Video-rate volumetric imaging by coded light-sheet array microscopy (Conference Presentation)

2019 ◽  
Author(s):  
Yuxuan Ren ◽  
Jianglai Wu ◽  
Dickson M. D. Siu ◽  
Kenneth K. Y. Wong ◽  
Kevin K. Tsia
Keyword(s):  
Light Sheet ◽  
Volumetric Imaging

scholarly journals 3D visualization of macromolecule synthesis

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Timothy J Duerr ◽  
Ester Comellas ◽  
Eun Kyung Jeon ◽  
Johanna E Farkas ◽  
Marylou Joetzjer ◽  
...  
Keyword(s):  
3D Visualization ◽  
Fluorescent Microscopy ◽  
Three Dimensional ◽  
Light Sheet ◽  
Macromolecular Synthesis ◽  
Volumetric Imaging ◽  
Processing Pipeline ◽  
Powerful Means

Measuring nascent macromolecular synthesis in vivo is key to understanding how cells and tissues progress through development and respond to external cues. Here we perform in vivo injection of alkyne- or azide-modified analogs of thymidine, uridine, methionine, and glucosamine to label nascent synthesis of DNA, RNA, protein, and glycosylation. Three-dimensional volumetric imaging of nascent macromolecule synthesis was performed in axolotl salamander tissue using whole-mount click chemistry-based fluorescent staining followed by light sheet fluorescent microscopy. We also developed an image processing pipeline for segmentation and classification of morphological regions of interest and individual cells, and we apply this pipeline to the regenerating humerus. We demonstrate our approach is sensitive to biological perturbations by measuring changes in DNA synthesis after limb denervation. This method provides a powerful means to quantitatively interrogate macromolecule synthesis in heterogenous tissues at the organ, cellular, and molecular levels of organization.

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 Subvoxel light-sheet microscopy for high-resolution high-throughput volumetric imaging of large biomedical specimens

2019 ◽  
Vol 1 (01) ◽  
pp. 1 ◽  
Author(s):  
Peng Fei ◽  
Jun Nie ◽  
Juhyun Lee ◽  
Yichen Ding ◽  
Shuoran Li ◽  
...  
Keyword(s):  
High Resolution ◽  
High Throughput ◽  
Light Sheet ◽  
Volumetric Imaging ◽  
Light Sheet Microscopy
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