scholarly journals Combining sample expansion and light sheet microscopy for the volumetric imaging of virus-infected cells with super-resolution

2020 ◽  
Vol 11 (9) ◽  
pp. 5032
Author(s):  
Luca Mascheroni ◽  
Katharina M. Scherer ◽  
James D. Manton ◽  
Edward Ward ◽  
Oliver Dibben ◽  
...  
Keyword(s):  
Super Resolution ◽  
Light Sheet ◽  
Volumetric Imaging ◽  
Light Sheet Microscopy ◽  
Infected Cells

scholarly journals Combining sample expansion and light sheet microscopy for the volumetric imaging of virus-infected cells with optical super-resolution

2020 ◽  
Author(s):  
Luca Mascheroni ◽  
Katharina M. Scherer ◽  
James D. Manton ◽  
Edward Ward ◽  
Oliver Dibben ◽  
...  
Keyword(s):  
Super Resolution ◽  
Light Sheet ◽  
Image Resolution ◽  
Preparation Technique ◽  
Volumetric Imaging ◽  
Light Sheet Microscopy ◽  
Sophisticated Equipment ◽  
Host Cell Interactions ◽  
Infected Cells ◽  
Cellular Organelles

AbstractExpansion microscopy is a sample preparation technique that enables the optical imaging of biological specimens at super-resolution owing to their physical magnification, which is achieved through water-absorbing polymers. The technique uses readily available chemicals and does not require sophisticated equipment, thus offering super-resolution to laboratories that are not microscopy-specialised. Here we present a protocol combining sample expansion with light sheet microscopy to generate high-contrast, high-resolution 3D reconstructions of whole virus-infected cells. The results are superior to those achievable with comparable imaging modalities and reveal details of the infection cycle that are not discernible before expansion. An image resolution of approximately 95 nm could be achieved in samples labelled in 3 colours. We clearly resolve the concentration of viral nucleoprotein on the surface of vesicular structures within the cell and their positioning relative to cellular organelles. We provide detailed guidance and a video protocol for the optimal application of the method and demonstrate its potential to study virus-host cell interactions.

Efficient super‐resolution volumetric imaging by radial fluctuation Bayesian analysis light‐sheet microscopy

2020 ◽  
Vol 13 (8) ◽  
Author(s):  
Rong Chen ◽  
Yuxuan Zhao ◽  
Mengna Li ◽  
Yarong Wang ◽  
Luoying Zhang ◽  
...  
Keyword(s):  
Bayesian Analysis ◽  
Super Resolution ◽  
Light Sheet ◽  
Volumetric Imaging ◽  
Light Sheet Microscopy

scholarly journals Circumvention of common labeling artifacts using secondary nanobodies

2019 ◽  
Author(s):  
Shama Sograte-Idrissi ◽  
Thomas Schlichthaerle ◽  
Carlos J. Duque-Afonso ◽  
Mihai Alevra ◽  
Sebastian Strauss ◽  
...  
Keyword(s):  
Super Resolution ◽  
Light Sheet ◽  
Common Procedure ◽  
Tissue Samples ◽  
Localization Accuracy ◽  
Light Sheet Microscopy ◽  
Large Size ◽  
Resolution Imaging ◽  
Single Domain Antibodies ◽  
Secondary Antibodies

AbstractThe most common procedure to reveal the location of specific (sub)cellular elements in biological samples is via immunostaining followed by optical imaging. This is typically performed with target-specific primary antibodies (1.Abs), which are revealed by fluorophore-conjugated secondary antibodies (2.Abs). However, at high resolution this methodology can induce a series of artifacts due to the large size of antibodies, their bivalency, and their polyclonality. Here we use STED and DNA-PAINT super-resolution microscopy or light sheet microscopy on cleared tissue to show how monovalent secondary reagents based on camelid single-domain antibodies (nanobodies; 2.Nbs) attenuate these artifacts. We demonstrate that monovalent 2.Nbs have four additional advantages: 1) they increase localization accuracy with respect to 2.Abs; 2) they allow direct pre-mixing with 1.Abs before staining, reducing experimental time, and enabling the use of multiple 1.Abs from the same species; 3) they penetrate thick tissues efficiently; and 4) they avoid the artificial clustering seen with 2.Abs both in live and in poorly fixed samples. Altogether, this suggests that 2.Nbs are a valuable alternative to 2.Abs, especially when super-resolution imaging or staining of thick tissue samples are involved.

Deep-learning on-chip light-sheet microscopy enabling video-rate volumetric imaging of dynamic biological specimens

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Xiaopeng Chen ◽  
Junyu Ping ◽  
Yixuan Sun ◽  
Chengqiang Yi ◽  
Sijian Liu ◽  
...  
Keyword(s):  
Deep Learning ◽  
Light Scattering ◽  
Light Sheet ◽  
High Contrast ◽  
Spatiotemporal Resolution ◽  
Volumetric Imaging ◽  
Light Sheet Microscopy ◽  
High Spatiotemporal Resolution ◽  
On Chip

Volumetric imaging of dynamic signals in a large, moving, and light-scattering specimen is extremely challenging, owing to the requirement on high spatiotemporal resolution and difficulty in obtaining high-contrast signals. Here...

scholarly journals Diagonally Scanned Light-Sheet Microscopy for Fast Volumetric Imaging of Adherent Cells

2016 ◽  
Vol 110 (6) ◽  
pp. 1456-1465 ◽  
Author(s):  
Kevin M. Dean ◽  
Philippe Roudot ◽  
Carlos R. Reis ◽  
Erik S. Welf ◽  
Marcel Mettlen ◽  
...  
Keyword(s):  
Light Sheet ◽  
Adherent Cells ◽  
Volumetric Imaging ◽  
Light Sheet Microscopy

scholarly journals A hybrid open-top light-sheet microscope for multi-scale imaging of cleared tissues

2021 ◽  
Author(s):  
Adam Glaser ◽  
Kevin Bishop ◽  
Lindsey Barner ◽  
Etsuo Susaki ◽  
Shimpei Kubota ◽  
...  
Keyword(s):  
Hybrid System ◽  
High Throughput ◽  
Light Sheet ◽  
Refractive Indices ◽  
Volumetric Imaging ◽  
Tissue Clearing ◽  
Sample Geometry ◽  
Multi Scale ◽  
Light Sheet Microscopy ◽  
User Friendly

Abstract Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues. However, there is a need for a user-friendly system that can address imaging applications with varied requirements in terms of resolution (mesoscopic to sub-micrometer), sample geometry (size, shape, and number), and compatibility with tissue-clearing protocols and sample holders of various refractive indices. We present a ‘hybrid’ system that combines a novel non-orthogonal dual-objective and conventional (orthogonal) open-top light-sheet architecture for versatile multi-scale volumetric imaging.

scholarly journals 3D Interferometric Lattice Light-Sheet Imaging

2020 ◽  
Author(s):  
Bin Cao ◽  
Guanshi Wang ◽  
Jieru Li ◽  
Alexandros Pertsinidis
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Light Sheet ◽  
Detection Sensitivity ◽  
Live Cells ◽  
Background Suppression ◽  
Axial Resolution ◽  
Volumetric Imaging ◽  
Spatio Temporal ◽  
And Function

Understanding cellular structure and function requires live-cell imaging with high spatio-temporal resolution and high detection sensitivity. Direct visualization of molecular processes using single-molecule/super-resolution techniques has thus been transformative. However, extracting the highest-resolution 4D information possible from weak and dynamic fluorescence signals in live cells remains challenging. For example, some of the highest spatial resolution methods, e.g. interferometric (4Pi) approaches1-6 can be slow, require high peak excitation intensities that accelerate photobleaching or suffer from increased out-of-focus background. Selective-plane illumination (SPIM)7-12 reduces background, but most implementations typically feature modest spatial, especially axial, resolution. Here we develop 3D interferometric lattice light-sheet (3D-iLLS) imaging, a technique that overcomes many of these limitations. 3D-iLLS provides, by virtue of SPIM, low light levels and photobleaching, while providing increased background suppression and significantly improved volumetric imaging/sectioning capabilities through 4Pi interferometry. We demonstrate 3D-iLLS with axial resolution and single-particle localization precision down to <100nm (FWHM) and <10nm (1σ) respectively. 3D-iLLS paves the way for a fuller elucidation of sub-cellular phenomena by enhanced 4D resolution and SNR live imaging.

scholarly journals Combination of photoactivation with lattice light-sheet imaging reveals PA-Rac1 generates untemplated, lamellar ruffles

2020 ◽  
Author(s):  
Finian Leyden ◽  
Sanjeev Uthishtran ◽  
U K Moorthi ◽  
H M York ◽  
A Patil ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Dorsal Surface ◽  
Experimental System ◽  
Light Sheet ◽  
Small Gtpase ◽  
Local Concentration ◽  
Volumetric Imaging ◽  
Light Sheet Microscopy ◽  
Membrane Protrusions ◽  
A Cell

ABSTRACTMembrane protrusions that occur on the dorsal surface of a cell are an excellent experimental system to study actin machinery at work in a living cell. Small GTPase Rac1 controls the membrane protrusions that form and encapsulate extracellular volumes to perform pinocytic or phagocytic functions. Here, capitalizing on rapid volumetric imaging capabilities of lattice light-sheet microscopy (LLSM), we describe optogenetic approaches using photoactivable Rac1 (PA-Rac1) for controlled ruffle generation. We demonstrate that PA-Rac1 activation needs to be continuous, suggesting a threshold local concentration for sustained actin polymerization leading to ruffling. We show that Rac1 activation leads to actin assembly at the dorsal surface of the cell membrane that result in sheet-like protrusion formation without any requirement of a template. Further, this approach can be used to study the complex morpho-dynamics of the protrusions or to investigate specific proteins that may be enriched in the ruffles. Deactivating PA-Rac1 leads to complex contractile processes resulting in formation of macropinosomes. Using multicolour imaging in combination with these approaches, we find that Myo1e specifically is enriched in the ruffles.

scholarly journals A polymer gel index-matched to water enables diverse applications in fluorescence microscopy

2020 ◽  
Author(s):  
Xiaofei Han ◽  
Yijun Su ◽  
Hamilton White ◽  
Kate M. O’Neill ◽  
Nicole Y. Morgan ◽  
...  
Keyword(s):  
Surface Passivation ◽  
Super Resolution ◽  
Light Sheet ◽  
Polymer Gel ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Neural Structure ◽  
Uv Curable ◽  
Isotropic Resolution ◽  
Light Sheet Microscopy

AbstractWe demonstrate diffraction-limited and super-resolution imaging through thick layers (tens-hundreds of microns) of BIO-133, a biocompatible, UV-curable, commercially available polymer with a refractive index (RI) matched to water. We show that cells can be directly grown on BIO-133 substrates without the need for surface passivation and use this capability to perform extended time-lapse volumetric imaging of cellular dynamics 1) at isotropic resolution using dual-view light-sheet microscopy, and 2) at super-resolution using instant structured illumination microscopy. BIO-133 also enables immobilization of 1) Drosophila tissue, allowing us to track membrane puncta in pioneer neurons, and 2) Caenorhabditis elegans, which allows us to image and inspect fine neural structure and to track pan-neuronal calcium activity over hundreds of volumes. Finally, BIO-133 is compatible with other microfluidic materials, enabling optical and chemical perturbation of immobilized samples, as we demonstrate by performing drug and optogenetic stimulation on cells and C. elegans.

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