Global morphogenetic flow is accurately predicted by the spatial distribution of myosin motors

During embryogenesis tissue layers undergo morphogenetic flow rearranging and folding into specific shapes. While developmental biology has identified key genes and local cellular processes, global coordination of tissue remodeling at the organ scale remains unclear. Here, we combine in toto light-sheet microscopy of the Drosophila embryo with quantitative analysis and physical modeling to relate cellular flow with the patterns of force generation during the gastrulation process. We find that the complex spatio-temporal flow pattern can be predicted from the measured meso-scale myosin density and anisotropy using a simple, effective viscous model of the tissue, achieving close to 90% accuracy with one time dependent and two constant parameters. Our analysis uncovers the importance of a) spatial modulation of myosin distribution on the scale of the embryo and b) the non-locality of its effect due to mechanical interaction of cells, demonstrating the need for the global perspective in the study of morphogenetic flow.

Download Full-text

Deformation Measurements of Neuronal Excitability Using Incoherent Holography Lattice Light-Sheet Microscopy (IHLLS)

Photonics ◽

10.3390/photonics8090383 ◽

2021 ◽

Vol 8 (9) ◽

pp. 383

Author(s):

Mariana Potcoava ◽

Jonathan Art ◽

Simon Alford ◽

Christopher Mann

Keyword(s):

Cell Membrane ◽

Structural Changes ◽

Neuronal Excitability ◽

Light Sheet ◽

Phase Imaging ◽

Excitable Cells ◽

Full Field ◽

Cellular Processes ◽

Light Sheet Microscopy ◽

Fluorescent Markers

Stimuli to excitable cells and various cellular processes can cause cell surface deformations; for example, when excitable cell membrane potentials are altered during action potentials. However, these cellular changes may be at or below the diffraction limit (in dendrites the structures measured are as small as 1 µm), and imaging by traditional methods is challenging. Using dual lenses incoherent holography lattice light-sheet (IHLLS-2L) detection with holographic phase imaging of selective fluorescent markers, we can extract the full-field cellular morphology or structural changes of the object’s phase in response to external stimulus. This approach will open many new possibilities in imaging neuronal activity and, overall, in light sheet imaging. In this paper, we present IHLLS-2L as a well-suited technique for quantifying cell membrane deformation in neurons without the actuation of a sample stage or detection microscope objective.

Download Full-text

Rapid Whole Cell Imaging Reveals A Calcium-APPL1-Dynein Nexus That Regulates Cohort Trafficking of Stimulated EGF Receptors

10.1101/481796 ◽

2018 ◽

Author(s):

H M York ◽

A Patil ◽

U K Moorthi ◽

A Kaur ◽

A Bhowmik ◽

...

Keyword(s):

Signal Processing ◽

Cell Imaging ◽

Light Sheet ◽

Endosomal Trafficking ◽

Egf Receptors ◽

Whole Cell ◽

Cellular Processes ◽

Light Sheet Microscopy ◽

Signaling Receptors ◽

Cell Surface Signaling

ABSTRACTMulticellular life processes such as proliferation and differentiation depend on cell surface signaling receptors that bind ligands generally referred to as growth factors. Recently, it has emerged that the endosomal system provides rich signal processing capabilities for responses elicited by these factors [1-3]. At the single cell level, endosomal trafficking becomes a critical component of signal processing, as exemplified by the epidermal growth factor (EGF) receptors of the receptor tyrosine kinase family. EGFRs, once activated by EGF, are robustly trafficked to the phosphatase-enriched peri-nuclear region (PNR), where they are dephosphorylated [4-8]. However, the details of the mechanisms regulating the movements of stimulated EGFR in time and space, i.e., towards the PNR, are not known. What endosomal regulators provide specificity to EGFR? Do modifications to the receptor upon stimulation regulate its trafficking? To understand the events leading to EGFR translocation, and especially the early endosomal dynamics that immediately follow EGFR internalization, requires the real-time, long-term, whole-cell imaging of multiple elements. Here, exploiting the advantages of lattice light-sheet microscopy [9], we show that the binding of EGF by its receptor, EGFR, triggers a transient calcium increase that peaks by 30 s, causing the desorption of APPL1 from pre-existing endosomes within one minute, the rebinding of liberated APPL1 to EGFR within three minutes, and the dynein-dependent translocation of APPL1-EGF-bearing endosomes to the PNR within five minutes. The novel, cell spanning, fast acting network that we reveal integrates a cascade of events dedicated to the cohort movement of activated EGFR receptors. Our findings support the intriguing proposal that certain endosomal pathways have shed some of the stochastic strategies of traditional trafficking, and have evolved behaviors whose predictability is better suited to signaling [10, 11]. Work presented here demonstrates that our whole cell imaging approach can be a powerful tool in revealing critical transient interactions in key cellular processes such as receptor trafficking.

Download Full-text

High-Resolution, Large Field-of-View, and Multi-View Single Objective Light-Sheet Microscopy

10.1101/2020.09.22.309229 ◽

2020 ◽

Author(s):

Bin Yang ◽

Alfred Millett-Sikking ◽

Merlin Lange ◽

Ahmet Can Solak ◽

Hirofumi Kobayashi ◽

...

Keyword(s):

High Resolution ◽

Light Sheet ◽

Field Of View ◽

Large Field ◽

Sample Rotation ◽

Light Sheet Microscopy ◽

Spatio Temporal ◽

Large Living ◽

Single Objective ◽

Imaging Speed

Light-sheet microscopy has become the preferred method for long-term imaging of large living samples because of its low photo-invasiveness and good optical sectioning capabilities. Unfortunately, refraction and scattering often pose obstacles to light-sheet propagation and limit imaging depth. This is typically addressed by imaging multiple complementary views to obtain high and uniform image quality throughout the sample. However, multi-view imaging often requires complex multi-objective configurations that complicate sample mounting, or sample rotation that decreases imaging speed. Recent developments in single-objective light-sheet microscopy have shown that it is possible to achieve high spatio-temporal resolution with a single objective for both illumination and detection. Here we describe a single-objective light-sheet microscope that achieves: (i) high-resolution and large field-of-view imaging via a custom remote focusing objective; (ii) simpler design and ergonomics by remote placement of coverslips; (iii) fast volumetric imaging by means of light-sheet stabilised stage scanning – a novel scanning modality that extends the imaging volume without compromising imaging speed nor quality; (iv) multi-view imaging by means of dual orthogonal light-sheet illumination. Finally, we demonstrate the speed, field of view and resolution of our novel instrument by imaging zebrafish tail development.

Download Full-text

u-track 3D: measuring and interrogating intracellular dynamics in three dimensions

10.1101/2020.11.30.404814 ◽

2020 ◽

Cited By ~ 1

Author(s):

Philippe Roudot ◽

Wesley R. Legant ◽

Qiongjing Zou ◽

Kevin M. Dean ◽

Erik S. Welf ◽

...

Keyword(s):

Particle Tracking ◽

Region Of Interest ◽

Confidence Score ◽

Light Sheet ◽

Three Dimensions ◽

Dense Particle ◽

Cellular Processes ◽

Light Sheet Microscopy ◽

Large Sets ◽

Complete Cell

AbstractParticle tracking is a ubiquitous task in the study of dynamic molecular and cellular processes by live microscopy. Light-sheet microscopy has recently opened a path to acquiring complete cell volumes for investigation in 3-dimensions (3D). However, hypothesis formulation and quantitative analysis have remained difficult due to fundamental challenges in the visualization and the verification of large sets of 3D particle trajectories. Here we describe u-track 3D, a software package that addresses these two challenges with three algorithmic innovations. Building on the established framework of globally optimal particle association in space and time implemented in the u-track package and recent advances in gaining association robustness in the case of erratic motion, we first report a complete and versatile pipeline for particle tracking. We then present the concept of dynamic region of interest (dynROI), which allows an experimenter to interact with dynamic 3D processes in 2D views amenable to visual inspection. Third, we present an estimator of trackability, which provides for every trajectory a confidence score, thereby overcoming the challenges of visual validation of trajectories in dense particle fields. With these combined strategies, u-track 3D provides a framework for the unbiased study of molecular processes in complex volumetric sequences.

Download Full-text

Multi-view light-sheet imaging and tracking with the MaMuT software reveals the cell lineage of a direct developing arthropod limb

eLife ◽

10.7554/elife.34410 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 52

Author(s):

Carsten Wolff ◽

Jean-Yves Tinevez ◽

Tobias Pietzsch ◽

Evangelia Stamataki ◽

Benjamin Harich ◽

...

Keyword(s):

Limb Development ◽

Expression Patterns ◽

Cell Lineage ◽

Light Sheet ◽

Limb Bud ◽

Spatial Modulation ◽

Spatiotemporal Resolution ◽

Cell Behaviors ◽

Light Sheet Microscopy ◽

Anterior Posterior

During development, coordinated cell behaviors orchestrate tissue and organ morphogenesis. Detailed descriptions of cell lineages and behaviors provide a powerful framework to elucidate the mechanisms of morphogenesis. To study the cellular basis of limb development, we imaged transgenic fluorescently-labeled embryos from the crustacean Parhyale hawaiensis with multi-view light-sheet microscopy at high spatiotemporal resolution over several days of embryogenesis. The cell lineage of outgrowing thoracic limbs was reconstructed at single-cell resolution with new software called Massive Multi-view Tracker (MaMuT). In silico clonal analyses suggested that the early limb primordium becomes subdivided into anterior-posterior and dorsal-ventral compartments whose boundaries intersect at the distal tip of the growing limb. Limb-bud formation is associated with spatial modulation of cell proliferation, while limb elongation is also driven by preferential orientation of cell divisions along the proximal-distal growth axis. Cellular reconstructions were predictive of the expression patterns of limb development genes including the BMP morphogen Decapentaplegic.

Download Full-text

Two-Photon Scanned Light Sheet Microscopy Reveals Spatio-Temporal Organization of Cells and Proteins in Developing Embryos

Biophysical Journal ◽

10.1016/j.bpj.2011.11.3366 ◽

2012 ◽

Vol 102 (3) ◽

pp. 618a

Author(s):

Thai V. Truong ◽

Scott E. Fraser

Keyword(s):

Light Sheet ◽

Temporal Organization ◽

Two Photon ◽

Light Sheet Microscopy ◽

Spatio Temporal

Download Full-text

E3 ubiquitin ligases

Essays in Biochemistry ◽

10.1042/bse0410015 ◽

2005 ◽

Vol 41 ◽

pp. 15-30 ◽

Cited By ~ 123

Author(s):

Helen C. Ardley ◽

Philip A. Robinson

Keyword(s):

Protein Complexes ◽

Loss Of Function ◽

Direct Role ◽

Cellular Processes ◽

Substrate Protein ◽

Protein Ubiquitination ◽

C Terminus ◽

Full Complement ◽

Spatio Temporal ◽

Eukaryotic Organisms

The selectivity of the ubiquitin–26 S proteasome system (UPS) for a particular substrate protein relies on the interaction between a ubiquitin-conjugating enzyme (E2, of which a cell contains relatively few) and a ubiquitin–protein ligase (E3, of which there are possibly hundreds). Post-translational modifications of the protein substrate, such as phosphorylation or hydroxylation, are often required prior to its selection. In this way, the precise spatio-temporal targeting and degradation of a given substrate can be achieved. The E3s are a large, diverse group of proteins, characterized by one of several defining motifs. These include a HECT (homologous to E6-associated protein C-terminus), RING (really interesting new gene) or U-box (a modified RING motif without the full complement of Zn2+-binding ligands) domain. Whereas HECT E3s have a direct role in catalysis during ubiquitination, RING and U-box E3s facilitate protein ubiquitination. These latter two E3 types act as adaptor-like molecules. They bring an E2 and a substrate into sufficiently close proximity to promote the substrate's ubiquitination. Although many RING-type E3s, such as MDM2 (murine double minute clone 2 oncoprotein) and c-Cbl, can apparently act alone, others are found as components of much larger multi-protein complexes, such as the anaphase-promoting complex. Taken together, these multifaceted properties and interactions enable E3s to provide a powerful, and specific, mechanism for protein clearance within all cells of eukaryotic organisms. The importance of E3s is highlighted by the number of normal cellular processes they regulate, and the number of diseases associated with their loss of function or inappropriate targeting.

Download Full-text

Faculty Opinions recommendation of Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1123365.584346 ◽

2008 ◽

Author(s):

Kees Jalink

Keyword(s):

Embryonic Development ◽

Light Sheet ◽

Early Embryonic Development ◽

Light Sheet Microscopy

Download Full-text

Achromatic terahertz Airy beam generation with dielectric metasurfaces

Nanophotonics ◽

10.1515/nanoph-2020-0536 ◽

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.

Download Full-text