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 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 Single-Molecule Imaging in Living Drosophila Embryos with Reflected Light-Sheet Microscopy

2016 ◽  
Vol 110 (4) ◽  
pp. 939-946 ◽  
Author(s):  
Ferdinand Greiss ◽  
Myrto Deligiannaki ◽  
Christophe Jung ◽  
Ulrike Gaul ◽  
Dieter Braun
Keyword(s):  
Single Molecule ◽  
Light Sheet ◽  
Single Molecule Imaging ◽  
Light Sheet Microscopy ◽  
Reflected Light ◽  
Drosophila Embryos

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

Rapid high resolution 3D imaging of expanded biological specimens with lattice light sheet microscopy

Methods ◽  
2020 ◽  
Vol 174 ◽  
pp. 11-19 ◽  
Author(s):  
Yun-Chi Tsai ◽  
Wei-Chun Tang ◽  
Christine Siok Lan Low ◽  
Yen-Ting Liu ◽  
Jyun-Sian Wu ◽  
...  
Keyword(s):  
High Resolution ◽  
3D Imaging ◽  
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

3D imaging of cleared human skin biopsies using light-sheet microscopy: A new way to visualize in-depth skin structure

2018 ◽  
Vol 24 (2) ◽  
pp. 294-303 ◽  
Author(s):  
S. Abadie ◽  
C. Jardet ◽  
J. Colombelli ◽  
B. Chaput ◽  
A. David ◽  
...  
Keyword(s):  
Human Skin ◽  
3D Imaging ◽  
Light Sheet ◽  
Skin Structure ◽  
Light Sheet Microscopy ◽  
Skin Biopsies

scholarly journals 3D Imaging of BABB Cleared Whole-Mount Organs Using Light Sheet Microscopy

2018 ◽  
Vol 24 (S1) ◽  
pp. 1390-1391
Author(s):  
Kingsley A. Boateng ◽  
Austin Cyphersmith ◽  
Glenn A. Fried ◽  
Barghav S. Sivaguru ◽  
Xiaochen Lu ◽  
...  
Keyword(s):  
3D Imaging ◽  
Light Sheet ◽  
Light Sheet Microscopy ◽  
Whole Mount

scholarly journals Label-free and Multimodal Second Harmonic Generation Light Sheet Microscopy

2020 ◽  
Author(s):  
Niall Hanrahan ◽  
Simon I. R. Lane ◽  
Peter Johnson ◽  
Konstantinos Bourdakos ◽  
Christopher Brereton ◽  
...  
Keyword(s):  
Second Harmonic Generation ◽  
Harmonic Generation ◽  
Biological Samples ◽  
3D Imaging ◽  
Second Harmonic ◽  
Three Dimensional ◽  
Light Sheet ◽  
Optical Methods ◽  
Label Free ◽  
Light Sheet Microscopy

AbstractLight sheet microscopy (LSM) has emerged as one of most profound three dimensional (3D) imaging tools in the life sciences over the last decade. However, LSM is currently performed with fluorescence detection on one- or multi-photon excitation. Label-free LSM imaging approaches have been rather limited. Second Harmonic Generation (SHG) imaging is a label-free technique that has enabled detailed investigation of collagenous structures, including its distribution and remodelling in cancers and respiratory tissue, and how these link to disease. SHG is generally regarded as having only forward- and back-scattering components, apparently precluding the orthogonal detection geometry used in Light Sheet Microscopy. In this work we demonstrate SHG imaging on a light sheet microscope (SHG-LSM) using a rotated Airy beam configuration that demonstrates a powerful new approach to direct, without any further processing or deconvolution, 3D imaging of harmonophores such as collagen in biological samples. We provide unambiguous identification of SHG signals on the LSM through its wavelength and polarisation sensitivity. In a multimodal LSM setup we demonstrate that SHG and two-photon signals can be acquired on multiple types of different biological samples. We further show that SHG-LSM is sensitive to changes in collagen synthesis within lung fibroblast 3D cell cultures. This work expands on the existing optical methods available for use with light sheet microscopy, adding a further label-free imaging technique which can be combined with other detection modalities to realise a powerful multi-modal microscope for 3D bioimaging.

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.

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