadhyaya, Arpita ; La Riviere, Patrick J. ; Shroff, HariLight-sheet fluorescence microscopy (LSFM) enables high-speed, high-resolution, and gentle imaging of live specimens over extended periods. Here we describe a technique that improves the spatiotemporal resolution and collection efficiency of LSFM without modifying the underlying microscope. By imaging samples on reflective coverslips, we enable simultaneous collection of four complementary views in 250 ms, doubling speed and improving information content relative to symmetric dual-view LSFM. We also report a modified deconvolution algorithm that removes associated epifluorescence contamination and fuses all views for resolution recovery. Furthermore, we enhance spatial resolution (to <300 nm in all three dimensions) by applying our method to single-view LSFM, permitting simultaneous acquisition of two high-resolution views otherwise difficult to obtain due to steric constraints at high numerical aperture. We demonstrate the broad applicability of our method in a variety of samples, studying mitochondrial, membrane, Golgi, and microtubule dynamics in cells and calcium activity in nematode embryos.
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ArticleSingle-fluorophore orientation determination with multiview polarized illumination : modeling and microscope design(Optical Society, 2017-12-01) Chandler, Talon ; Mehta, Shalin B. ; Shroff, Hari ; Oldenbourg, Rudolf ; La Riviere, Patrick J.We investigate the use of polarized illumination in multiview microscopes for determining the orientation of single-molecule fluorescence transition dipoles. First, we relate the orientation of single dipoles to measurable intensities in multiview microscopes and develop an information-theoretic metric—the solid-angle uncertainty—to compare the ability of multiview microscopes to estimate the orientation of single dipoles. Next, we compare a broad class of microscopes using this metric—single- and dual-view microscopes with varying illumination polarization, illumination numerical aperture (NA), detection NA, obliquity, asymmetry, and exposure. We find that multi-view microscopes can measure all dipole orientations, while the orientations measurable with single-view microscopes is halved because of symmetries in the detection process. We also find that choosing a small illumination NA and a large detection NA are good design choices, that multiview microscopes can benefit from oblique illumination and detection, and that asymmetric NA microscopes can benefit from exposure asymmetry.
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ArticleUncovering the effects of symbiosis and temperature on coral calcification(University of Chicago Press, 2022-01-10) Dellaert, Zoe ; Vargas, Phillip A. ; La Riviere, Patrick J. ; Roberson, Loretta M.We tested the impact of temperature and symbiont state on calcification in corals, using the facultatively symbiotic coral Astrangia poculata as a model system. Symbiotic and aposymbiotic colonies of A. poculata were reared in 15, 20, and 27 °C conditions. We used scanning electron microscopy to quantify how these physiological and environmental conditions impact skeletal structure. Buoyant weight data over time revealed that temperature significantly affects calcification rates. Scanning electron microscopy of A. poculata skeletons showed that aposymbiotic colonies appear to have a lower density of calcium carbonate in actively growing septal spines. We describe a novel approach to analyze the roughness and texture of scanning electron microscopy images. Quantitative analysis of the roughness of septal spines revealed that aposymbiotic colonies have a rougher surface than symbiotic colonies in tropical conditions (27 °C). This trend reversed at 15 °C, a temperature at which the symbionts of A. poculata may exhibit parasitic properties. Analysis of surface texture patterns showed that temperature impacts the spatial variance of crystals on the spine surface. Few published studies have examined the skeleton of A. poculata by using scanning electron microscopy. Our approach provides a way to study detailed changes in skeletal microstructure in response to environmental parameters and can serve as a proxy for more expensive and time-consuming analyses. Utilizing a facultatively symbiotic coral that is native to both temperate and tropical regions provides new insights into the impact of both symbiosis and temperature on calcification in corals.
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ArticleThree-dimensional structured illumination microscopy with enhanced axial resolution(Nature Research, 2023-01-26) Li, Xuesong ; Wu, Yicong ; Su, Yijun ; Rey-Suarez, Ivan ; Matthaeus, Claudia ; Updegrove, Taylor B. ; Wei, Zhuang ; Zhang, Lixia ; Sasaki, Hideki ; Li, Yue ; Guo, Min ; Giannini, John P. ; Vishwasrao, Harshad D. ; Chen, Jiji ; Lee, Shih-Jong J. ; Shao, Lin ; Liu, Huafeng ; Ramamurthi, Kumaran S. ; Taraska, Justin W. ; Upadhyaya, Arpita ; La Riviere, Patrick ; Shroff, HariThe axial resolution of three-dimensional structured illumination microscopy (3D SIM) is limited to ∼300 nm. Here we present two distinct, complementary methods to improve axial resolution in 3D SIM with minimal or no modification to the optical system. We show that placing a mirror directly opposite the sample enables four-beam interference with higher spatial frequency content than 3D SIM illumination, offering near-isotropic imaging with ∼120-nm lateral and 160-nm axial resolution. We also developed a deep learning method achieving ∼120-nm isotropic resolution. This method can be combined with denoising to facilitate volumetric imaging spanning dozens of timepoints. We demonstrate the potential of these advances by imaging a variety of cellular samples, delineating the nanoscale distribution of vimentin and microtubule filaments, observing the relative positions of caveolar coat proteins and lysosomal markers and visualizing cytoskeletal dynamics within T cells in the early stages of immune synapse formation.