scholarly journals Single-Molecule Light-Sheet Microscopy with Local Nanopipette Delivery

2021 ◽  
Vol 93 (8) ◽  
pp. 4092-4099
Author(s):  
Bing Li ◽  
Aleks Ponjavic ◽  
Wei-Hsin Chen ◽  
Lee Hopkins ◽  
Craig Hughes ◽  
...  
Keyword(s):  
Single Molecule ◽  
Light Sheet ◽  
Light Sheet Microscopy

scholarly journals Single Molecule Imaging in Live Embryos Using Lattice Light-Sheet Microscopy

2018 ◽  
pp. 541-559 ◽  
Author(s):  
Mustafa Mir ◽  
Armando Reimer ◽  
Michael Stadler ◽  
Astou Tangara ◽  
Anders S. Hansen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Light Sheet ◽  
Single Molecule Imaging ◽  
Light Sheet Microscopy

scholarly journals Single-molecule light-sheet microscopy with local nanopipette delivery

2020 ◽  
Author(s):  
B. Li ◽  
A. Ponjavic ◽  
W. H. Chen ◽  
L. Hopkins ◽  
C. Hughes ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Single Molecule ◽  
Single Molecules ◽  
Light Sheet ◽  
Local Delivery ◽  
Live Cells ◽  
Biological Processes ◽  
Light Sheet Microscopy ◽  
Wide Range

AbstractDetection of single molecules in biological systems has rapidly increased in resolution over the past decade. However, delivery of single molecules has remained a challenge. Currently there is no effective method that can both introduce a precise amount of molecules onto or into a single cell at a defined position, and then image the cellular response. Here we have combined light sheet microscopy with local delivery, using a nanopipette, to accurately deliver individual proteins to a defined position. We call this method local delivery selective plane illumination microscopy (ldSPIM). ldSPIM uses a nanopipette and the ionic feedback current at the nanopipette tip to control the position from which molecules are delivered. The number of proteins delivered can be controlled by varying the voltage applied. For single-molecule detection, we implemented single-objective SPIM using a reflective atomic force microscopy cantilever to create a 2µm thin sheet. Using this setup, we demonstrate that ldSPIM can deliver single fluorescently-labeled proteins onto the plasma membrane of HK293 cells or into the cytoplasm. Next, we deposited aggregates of amyloid-β, which causes proteotoxicity relevant to Alzheimer’s disease, onto a single macrophage stably expressing a MyDD88-eGFP fusion construct. Whole-cell imaging in 3D mode enables live detection of MyDD88 accumulation and formation of MyDDosome signaling complexes, as a result of aggregate-induced triggering of toll-like receptor 4. Overall, we demonstrate a novel multifunctional imaging system capable of precise delivery of single proteins to a specific location on the cell surface or inside the cytoplasm and high-speed 3D detection at single-molecule resolution within live cells.Statement of SignificanceThis paper describes and validates a new method to study biological processes based on the controlled local delivery of molecules onto or into the cell, combined with single molecule imaging using light sheet microscopy. we not only demonstrate the instrument’s capability of delivering controlled numbers of molecules to a defined position, down to the level of single molecules, but also its potential in study of the triggering of the innate immune response by protein aggregates, a key process in the development of neurodegenerative diseases such as Alzheimer’s disease. The same approach could be applied to a wide range of other important biological processes allowing them to be followed in live cells in real-time, hence it will be of great interest to the biophysical community.

scholarly journals Dynamic multifactor hubs interact transiently with sites of active transcription in Drosophila embryos

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Mustafa Mir ◽  
Michael R Stadler ◽  
Stephan A Ortiz ◽  
Colleen E Hannon ◽  
Melissa M Harrison ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Single Molecule ◽  
High Speed ◽  
Light Sheet ◽  
Editorial Note ◽  
Regulation Of Transcription ◽  
Light Sheet Microscopy ◽  
4D Imaging ◽  
Active Transcription ◽  
Drosophila Embryos

The regulation of transcription requires the coordination of numerous activities on DNA, yet how transcription factors mediate these activities remains poorly understood. Here, we use lattice light-sheet microscopy to integrate single-molecule and high-speed 4D imaging in developing Drosophila embryos to study the nuclear organization and interactions of the key transcription factors Zelda and Bicoid. In contrast to previous studies suggesting stable, cooperative binding, we show that both factors interact with DNA with surprisingly high off-rates. We find that both factors form dynamic subnuclear hubs, and that Bicoid binding is enriched within Zelda hubs. Remarkably, these hubs are both short lived and interact only transiently with sites of active Bicoid-dependent transcription. Based on our observations, we hypothesize that, beyond simply forming bridges between DNA and the transcription machinery, transcription factors can organize other proteins into hubs that transiently drive multiple activities at their gene targets.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (<xref ref-type="decision-letter" rid="SA1">see decision letter</xref>).

Single-Molecule Super-Resolution Light-Sheet Microscopy

ChemPhysChem ◽  
2014 ◽  
Vol 15 (4) ◽  
pp. 577-586 ◽  
Author(s):  
Ying S. Hu ◽  
Maxwell Zimmerley ◽  
Yu Li ◽  
Robin Watters ◽  
Hu Cang
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Light Sheet ◽  
Light Sheet Microscopy

scholarly journals Fast 3D imaging of giant unilamellar vesicles using reflected light‐sheet microscopy with single molecule sensitivity

2021 ◽  
Author(s):  
Anita Jannasch ◽  
Sven A. Szilagyi ◽  
Moritz Burmeister ◽  
Q. Tyrell Davis ◽  
Gero L. Hermsdorf ◽  
...  
Keyword(s):  
Single Molecule ◽  
3D Imaging ◽  
Light Sheet ◽  
Giant Unilamellar Vesicles ◽  
Unilamellar Vesicles ◽  
Light Sheet Microscopy ◽  
Reflected Light

scholarly journals Fast 3D imaging of giant unilamellar vesicles using reflected light-sheet microscopy with single molecule sensitivity

2020 ◽  
Author(s):  
Sven A. Szilagyi ◽  
Moritz Burmeister ◽  
Q. Tyrell Davis ◽  
Gero L. Hermsdorf ◽  
Suman De ◽  
...  
Keyword(s):  
Single Molecule ◽  
Signal To Noise Ratio ◽  
Numerical Aperture ◽  
Light Sheet ◽  
Living Cells ◽  
High Signal ◽  
Active Optics ◽  
Single Molecule Level ◽  
Light Sheet Microscopy ◽  
Reflected Light

AbstractObservation of highly dynamic processes inside living cells at the single molecule level is key for a quantitative understanding of biological systems. However, imaging of single molecules in living cells usually is limited by the spatial and temporal resolution, photobleaching and the signal-to-background ratio. To overcome these limitations, light-sheet microscopes with thin selective plane illumination have recently been developed. For example, a reflected light-sheet design combines the illumination by a thin light-sheet with a high numerical aperture objective for single-molecule detection. Here, we developed a reflected light-sheet microscope with active optics for fast, high contrast, two-color acquisition of z-stacks. We demonstrate fast volume scanning by imaging a two-color giant unilamellar vesicle (GUV) hemisphere. In addition, the high signal-to-noise ratio enabled the imaging and tracking of single lipids in the cap of a GUV. In the long term, the enhanced reflected scanning light sheet microscope enables fast 3D scanning of artificial membrane systems and cells with single-molecule sensitivity and thereby will provide quantitative and molecular insight into the operation of cells.

scholarly journals Recent Progress in Light Sheet Microscopy for Biological Applications

2018 ◽  
Vol 72 (8) ◽  
pp. 1137-1169 ◽  
Author(s):  
Krishnendu Chatterjee ◽  
Feby Wijaya Pratiwi ◽  
Frances Camille M. Wu ◽  
Peilin Chen ◽  
Bi-Chang Chen
Keyword(s):  
Developmental Biology ◽  
Fluorescence Microscopy ◽  
Single Cell ◽  
Single Molecule ◽  
Super Resolution ◽  
Light Sheet ◽  
High Signal ◽  
Spatiotemporal Resolution ◽  
Light Sheet Microscopy ◽  
Resolution Imaging

The introduction of light sheet fluorescence microscopy (LSFM) has overcome the challenges in conventional optical microscopy. Among the recent breakthroughs in fluorescence microscopy, LSFM had been proven to provide a high three-dimensional spatial resolution, high signal-to-noise ratio, fast imaging acquisition rate, and minuscule levels of phototoxic and photodamage effects. The aforementioned auspicious properties are crucial in the biomedical and clinical research fields, covering a broad range of applications: from the super-resolution imaging of intracellular dynamics in a single cell to the high spatiotemporal resolution imaging of developmental dynamics in an entirely large organism. In this review, we provided a systematic outline of the historical development of LSFM, detailed discussion on the variants and improvements of LSFM, and delineation on the most recent technological advancements of LSFM and its potential applications in single molecule/particle detection, single-molecule super-resolution imaging, imaging intracellular dynamics of a single cell, multicellular imaging: cell–cell and cell–matrix interactions, plant developmental biology, and brain imaging and developmental biology.

scholarly journals Superresolution and Single Molecule Imaging of Transcription by Reflected Light Sheet Microscopy

2012 ◽  
Vol 102 (3) ◽  
pp. 224a
Author(s):  
J. Christof M. Gebhardt ◽  
Rahul Roy ◽  
David Suter ◽  
Ziqing Zhao ◽  
Alec Chapman ◽  
...  
Keyword(s):  
Single Molecule ◽  
Light Sheet ◽  
Single Molecule Imaging ◽  
Light Sheet Microscopy ◽  
Reflected Light

scholarly journals Light Sheet Microscopy for Single Molecule Tracking in Living Tissue

PLoS ONE ◽  
2010 ◽  
Vol 5 (7) ◽  
pp. e11639 ◽  
Author(s):  
Jörg Gerhard Ritter ◽  
Roman Veith ◽  
Andreas Veenendaal ◽  
Jan Peter Siebrasse ◽  
Ulrich Kubitscheck
Keyword(s):  
Single Molecule ◽  
Light Sheet ◽  
Living Tissue ◽  
Single Molecule Tracking ◽  
Light Sheet Microscopy
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