Sample Preparation and Mounting of Drosophila Embryos for Multiview Light Sheet Microscopy

2016 ◽  
pp. 189-202 ◽  
Author(s):  
Christopher Schmied ◽  
Pavel Tomancak
Keyword(s):  
Sample Preparation ◽  
Light Sheet ◽  
Light Sheet Microscopy ◽  
Drosophila Embryos

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>).

Dual-color on-chip light sheet microscopy of drosophila embryos

2020 ◽  
Author(s):  
Petra Paiè ◽  
Roberto Memeo ◽  
Federico Sala ◽  
Michele Castriotta ◽  
Thomas Vaccari ◽  
...  
Keyword(s):  
Light Sheet ◽  
Light Sheet Microscopy ◽  
Dual Color ◽  
On Chip ◽  
Drosophila Embryos

scholarly journals Attractive internuclear force drives the collective behavior of nuclear arrays in Drosophila embryos

2021 ◽  
Vol 17 (11) ◽  
pp. e1009605
Author(s):  
Xiaoxuan Wu ◽  
Kakit Kong ◽  
Wenlei Xiao ◽  
Feng Liu
Keyword(s):  
Force Field ◽  
Collective Behavior ◽  
Collective Motion ◽  
Standing Waves ◽  
Light Sheet ◽  
Developmental Patterns ◽  
Collective Behaviors ◽  
Light Sheet Microscopy ◽  
Anterior Posterior ◽  
Drosophila Embryos

The collective behavior of the nuclear array in Drosophila embryos during nuclear cycle (NC) 11 to NC14 is crucial in controlling cell size, establishing developmental patterns, and coordinating morphogenesis. After live imaging on Drosophila embryos with light sheet microscopy, we extract the nuclear trajectory, speed, and internuclear distance with an automatic nuclear tracing method. We find that the nuclear speed shows a period of standing waves along the anterior-posterior (AP) axis after each metaphase as the nuclei collectively migrate towards the embryo poles and partially move back. And the maximum nuclear speed dampens by 28%-45% in the second half of the standing wave. Moreover, the nuclear density is 22–42% lower in the pole region than the middle of the embryo during the interphase of NC12-NC14. To find mechanical rules controlling the collective motion and packing patterns of the nuclear array, we use a deep neural network (DNN) to learn the underlying force field from data. We apply the learned spatiotemporal attractive force field in the simulations with a particle-based model. And the simulations recapitulate nearly all the observed characteristic collective behaviors of nuclear arrays in Drosophila embryos.

scholarly journals CLARITY and Light-Sheet microscopy sample preparation in application to human cerebral organoids

2022 ◽  
Vol 25 (8) ◽  
pp. 889-895
Author(s):  
T. A. Shnaider ◽  
I. E. Pristyazhnyuk
Keyword(s):  
Sample Preparation ◽  
Three Dimensional ◽  
Immunohistochemical Analysis ◽  
Light Sheet ◽  
Biological Research ◽  
Tissue Preparation ◽  
Universal Method ◽  
Internal Organization ◽  
Light Sheet Microscopy ◽  
Biological Objects

Cerebral organoids are three-dimensional cell-culture systems that represent a unique experimental model reconstructing early events of human neurogenesis in vitro in health and various pathologies. The most commonly used approach to studying the morphological parameters of organoids is immunohistochemical analysis; therefore, the three-dimensional cytoarchitecture of organoids, such as neural networks or asymmetric internal organization, is difficult to reconstruct using routine approaches. Immunohistochemical analysis of biological objects is a universal method in biological research. One of the key stages of this method is the production of cryo- or paraffin serial sections of samples, which is a very laborious and time-consuming process. In addition, slices represent only a tiny part of the object under study; three-dimensional reconstruction from the obtained serial images is an extremely complex process and often requires expensive special programs for image processing. Unfortunately, staining and microscopic examination of samples are difficult due to their low permeability and a high level of autofluorescence. Tissue cleaning technologies combined with Light-Sheet microscopy allows these challenges to be overcome. CLARITY is one of the tissue preparation techniques that makes it possible to obtain opaque biological objects transparent while maintaining the integrity of their internal structures. This method is based on a special sample preparation, during which lipids are removed from cells and replaced with hydrogel compounds such as acrylamide, while proteins and nucleic acids remain intact. CLARITY provides researchers with a unique opportunity to study three-dimensional biological structures while preserving their internal organization, including whole animals or embryos, individual organs and artificially grown organoids, in particular cerebral organoids. This protocol summarizes an optimization of CLARITY conditions for human brain organoids and the preparation of Light-Sheet microscopy samples.

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

2018 ◽  
Author(s):  
Mustafa Mir ◽  
Michael R. Stadler ◽  
Stephan A. Ortiz ◽  
Melissa M. Harrison ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Single Molecule ◽  
High Speed ◽  
Nuclear Organization ◽  
Light Sheet ◽  
Regulation Of Transcription ◽  
Light Sheet Microscopy ◽  
4D Imaging ◽  
Active Transcription ◽  
Drosophila Embryos

AbstractThe regulation of transcription requires the coordination of numerous activities on DNA, yet it remains poorly understood how transcription factors facilitate these multiple functions. 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 patterning 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.

scholarly journals Achromatic terahertz Airy beam generation with dielectric metasurfaces

Nanophotonics ◽  
2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Qingqing Cheng ◽  
Juncheng Wang ◽  
Ling Ma ◽  
Zhixiong Shen ◽  
Jing Zhang ◽  
...  
Keyword(s):  
Biomedical Imaging ◽  
Frequency Dispersion ◽  
Light Sheet ◽  
Photonic Devices ◽  
Self Healing ◽  
Beam Focusing ◽  
Light Sheet Microscopy ◽  
Airy Beam ◽  
Potential Applications ◽  
Airy Beams

AbstractAiry beams exhibit intriguing properties such as nonspreading, self-bending, and self-healing and have attracted considerable recent interest because of their many potential applications in photonics, such as to beam focusing, light-sheet microscopy, and biomedical imaging. However, previous approaches to generate Airy beams using photonic structures have suffered from severe chromatic problems arising from strong frequency dispersion of the scatterers. Here, we design and fabricate a metasurface composed of silicon posts for the frequency range 0.4–0.8 THz in transmission mode, and we experimentally demonstrate achromatic Airy beams exhibiting autofocusing properties. We further show numerically that a generated achromatic Airy-beam-based metalens exhibits self-healing properties that are immune to scattering by particles and that it also possesses a larger depth of focus than a traditional metalens. Our results pave the way to the realization of flat photonic devices for applications to noninvasive biomedical imaging and light-sheet microscopy, and we provide a numerical demonstration of a device protocol.

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