14 research outputs found

    Axially overlapped multi-focus light sheet with enlarged field of view

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    Light sheet fluorescence microscopy provides optical sectioning and is widely used in volumetric imaging of large specimens. However, the axial resolution and the lateral Field of View (FoV) of the system, defined by the light sheet, typically limit each other due to the spatial band product of the excitation objective. Here, we develop a simple multi-focus scheme to extend the FoV, where a Gaussian light sheet can be focused at three or more consecutive positions. Axially overlapped multiple light sheets significantly enlarge the FoV with improved uniformity and negligible loss in axial resolution. By measuring the point spread function of fluorescent beads, we demonstrated that the obtained light sheet has a FoV of 450 μm and a maximum axial FWHM of 7.5 μm. Compared with the conventional single-focus one, the multi-focus Gaussian light sheet displays a significantly improved optical sectioning ability over the full FoV when imaging cells and zebrafish. Light sheet fluorescence microscopy (LSFM) has become an indispensable tool in volumetric imaging, with the advances in high spatiotemporal resolution and low photo-toxicity to the fluorescent sample. The open-source design with detailed instructions encourages DIY setups, which has significantly accelerated the wide-range adoption and applications of LSFM. Pioneering works, including OpenSPIM,1 OpenSpin,2 and the recent mesoSPIM,3 provide detailed protocols for building and using the microscopes. These joint efforts further allow the biology labs to build their LSFM system for a specific application, including more complex schemes, such as multiview excitation or detection configurations.4

    High-dimensional super-resolution imaging reveals heterogeneity and dynamics of subcellular lipid membranes.

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    Lipid membranes are found in most intracellular organelles, and their heterogeneities play an essential role in regulating the organelles' biochemical functionalities. Here we report a Spectrum and Polarization Optical Tomography (SPOT) technique to study the subcellular lipidomics in live cells. Simply using one dye that universally stains the lipid membranes, SPOT can simultaneously resolve the membrane morphology, polarity, and phase from the three optical-dimensions of intensity, spectrum, and polarization, respectively. These high-throughput optical properties reveal lipid heterogeneities of ten subcellular compartments, at different developmental stages, and even within the same organelle. Furthermore, we obtain real-time monitoring of the multi-organelle interactive activities of cell division and successfully reveal their sophisticated lipid dynamics during the plasma membrane separation, tunneling nanotubules formation, and mitochondrial cristae dissociation. This work suggests research frontiers in correlating single-cell super-resolution lipidomics with multiplexed imaging of organelle interactome

    Super-resolution imaging of fluorescent dipoles via polarized structured illumination microscopy

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    © 2019, The Author(s). Fluorescence polarization microscopy images both the intensity and orientation of fluorescent dipoles and plays a vital role in studying molecular structures and dynamics of bio-complexes. However, current techniques remain difficult to resolve the dipole assemblies on subcellular structures and their dynamics in living cells at super-resolution level. Here we report polarized structured illumination microscopy (pSIM), which achieves super-resolution imaging of dipoles by interpreting the dipoles in spatio-angular hyperspace. We demonstrate the application of pSIM on a series of biological filamentous systems, such as cytoskeleton networks and λ-DNA, and report the dynamics of short actin sliding across a myosin-coated surface. Further, pSIM reveals the side-by-side organization of the actin ring structures in the membrane-associated periodic skeleton of hippocampal neurons and images the dipole dynamics of green fluorescent protein-labeled microtubules in live U2OS cells. pSIM applies directly to a large variety of commercial and home-built SIM systems with various imaging modality

    Super-resolution fluorescence polarization microscopy

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    © 2018 The Author(s). Fluorescence polarization is related to the dipole orientation of chromophores, making fluorescence polarization microscopy possible to reveal structures and functions of tagged cellular organelles and biological macromolecules. Several recent super resolution techniques have been applied to fluorescence polarization microscopy, achieving dipole measurement at nanoscale. In this review, we summarize both diffraction limited and super resolution fluorescence polarization microscopy techniques, as well as their applications in biological imaging

    Super-resolution dipole orientation mapping via polarization demodulation

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    © The Author(s) 2016. Fluorescence polarization microscopy (FPM) aims to detect the dipole orientation of fluorophores and to resolve structural information for labeled organelles via wide-field or confocal microscopy. Conventional FPM often suffers from the presence of a large number of molecules within the diffraction-limited volume, with averaged fluorescence polarization collected from a group of dipoles with different orientations. Here, we apply sparse deconvolution and least-squares estimation to fluorescence polarization modulation data and demonstrate a super-resolution dipole orientation mapping (SDOM) method that resolves the effective dipole orientation from a much smaller number of fluorescent molecules within a sub-diffraction focal area. We further apply this method to resolve structural details in both fixed and live cells. For the first time, we show that different borders of a dendritic spine neck exhibit a heterogeneous distribution of dipole orientation. Furthermore, we illustrate that the dipole is always perpendicular to the direction of actin filaments in mammalian kidney cells and radially distributed in the hourglass structure of the septin protein under specific labelling. The accuracy of the dipole orientation can be further mapped using the orientation uniform factor, which shows the superiority of SDOM compared with its wide-field counterpart as the number of molecules is decreased within the smaller focal area. Using the inherent feature of the orientation dipole, the SDOM technique, with its fast imaging speed (at sub-second scale), can be applied to a broad range of fluorescently labeled biological systems to simultaneously resolve the valuable dipole orientation information with super-resolution imaging

    Versatile Application of Fluorescent Quantum Dot Labels in Super-resolution Fluorescence Microscopy

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    © 2016 American Chemical Society. Quantum dots (QDs) are well known as bright and photostable inorganic fluorescent probes for microscopy imaging, with many attractive features superior to those found in organic dyes. However, their broadband excitation spectrum and emission blinking property have limited the applicability of QDs in modern super-resolution microscopy techniques. In this work, we systematically investigate practical approaches to overcoming these drawbacks and provide examples of their use across many commercially available super-resolution microscopy systems now accessible to biologists, with examples across the major super-resolution techniques. This work further maps out how QDs can be further engineered to facilitate their applications in the respective super-resolution microscopy techniques

    Developing novel methods to image and visualize 3D genomes

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    © 2018, The Author(s). To investigate three-dimensional (3D) genome organization in prokaryotic and eukaryotic cells, three main strategies are employed, namely nuclear proximity ligation-based methods, imaging tools (such as fluorescence in situ hybridization (FISH) and its derivatives), and computational/visualization methods. Proximity ligation-based methods are based on digestion and re-ligation of physically proximal cross-linked chromatin fragments accompanied by massively parallel DNA sequencing to measure the relative spatial proximity between genomic loci. Imaging tools enable direct visualization and quantification of spatial distances between genomic loci, and advanced implementation of (super-resolution) microscopy helps to significantly improve the resolution of images. Computational methods are used to map global 3D genome structures at various scales driven by experimental data, and visualization methods are used to visualize genome 3D structures in virtual 3D space-based on algorithms. In this review, we focus on the introduction of novel imaging and visualization methods to study 3D genomes. First, we introduce the progress made recently in 3D genome imaging in both fixed cell and live cells based on long-probe labeling, short-probe labeling, RNA FISH, and the CRISPR system. As the fluorescence-capturing capability of a particular microscope is very important for the sensitivity of bioimaging experiments, we also introduce two novel super-resolution microscopy methods, SDOM and low-power super-resolution STED, which have potential for time-lapse super-resolution live-cell imaging of chromatin. Finally, we review some software tools developed recently to visualize proximity ligation-based data. The imaging and visualization methods are complementary to each other, and all three strategies are not mutually exclusive. These methods provide powerful tools to explore the mechanisms of gene regulation and transcription in cell nuclei