Living and fixed neurons observed in brain slices by laser-scanned confocal light microscopy

1992 ◽  
Vol 50 (1) ◽  
pp. 472-473
Author(s):  
James N. Turner ◽  
Donald H. Szarowski ◽  
Karen L. Smith ◽  
John W Swann
Keyword(s):  
Light Microscopy ◽  
Light Microscope ◽  
Fluorescent Dyes ◽  
Three Dimensional ◽  
Brain Slices ◽  
Data Sets ◽  
Direction Discrimination ◽  
Large Numbers ◽  
Entire Thickness

Thick slices of brain tissue are studied in vitro because neurons deep in the slice maintain physiologic contact with large numbers of other neurons, and are thought to function in a manner similar to that of in tact brain. The three-dimensional (3-D) morphology and electrophysiology of these cells can be studied and correlated. The confocal light microscope with its z-direction discrimination forms optical sections through the entire thickness of the slice, and stereo pairs or full 3-D reconstructions can be displayed using the optical sections as data sets. Individual neurons injected with fluorescent dyes or peroxidase based stains are imaged in either the fluorescent or reflection modes.

scholarly journals Chemically-defined induction of a primitive endoderm and epiblast-like niche supports post-implantation progression from blastoids

2019 ◽  
Author(s):  
Erik J. Vrij ◽  
Yvonne S. Scholte op Reimer ◽  
Javier Frias Aldeguer ◽  
Isabel Misteli Guerreiro ◽  
Jop Kind ◽  
...  
Keyword(s):  
Small Molecules ◽  
Three Dimensional ◽  
Embryoid Bodies ◽  
Primitive Endoderm ◽  
Amniotic Cavity ◽  
Large Numbers ◽  
Body Modifications ◽  
Free Serum ◽  
Post Implantation

AbstractThe early mammalian conceptus (blastocyst) contains two supporting extraembryonic tissues - the trophectoderm and the primitive endoderm (PrE) - that encase and guide the epiblast (Epi) to eventually form the all body. Modifications of the conceptus exposed key genes regulating these tissues co-development. However, the combinations of signalling pathways underlying the interplay of PrE and Epi remains elusive. Stem cell-based models including embryoid bodies and blastoids can be generated in large numbers and subjected to high-content screens. Here, we use combinatorial screens of proteins, GPCR ligands and small molecules to rapidly (72 hours) and efficiently (80%) guide embryoid bodies to form a three-dimensional PrE-/Epiblast-like niche in chemically-defined conditions (gel-free, serum-free). This bipotent niche spontaneously progresses, without growth factors, to form a pro-amniotic cavity surrounded by a polarized Epi covered with parietal and visceral endoderm-like cells. In blastoids, these molecules enhance the ratio and number of Gata6+/Nanog+ cells and promote the survival, expansion and morphogenesis of a post-implantation-like Epi in vitro. Altogether, modelling early development in chemically-defined conditions delineates the pathways sufficient to form a functional PrE/Epiblast niche that fuels post-implantation development.

scholarly journals Light microscope observation of circulating human lymphocytes cultured in vitro

2010 ◽  
Vol 53 (5) ◽  
pp. 1097-1100
Author(s):  
Naila Francis Paulo de Oliveira ◽  
Mary Anne Heidi Dolder ◽  
Selma Candelária Genari
Keyword(s):  
Tissue Culture ◽  
Light Microscopy ◽  
Light Microscope ◽  
Human Lymphocytes ◽  
Good Choice ◽  
Blood Samples ◽  
Microscopy Technique ◽  
Blood Smears ◽  
Percoll Density Gradient

The purpose of this work was to study the isolation and a light microscopy technique for cultured lymphocytes. Blood samples were obtained by venipuncture with an anticoagulant added and centrifuged in a Percoll density gradient to separate the leukocytes. Lymphocytes were placed in 25 cm ³ tissue culture flasks at 37ºC. After culturing, they were fixed and stained with the methods used for blood smears. Results showed that not all fixing solutions and stains were an equally good choice for cultured lymphocytes.

scholarly journals 3DStock: A new kind of three-dimensional model of the building stock of England and Wales, for use in energy analysis

2016 ◽  
Vol 44 (2) ◽  
pp. 227-255 ◽  
Author(s):  
Stephen Evans ◽  
Rob Liddiard ◽  
Philip Steadman
Keyword(s):  
Energy Use ◽  
Three Dimensional ◽  
Data Sets ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
Building Stock ◽  
England And Wales ◽  
Large Numbers ◽  
New Type ◽  
Gas Consumption

This article describes the development of a new three-dimensional model of the British building stock, called ‘3DStock’. The model differs from other 3D urban and stock models, in that it represents explicitly and in detail the spatial relationships between ‘premises’ and ‘buildings’. It also represents the pattern of activities on different floors within buildings. The geometrical/geographical structure of the model is assembled automatically from two existing national data sets. Additional data from other sources including figures for electricity and gas consumption are then attached. Some sample results are given for energy use intensities. The first purpose of the model is in the analysis of energy use in the building stock. With actual energy data for very large numbers of premises, it is possible to take a completely new type of statistical approach, in which consumption can be related to a range of characteristics including activity, built form, construction and materials. Models have been built to date of the London Borough of Camden and the cities of Leicester, Tamworth and Swindon. Work is in progress to extend the modelling to other parts of Britain. Because of the coverage of the data, this will be limited however to England and Wales.

scholarly journals Microfabricated disk technology: rapid scale up in midbrain organoid generation

2021 ◽  
Author(s):  
Nguyen-Vi Mohamed ◽  
Paula Lepine ◽  
Maria Lacalle-Aurioles ◽  
Julien Sirois ◽  
Meghna Mathur ◽  
...  
Keyword(s):  
Scale Up ◽  
Three Dimensional ◽  
Require Time ◽  
Large Numbers ◽  
Midbrain Region ◽  
Brain Organoid ◽  
Induced Pluripotent ◽  
The Brain

By providing a three-dimensional in vitro culture system with key features of the substantia nigra region in the brain, 3D neuronal organoids derived from human induced pluripotent stem cells (iPSCs) provide living neuronal tissue resembling the midbrain region of the brain. However, a major limitation of conventional brain organoid culture is that it is often labor-intensive, requiring highly specialized personnel for moderate throughput. Additionally, the methods published for long-term cultures require time-consuming maintenance to generate brain organoids in large numbers. With the increasing need for human midbrain organoids (hMOs) to better understand and model Parkinson′s disease (PD) in a dish, there is a need to implement new workflows and methods to both generate and maintain hMOs, while minimizing batch to batch variation. In this study, we developed a method with microfabricated disks to scale up the generation of hMOs. This opens up the possibility to generate larger numbers of hMOs, in a manner that minimizes the amount of labor required, while decreasing variability and maintaining the viability of these hMOs over time. Taken together, producing hMOs in this manner opens up the potential for these to be used to further PD studies.

scholarly journals A three-dimensional approach to visualize pairwise morphological variation and its application to fragmentary palaeontological specimens

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e10545
Author(s):  
Matt A. White ◽  
Nicolás E. Campione
Keyword(s):  
Morphological Variation ◽  
Three Dimensional ◽  
Large Data ◽  
Age Difference ◽  
Data Sets ◽  
Human Errors ◽  
Terrestrial Vertebrate ◽  
Three Dimensional Mapping ◽  
Large Numbers ◽  
High Level

Classifying isolated vertebrate bones to a high level of taxonomic precision can be difficult. Many of Australia’s Cretaceous terrestrial vertebrate fossil-bearing deposits, for example, produce large numbers of isolated bones and very few associated or articulated skeletons. Identifying these often fragmentary remains beyond high-level taxonomic ranks, such as Ornithopoda or Theropoda, is difficult and those classified to lower taxonomic levels are often debated. The ever-increasing accessibility to 3D-based comparative techniques has allowed palaeontologists to undertake a variety of shape analyses, such as geometric morphometrics, that although powerful and often ideal, require the recognition of diagnostic landmarks and the generation of sufficiently large data sets to detect clusters and accurately describe major components of morphological variation. As a result, such approaches are often outside the scope of basic palaeontological research that aims to simply identify fragmentary specimens. Herein we present a workflow in which pairwise comparisons between fragmentary fossils and better known exemplars are digitally achieved through three-dimensional mapping of their surface profiles and the iterative closest point (ICP) algorithm. To showcase this methodology, we compared a fragmentary theropod ungual (NMV P186153) from Victoria, Australia, identified as a neovenatorid, with the manual unguals of the megaraptoran Australovenator wintonensis (AODF604). We discovered that NMV P186153 was a near identical match to AODF604 manual ungual II-3, differing only in size, which, given their 10–15Ma age difference, suggests stasis in megaraptoran ungual morphology throughout this interval. Although useful, our approach is not free of subjectivity; care must be taken to eliminate the effects of broken and incomplete surfaces and identify the human errors incurred during scaling, such as through replication. Nevertheless, this approach will help to evaluate and identify fragmentary remains, adding a quantitative perspective to an otherwise qualitative endeavour.

Microtubules in immature oocytes of Xenopus laevis

1985 ◽  
Vol 77 (1) ◽  
pp. 129-141
Author(s):  
S.R. Heidemann ◽  
M.A. Hamborg ◽  
J.E. Balasz ◽  
S. Lindley
Keyword(s):  
Light Microscopy ◽  
Microscopic Observation ◽  
Electron Microscopic Observation ◽  
Microtubule Assembly ◽  
Electron Microscopic ◽  
Immature Oocytes ◽  
Large Numbers ◽  
Brain Microtubules ◽  
Brain Tubulin

Previous work indicated that immature oocytes of Xenopus were incapable of assembling microtubules but that competence was achieved during maturation. We report here that small numbers of microtubules do exist in immature oocytes. Consistent with this finding, ultrastructural observations indicate that brain microtubules injected into immature oocytes persist in large numbers for at least 30 min. We report that the tubulin dimers of mature and immature oocytes are equally capable of assembling with brain tubulin in vitro. We confirmed previous results that injection of taxol into immature oocytes has no effect when assayed by light microscopy. However, ultrastructural observations suggest that some microtubule assembly is stimulated by taxol. We tested for the ability of immature oocytes to elongate microtubules from ‘seeds’ by injecting deciliated pellicles of Tetrahymena. No elongation was observed either by light or electron microscopic observation. We conclude that the immature oocyte is capable of very limited microtubule assembly and that a marked increase in assembly competence occurs during maturation. Our data suggest that the change in assembly competence during maturation is due to the release, activation or synthesis of a stimulatory co-factor.

scholarly journals Evaluation of equine oocyte developmental competence using polarized light microscopy

Reproduction ◽  
2017 ◽  
Vol 153 (6) ◽  
pp. 775-784 ◽  
Author(s):  
A Bertero ◽  
F Ritrovato ◽  
F Evangelista ◽  
V Stabile ◽  
R Fortina ◽  
...  
Keyword(s):  
Light Microscopy ◽  
Zona Pellucida ◽  
Light Microscope ◽  
Polarized Light ◽  
Developmental Competence ◽  
Immature Oocytes ◽  
Oocyte Developmental Competence ◽  
Morphological Evaluation ◽  
Meiotic Competence

The purpose of this study was to observe in vitro-matured equine oocytes with an objective computerized technique that involves the use of a polarized light microscope (PLM) in addition to the subjective morphological evaluation obtained using a classic light microscope (LM). Equine cumulus-oocyte complexes (COCs, n = 922) were subjected to different in vitro maturation times (24, 36 or 45 h), however, only 36-h matured oocytes were analyzed using CLM. The 36-h matured oocytes that reached maturity were parthenogenetically activated to evaluate the quality and meiotic competence. Average maturation percentages per session in groups 1, 2 and 3 (24-, 36- and 45-h matured oocytes respectively) were 29.31 ± 13.85, 47.01 ± 9.90 and 36.62 ± 5.28%, whereas the average percentages of immature oocytes per session were 28.78 ± 20.17, 7.83 ± 5.51 and 22.36 ± 8.39% respectively. The zona pellucida (ZP) birefringent properties were estimated and correlated with activation outcome. ZP thickness and retardance of the inner layer of the zona pellucida (IL-ZP) were significantly increased in immature oocytes compared with mature oocytes (P < 0.001 and P < 0.01 respectively). The comparison between parthenogenetically activated and non-activated oocytes showed a significant increase in the area and thickness of the IL-ZP in parthenogenetically activated oocytes (P < 0.01). These results show that the 36-h in vitro maturation (IVM) protocol allowed equine oocytes to reach maturity, and PLM observation of ZP can be used to distinguish mature and immature oocytes as well as activated and non-activated oocytes.

scholarly journals Three dimensional microelectrodes enable high signal and spatial resolution for neural seizure recordings in brain slices and freely behaving animals

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
P. Wijdenes ◽  
K. Haider ◽  
C. Gavrilovici ◽  
B. Gunning ◽  
M. D. Wolff ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
Signal To Noise Ratio ◽  
Brain Activity ◽  
Three Dimensional ◽  
Brain Slices ◽  
High Signal ◽  
Diagnostic Potential ◽  
Freely Behaving

AbstractNeural recordings made to date through various approaches—both in-vitro or in-vivo—lack high spatial resolution and a high signal-to-noise ratio (SNR) required for detailed understanding of brain function, synaptic plasticity, and dysfunction. These shortcomings in turn deter the ability to further design diagnostic, therapeutic strategies and the fabrication of neuro-modulatory devices with various feedback loop systems. We report here on the simulation and fabrication of fully configurable neural micro-electrodes that can be used for both in vitro and in vivo applications, with three-dimensional semi-insulated structures patterned onto custom, fine-pitch, high density arrays. These microelectrodes were interfaced with isolated brain slices as well as implanted in brains of freely behaving rats to demonstrate their ability to maintain a high SNR. Moreover, the electrodes enabled the detection of epileptiform events and high frequency oscillations in an epilepsy model thus offering a diagnostic potential for neurological disorders such as epilepsy. These microelectrodes provide unique opportunities to study brain activity under normal and various pathological conditions, both in-vivo and in in-vitro, thus furthering the ability to develop drug screening and neuromodulation systems that could accurately record and map the activity of large neural networks over an extended time period.

Scanning Microscopy of Human Blood and Marrow Smears

1968 ◽  
Vol 26 ◽  
pp. 166-167
Author(s):  
L. W. McDonald ◽  
T. L. Hayes
Keyword(s):  
Bone Marrow ◽  
Light Microscopy ◽  
Light Microscope ◽  
Tap Water ◽  
Single Cells ◽  
Three Dimensional ◽  
Staining Procedure ◽  
Microscope Slide ◽  
Cover Glass ◽  
Lymphatic Leukemia

The scanning electron microscope (SEM) permits direct observation of standard light microscope slide and cover slip preparations. Single cells can be located and identified in the two instruments and the higher resolution, three dimensional image of the SEM can be correlated with the classic cytological information contained in the light microscope image.Samples of peripheral blood and bone marrow were obtained from patients with chronic lymphatic leukemia and polycythemia. A drop of either blood or bone marrow from the syringe used for drawing the specimen was placed on alcohol cleaned, 22 mm square cover glasses. A second cover glass was placed over the drop of blood or marrow and the two cover glasses drawn apart in the manner usual for preparing blood and marrow for examination by light microscopy. These cover slips were rapidly air dried and then stained for examination by light microscopy. The staining procedure consisted of flooding the cover glass with Jenner's stain for 5 minutes, adding distilled water to cover the glass for another 5 minutes and then washing in tap water. Following this a Giemsa stain made up freshly from the stock solution was applied to the cover glass for 10 minutes. The cover glass was then washed and air dried. A thin coating of platinum-paladium was applied in a vacuum evaporator to facilitate electrical conduction.

scholarly journals High-Throughput Flow Cytometry Drug Combination Discovery with Novel Synergy Analysis Software, SynScreen

2018 ◽  
Vol 23 (7) ◽  
pp. 751-760
Author(s):  
Dominique R. Perez ◽  
Bruce S. Edwards ◽  
Larry A. Sklar ◽  
Alexandre Chigaev
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
Drug Combination ◽  
Three Dimensional ◽  
Drug Combinations ◽  
Data Sets ◽  
Software Application ◽  
Dose Responses ◽  
Analysis Models

Classical therapeutic regimens are subject to toxicity, low efficacy, and/or the development of drug resistance. Thus, the discovery of synergistic drug combinations would permit treatment with lower, tolerable dosages of each agent and restored sensitivity. We describe the development and use of the SynScreen software application, which allows for visual and mathematical determinations of compound concentrations that produce super-additive effects. This software uses nonlinear regression fits of dose responses to determine synergism by the Bliss independence and Loewe additivity analysis models. We demonstrate the utility of SynScreen with data analysis from in vitro high-throughput flow cytometry (HTFC) combination screens with repurposed drugs and multiplexed synergy analysis of multiple biologic parameters in parallel. The applicability of SynScreen was confirmed by testing open-source data sets used in published drug combination literature. A key benefit of SynScreen for high-throughput drug combination screening is that observed measurements are graphically depicted in comparison with a three-dimensional surface that represents the theoretical responses at which Bliss additivity would occur. These images and summary tables for the calculated drug interactions are automatically exported. This allows for substantial data sets to be visually assessed, expediting the quick identification of efficacious drug combinations and thereby facilitating the design of confirmatory studies and clinical trials.

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