The Experts below are selected from a list of 383601 Experts worldwide ranked by ideXlab platform
W E Moerner - One of the best experts on this subject based on the ideXlab platform.
-
tilted light sheet microscopy with 3d point spread functions for single molecule super resolution Imaging in mammalian cells
Proceedings of SPIE--the International Society for Optical Engineering, 2018Co-Authors: Annakarin Gustavsson, Yoav Shechtman, Petar N Petrov, Maurice Y Lee, W E MoernerAbstract:To obtain a complete picture of subcellular nanostructures, cells must be imaged with high resolution in all three dimensions (3D). Here, we present tilted light sheet microscopy with 3D point spread functions (TILT3D), an Imaging platform that combines a novel, tilted light sheet illumination strategy with engineered long axial range point spread functions (PSFs) for low-background, 3D super localization of single molecules as well as 3D Super-Resolution Imaging in thick cells. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The axial positions of the single molecules are encoded in the shape of the PSF rather than in the position or thickness of the light sheet, and the light sheet can therefore be formed using simple optics. The result is flexible and user-friendly 3D Super-Resolution Imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D Super-Resolution Imaging in mammalian cells by Imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed Tetrapod PSF for fiducial bead tracking and live axial drift correction. We envision TILT3D to become an important tool not only for 3D Super-Resolution Imaging, but also for live whole-cell single-particle and single-molecule tracking.
-
maximally informative point spread functions for 3d super resolution Imaging
Conference on Lasers and Electro-Optics, 2015Co-Authors: Yoav Shechtman, Steffen J Sahl, Adam S Backer, W E MoernerAbstract:We generate optimal point spread functions (PSFs) for 3D Super-Resolution Imaging, and demonstrate their application in biological conditions. These PSFs exhibit significantly improved localization precision and depth of field over the current state of the art.
-
the role of molecular dipole orientation in single molecule fluorescence microscopy and implications for super resolution Imaging
ChemPhysChem, 2014Co-Authors: Mikael P Backlund, Steffen J Sahl, Adam S Backer, Matthew D Lew, W E MoernerAbstract:Numerous methods for determining the orientation of single-molecule transition dipole moments from microscopic images of the molecular fluorescence have been developed in recent years. At the same time, techniques that rely on nanometer-level accuracy in the determination of molecular position, such as single-molecule Super-Resolution Imaging, have proven immensely successful in their ability to access unprecedented levels of detail and resolution previously hidden by the optical diffraction limit. However, the level of accuracy in the determination of position is threatened by insufficient treatment of molecular orientation. Here we review a number of methods for measuring molecular orientation using fluorescence microscopy, focusing on approaches that are most compatible with position estimation and single-molecule Super-Resolution Imaging. We highlight recent methods based on quadrated pupil Imaging and on double-helix point spread function microscopy and apply them to the study of fluorophore mobility on immunolabeled microtubules.
-
super resolution Imaging of the nucleoid associated protein hu in caulobacter crescentus
Biophysical Journal, 2011Co-Authors: Steven F Lee, Lucy Shapiro, Michael A. Thompson, Monica A Schwartz, W E MoernerAbstract:Little is known about the structure and function of most nucleoid-associated proteins (NAPs) in bacteria. One reason for this is that the distribution and structure of the proteins is obfuscated by the diffraction limit in standard wide-field and confocal fluorescence Imaging. In particular, the distribution of HU, which is the most abundant NAP, has received little attention. In this study, we investigate the distribution of HU in Caulobacter crescentus using a combination of Super-Resolution fluorescence Imaging and spatial point statistics. By simply increasing the laser power, single molecules of the fluorescent protein fusion HU2-eYFP can be made to blink on and off to achieve Super-Resolution Imaging with a single excitation source. Through quantification by Ripley's K-test and comparison with Monte Carlo simulations, we find the protein is slightly clustered within a mostly uniform distribution throughout the swarmer and stalked stages of the cell cycle but more highly clustered in predivisional cells. The methods presented in this letter should be of broad applicability in the future study of prokaryotic NAPs.
-
super resolution Imaging in live caulobacter crescentus cells using photoswitchable eyfp
Nature Methods, 2008Co-Authors: Julie S. Biteen, Lucy Shapiro, Michael A. Thompson, Nicole K Tselentis, Grant R Bowman, W E MoernerAbstract:The commonly used, monomeric EYFP enabled Imaging of intracellular protein structures beyond the optical resolution limit ('Super-Resolution' Imaging) in living cells. By combining photoinduced activation of single EYFP fusions and time-lapse Imaging, we obtained sub-40 nm resolution images of the filamentous superstructure of the bacterial actin protein MreB in live Caulobacter crescentus cells. These studies demonstrated that EYFP is a useful emitter for in vivo Super-Resolution Imaging.
Xiaowei Zhuang - One of the best experts on this subject based on the ideXlab platform.
-
isotropic three dimensional super resolution Imaging with a self bending point spread function
Nature Photonics, 2014Co-Authors: Joshua C Vaughan, Xiaowei Zhuang, Shu JiaAbstract:Airy beams maintain their intensity profiles over a large propagation distance without substantial diffraction and exhibit lateral bending during propagation1,2,3,4,5. This unique property has been exploited for the micromanipulation of particles6, the generation of plasma channels7 and the guidance of plasmonic waves8, but has not been explored for high-resolution optical microscopy. Here, we introduce a self-bending point spread function (SB-PSF) based on Airy beams for three-dimensional Super-Resolution fluorescence Imaging. We designed a side-lobe-free SB-PSF and implemented a two-channel detection scheme to enable unambiguous three-dimensional localization of fluorescent molecules. The lack of diffraction and the propagation-dependent lateral bending make the SB-PSF well suited for precise three-dimensional localization of molecules over a large Imaging depth. Using this method, we obtained Super-Resolution Imaging with isotropic three-dimensional localization precision of 10–15 nm over a 3 µm Imaging depth from ∼2,000 photons per localization. By exploiting a self-bending point spread function based on Airy beams, a three-dimensional Super-Resolution fluorescence Imaging is realized. A three-dimensional localization precision in the range 10–15 nm was obtained at an Imaging depth of 3 µm from ∼2,000 photons per localization.
-
Fast, three-dimensional Super-Resolution Imaging of live cells
Nature methods, 2011Co-Authors: Sara A. Jones, Sang-hee Shim, Xiaowei ZhuangAbstract:We report Super-Resolution fluorescence Imaging of live cells with high spatiotemporal resolution using stochastic optical reconstruction microscopy (STORM). By labeling proteins either directly or via SNAP tags with photoswitchable dyes, we obtained two-dimensional (2D) and 3D Super-Resolution images of living cells, using clathrin-coated pits and the transferrin cargo as model systems. Bright, fast-switching probes enabled us to achieve 2D Imaging at spatial resolutions of ∼25 nm and temporal resolutions as fast as 0.5 s. We also demonstrated live-cell 3D Super-Resolution Imaging. We obtained 3D spatial resolution of ∼30 nm in the lateral direction and ∼50 nm in the axial direction at time resolutions as fast as 1-2 s with several independent snapshots. Using photoswitchable dyes with distinct emission wavelengths, we also demonstrated two-color 3D Super-Resolution Imaging in live cells. These Imaging capabilities open a new window for characterizing cellular structures in living cells at the ultrastructural level.
-
multicolor super resolution Imaging with photo switchable fluorescent probes
Science, 2007Co-Authors: Mark W Bates, Xiaowei Zhuang, Bo Huang, Graham T DempseyAbstract:Recent advances in far-field optical nanoscopy have enabled fluorescence Imaging with a spatial resolution of 20 to 50 nanometers. Multicolor Super-Resolution Imaging, however, remains a challenging task. Here, we introduce a family of photo-switchable fluorescent probes and demonstrate multicolor stochastic optical reconstruction microscopy (STORM). Each probe consists of a photo-switchable "reporter" fluorophore that can be cycled between fluorescent and dark states, and an "activator" that facilitates photo-activation of the reporter. Combinatorial pairing of reporters and activators allows the creation of probes with many distinct colors. Iterative, color-specific activation of sparse subsets of these probes allows their localization with nanometer accuracy, enabling the construction of a Super-Resolution STORM image. Using this approach, we demonstrate multicolor Imaging of DNA model samples and mammalian cells with 20- to 30-nanometer resolution. This technique will facilitate direct visualization of molecular interactions at the nanometer scale.
Hiroshi Sasaki - One of the best experts on this subject based on the ideXlab platform.
-
multiplexed 3d super resolution Imaging of whole cells using spinning disk confocal microscopy and dna paint
Nature Communications, 2017Co-Authors: Florian Schueder, Juanita Laragutierrez, Brian J Beliveau, Sinem K Saka, Hiroshi SasakiAbstract:Single-molecule localization microscopy (SMLM) can visualize biological targets on the nanoscale, but complex hardware is required to perform SMLM in thick samples. Here, we combine 3D DNA points accumulation for Imaging in nanoscale topography (DNA-PAINT) with spinning disk confocal (SDC) hardware to overcome this limitation. We assay our achievable resolution with two- and three-dimensional DNA origami structures and demonstrate the general applicability by Imaging a large variety of cellular targets including proteins, DNA and RNA deep in cells. We achieve multiplexed 3D Super-Resolution Imaging at sample depths up to ~10 µm with up to 20 nm planar and 80 nm axial resolution, now enabling DNA-based Super-Resolution microscopy in whole cells using standard instrumentation.
Alberto Diaspro - One of the best experts on this subject based on the ideXlab platform.
-
live cell 3d super resolution Imaging in thick biological samples
Nature Methods, 2011Co-Authors: Francesca Cella Zanacchi, Zeno Lavagnino, Michela Perrone Donnorso, Alessio Del Bue, Laura Furia, Mario Faretta, Alberto DiasproAbstract:We demonstrate three-dimensional (3D) Super-Resolution live-cell Imaging through thick specimens (50-150 μm), by coupling far-field individual molecule localization with selective plane illumination microscopy (SPIM). The improved signal-to-noise ratio of selective plane illumination allows nanometric localization of single molecules in thick scattering specimens without activating or exciting molecules outside the focal plane. We report 3D Super-Resolution Imaging of cellular spheroids.
-
Live-cell 3D Super-Resolution Imaging in thick biological samples
Nature methods, 2011Co-Authors: Francesca Cella Zanacchi, Zeno Lavagnino, Michela Perrone Donnorso, Alessio Del Bue, Laura Furia, Mario Faretta, Alberto DiasproAbstract:The combination of light-sheet microscopy and localization-based Super-Resolution Imaging allows deep subdiffraction resolution Imaging in thick scattering specimens as demonstrated by three-dimensional Super-Resolution Imaging of proteins in live 150-μm-diameter cell spheroids.
Florian Schueder - One of the best experts on this subject based on the ideXlab platform.
-
multiplexed 3d super resolution Imaging of whole cells using spinning disk confocal microscopy and dna paint
Nature Communications, 2017Co-Authors: Florian Schueder, Juanita Laragutierrez, Brian J Beliveau, Sinem K Saka, Hiroshi SasakiAbstract:Single-molecule localization microscopy (SMLM) can visualize biological targets on the nanoscale, but complex hardware is required to perform SMLM in thick samples. Here, we combine 3D DNA points accumulation for Imaging in nanoscale topography (DNA-PAINT) with spinning disk confocal (SDC) hardware to overcome this limitation. We assay our achievable resolution with two- and three-dimensional DNA origami structures and demonstrate the general applicability by Imaging a large variety of cellular targets including proteins, DNA and RNA deep in cells. We achieve multiplexed 3D Super-Resolution Imaging at sample depths up to ~10 µm with up to 20 nm planar and 80 nm axial resolution, now enabling DNA-based Super-Resolution microscopy in whole cells using standard instrumentation.
