scholarly journals Hydrogel encapsulation of living organisms for long-term microscopy

2017 ◽  
Author(s):  
Kyra Burnett ◽  
Eric Edsinger ◽  
Dirk R. Albrecht
Keyword(s):  
Light Sheet ◽  
C Elegans ◽  
Light Sheet Microscopy ◽  
Confined Spaces ◽  
Single Cell Imaging ◽  
Volumetric Images ◽  
Living Organisms ◽  
Resolution Single ◽  
Hydrogel Encapsulation

AbstractImaging living organisms at high spatial resolution requires effective and innocuous immobilization. Long-term imaging, across development or behavioral states, places further demands on sample mounting with minimal perturbation of the organism. Here we present a simple and inexpensive method for rapid encapsulation of small animals of any developmental stage within a photocrosslinked polyethylene glycol (PEG) hydrogel, gently restricting movement within their confined spaces. Immobilized animals maintained a normal, uncompressed morphology in a hydrated environment and could be exposed to different aqueous chemicals. We focus in particular on the nematode C. elegans, an organism that is typically viewed with paralyzing reagents, nanobeads, adhesives, or microfluidic traps. The hydrogel is optically clear, non-autofluorescent, and nearly index-matched with water for use with light-sheet microscopy. We captured volumetric images of optogenetically-stimulated responses in multiple sensory neurons over 14 hours using a diSPIM light-sheet microscope, and immobilized worms were recoverable and viable after 24 hours encapsulation. We further imaged living pygmy squid hatchlings to demonstrate size scalability, characterized immobilization quality for various crosslinking parameters and identified paralytic-free conditions suitable for high-resolution single cell imaging. PEG hydrogel encapsulation enables continuous observation for hours of small living organisms, from yeast to zebrafish, and is compatible with multiple microscope mounting geometries.

Adaptive light-sheet microscopy for long-term, high-resolution imaging in living organisms

2016 ◽  
Vol 34 (12) ◽  
pp. 1267-1278 ◽  
Author(s):  
Loïc A Royer ◽  
William C Lemon ◽  
Raghav K Chhetri ◽  
Yinan Wan ◽  
Michael Coleman ◽  
...  
Keyword(s):  
High Resolution ◽  
Light Sheet ◽  
High Resolution Imaging ◽  
Light Sheet Microscopy ◽  
Resolution Imaging ◽  
Living Organisms

scholarly journals Soft-wired long-term memory in a natural recurrent neuronal network

2020 ◽  
Author(s):  
Miguel A. Casal ◽  
Santiago Galella ◽  
Oscar Vilarroya ◽  
Jordi Garcia-Ojalvo
Keyword(s):  
Neuronal Network ◽  
Neuronal Networks ◽  
Initial Conditions ◽  
Permutation Entropy ◽  
Long Term Memory ◽  
Term Memory ◽  
C Elegans ◽  
Living Organisms ◽  
Recurrent Connections

Neuronal networks provide living organisms with the ability to process information. They are also characterized by abundant recurrent connections, which give rise to strong feed-back that dictates their dynamics and endows them with fading (short-term) memory. The role of recurrence in long-term memory, on the other hand, is still unclear. Here we use the neuronal network of the roundworm C. elegans to show that recurrent architectures in living organisms can exhibit long-term memory without relying on specific hard-wired modules. A genetic algorithm reveals that the experimentally observed dynamics of the worm’s neuronal network exhibits maximal complexity (as measured by permutation entropy). In that complex regime, the response of the system to repeated presentations of a time-varying stimulus reveals a consistent behavior that can be interpreted as soft-wired long-term memory.A common manifestation of our ability to remember the past is the consistence of our responses to repeated presentations of stimuli across time. Complex chaotic dynamics is known to produce such reliable responses in spite of its characteristic sensitive dependence on initial conditions. In neuronal networks, complex behavior is known to result from a combination of (i) recurrent connections and (ii) a balance between excitation and inhibition. Here we show that those features concur in the neuronal network of a living organism, namely C. elegans. This enables long-term memory to arise in an on-line manner, without having to be hard-wired in the brain.

scholarly journals Systematic analysis of C. elegans embryo behavior reveals a stereotyped developmental progression and a rhythmic pattern of behavioral quiescence

2021 ◽  
Author(s):  
Evan L Ardiel ◽  
Andrew Lauziere ◽  
Stephen Xu ◽  
Brandon J Harvey ◽  
Ryan Christensen ◽  
...  
Keyword(s):  
Late Stage ◽  
Rhythmic Activity ◽  
Light Sheet ◽  
Rhythmic Pattern ◽  
Directed Motion ◽  
Systematic Analysis ◽  
C Elegans ◽  
Light Sheet Microscopy ◽  
Developmental Progression ◽  
Inactive Phase

Systematic analysis of rich behavioral recordings is being used to uncover how circuits encode complex behaviors. Here we apply the approach to embryos. What are the first embryonic behaviors and how do they evolve as early neurodevelopment ensues? To address these questions, we present a systematic description of behavioral maturation for Caenorhabditis elegans embryos. Posture libraries were derived from a genetically encoded motion capture suit imaged with light-sheet microscopy and annotated using custom semi-automated tracking software (Multiple Hypothesis Hypergraph Tracking; MHHT). Analysis of cell trajectories, postures, and behavioral motifs revealed a stereotyped developmental progression. Early movement is dominated by flipping between dorsal and ventral coiling, which gradually slows into a period of reduced motility. Late-stage embryos exhibit sinusoidal waves of dorsoventral bends, prolonged bouts of directed motion, and a rhythmic pattern of pausing, which we designate slow wave twitch (SWT). Synaptic transmission is required for late-stage motion but not for early flipping or the intervening inactive phase. A high-throughput behavioral assay and calcium imaging revealed that SWT is elicited by the rhythmic activity of a quiescence-promoting neuron (RIS). Similar periodic quiescent states are seen prenatally in divergent animals and may play an important role in promoting normal developmental outcomes.

scholarly journals Scalable image processing techniques for quantitative analysis of volumetric biological images from light-sheet microscopy

2019 ◽  
Author(s):  
Justin Swaney ◽  
Lee Kamentsky ◽  
Nicholas B Evans ◽  
Katherine Xie ◽  
Young-Gyun Park ◽  
...  
Keyword(s):  
Image Processing ◽  
Quantitative Analysis ◽  
Parallel Processing ◽  
Light Sheet ◽  
Reference Image ◽  
Initial Alignment ◽  
Biological Images ◽  
Light Sheet Microscopy ◽  
Volumetric Images ◽  
Dataset Size

AbstractHere we describe an image processing pipeline for quantitative analysis of terabyte-scale volumetric images of SHIELD-processed mouse brains imaged with light-sheet microscopy. The pipeline utilizes open-source packages for destriping, stitching, and atlas alignment that are optimized for parallel processing. The destriping step removes stripe artifacts, corrects uneven illumination, and offers over 100x speed improvements compared to previously reported algorithms. The stitching module builds upon Terastitcher to create a single volumetric image quickly from individual image stacks with parallel processing enabled by default. The atlas alignment module provides an interactive web-based interface that automatically calculates an initial alignment to a reference image which can be manually refined. The atlas alignment module also provides summary statistics of fluorescence for each brain region as well as region segmentations for visualization. The expected runtime of our pipeline on a whole mouse brain hemisphere is 1-2 d depending on the available computational resources and the dataset size.

scholarly journals Regulation of RNA granule dynamics by phosphorylation of serine-rich, intrinsically disordered proteins in C. elegans

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Jennifer T Wang ◽  
Jarrett Smith ◽  
Bi-Chang Chen ◽  
Helen Schmidt ◽  
Dominique Rasoloson ◽  
...  
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Light Sheet ◽  
Disordered Proteins ◽  
Liquid Droplets ◽  
P Granules ◽  
C Elegans ◽  
Rna Granules ◽  
Intrinsically Disordered ◽  
Light Sheet Microscopy

RNA granules have been likened to liquid droplets whose dynamics depend on the controlled dissolution and condensation of internal components. The molecules and reactions that drive these dynamics in vivo are not well understood. In this study, we present evidence that a group of intrinsically disordered, serine-rich proteins regulate the dynamics of P granules in C. elegans embryos. The MEG (maternal-effect germline defective) proteins are germ plasm components that are required redundantly for fertility. We demonstrate that MEG-1 and MEG-3 are substrates of the kinase MBK-2/DYRK and the phosphatase PP2APPTR−½. Phosphorylation of the MEGs promotes granule disassembly and dephosphorylation promotes granule assembly. Using lattice light sheet microscopy on live embryos, we show that GFP-tagged MEG-3 localizes to a dynamic domain that surrounds and penetrates each granule. We conclude that, despite their liquid-like behavior, P granules are non-homogeneous structures whose assembly in embryos is regulated by phosphorylation.

scholarly journals Lineage recording in human cerebral organoids

2021 ◽  
Author(s):  
Zhisong He ◽  
Ashley Maynard ◽  
Akanksha Jain ◽  
Tobias Gerber ◽  
Rebecca Petri ◽  
...  
Keyword(s):  
Single Cell ◽  
Time Window ◽  
Induced Pluripotent Stem Cell ◽  
Light Sheet ◽  
Human Organ ◽  
Light Sheet Microscopy ◽  
Induced Pluripotent ◽  
Fate Restriction ◽  
Gene Perturbation

AbstractInduced pluripotent stem cell (iPSC)-derived organoids provide models to study human organ development. Single-cell transcriptomics enable highly resolved descriptions of cell states within these systems; however, approaches are needed to directly measure lineage relationships. Here we establish iTracer, a lineage recorder that combines reporter barcodes with inducible CRISPR–Cas9 scarring and is compatible with single-cell and spatial transcriptomics. We apply iTracer to explore clonality and lineage dynamics during cerebral organoid development and identify a time window of fate restriction as well as variation in neurogenic dynamics between progenitor neuron families. We also establish long-term four-dimensional light-sheet microscopy for spatial lineage recording in cerebral organoids and confirm regional clonality in the developing neuroepithelium. We incorporate gene perturbation (iTracer-perturb) and assess the effect of mosaic TSC2 mutations on cerebral organoid development. Our data shed light on how lineages and fates are established during cerebral organoid formation. More broadly, our techniques can be adapted in any iPSC-derived culture system to dissect lineage alterations during normal or perturbed development.

scholarly journals Isoflurane Exposure in Juvenile Caenorhabditis elegans Causes Persistent Changes in Neuron Dynamics

2020 ◽  
Vol 133 (3) ◽  
pp. 569-582 ◽  
Author(s):  
Gregory S. Wirak ◽  
Christopher V. Gabel ◽  
Christopher W. Connor
Keyword(s):  
Caenorhabditis Elegans ◽  
Early Adulthood ◽  
Light Sheet ◽  
Loss Of Function ◽  
Behavioral Impairment ◽  
Mtor Inhibition ◽  
Late Adulthood ◽  
C Elegans ◽  
Light Sheet Microscopy ◽  
Activity Dynamics

Background Animal studies demonstrate that anesthetic exposure during neurodevelopment can lead to persistent behavioral impairment. The changes in neuronal function underlying these effects are incompletely understood. Caenorhabditis elegans is well suited for functional imaging of postanesthetic effects on neuronal activity. This study aimed to examine such effects within the neurocircuitry underlying C. elegans locomotion. Methods C. elegans were exposed to 8% isoflurane for 3 h during the neurodevelopmentally critical L1 larval stage. Locomotion was assessed during early and late adulthood. Spontaneous activity was measured within the locomotion command interneuron circuitry using confocal and light-sheet microscopy of the calcium-sensitive fluorophore GCaMP6s. Results C. elegans exposed to isoflurane demonstrated attenuation in spontaneous reversal behavior, persisting throughout the animal’s lifespan (reversals/min: untreated early adulthood, 1.14 ± 0.42, vs. isoflurane-exposed early adulthood, 0.83 ± 0.55; untreated late adulthood, 1.75 ± 0.64, vs. isoflurane-exposed late adulthood, 1.14 ± 0.68; P = 0.001 and 0.006, respectively; n > 50 animal tracks/condition). Likewise, isoflurane exposure altered activity dynamics in the command interneuron AVA, which mediates crawling reversals. The rate at which AVA transitions between activity states was found to be increased. These anesthetic-induced effects were more pronounced with age (off-to-on activity state transition time (s): untreated early adulthood, 2.5 ± 1.2, vs. isoflurane-exposed early adulthood, 1.9 ± 1.3; untreated late adulthood, 4.6 ± 3.0, vs. isoflurane-exposed late adulthood, 3.0 ± 2.4; P = 0.028 and 0.008, respectively; n > 35 traces acquired from more than 15 animals/condition). Comparable effects were observed throughout the command interneuron circuitry, indicating that isoflurane exposure alters transition rates between behavioral crawling states of the system overall. These effects were modulated by loss-of-function mutations within the FoxO transcription factor daf-16 and by rapamycin-mediated mechanistic Target of Rapamycin (mTOR) inhibition. Conclusions Altered locomotive behavior and activity dynamics indicate a persistent effect on interneuron dynamics and circuit function in C. elegansafter developmental exposure to isoflurane. These effects are modulated by a loss of daf-16 or mTOR activity, consistent with a pathologic activation of stress-response pathways. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

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