scholarly journals Cell geometry distinguishes migration-associated heterogeneity in two-dimensional systems

2021 ◽  
Author(s):  
Sagar S Varankar ◽  
Kishore Hari ◽  
Sharmila A Bapat ◽  
Mohit Kumar Jolly
Keyword(s):  
Statistical Analysis ◽  
Cell Biology ◽  
Time Lapse ◽  
Two Dimensional ◽  
Label Free ◽  
Functional Heterogeneity ◽  
Cell Geometry ◽  
Migration Assay ◽  
Migratory Status

Background: In vitro migration assays are a cornerstone of cell biology and have found extensive utility in research. Over the past decade, several variations of the two-dimensional (2D) migration assay have improved our understanding of this fundamental process. However, the ability of these approaches to capture the functional heterogeneity during migration and their accessibility to inexperienced users has been limited. Methods: We downloaded published time-lapse 2D cell migration datasets and subjected them to feature extraction with the Fiji software. We used the 'Analyze Particles' tool to extract ten cell geometry features (CGFs), which were grouped into 'shape, 'size and 'position' descriptors. Next, we defined the migratory status of cells using the 'MTrack2' plugin. All data obtained from Fiji were further subjected to rigorous statistical analysis with R version 4.0.2. Results: We observed consistent associative trends between size and shape descriptors and validated the robustness of our observations across four independent datasets. We used these descriptors to resolve the functional heterogeneity during migration by identifying and characterizing 'non-migrators (NM)' and 'migrators (M)'. Statistical analysis allowed us to identify considerable heterogeneity in the NM subset, that has not been previously reported. Interestingly, differences in 2D-packing appeared to affect CGF trends and heterogeneity of the migratory subsets for the datasets under investigation. Conclusion: We developed an analytical pipeline using open source tools, to identify and morphologically characterize functional migratory subsets from label-free, time-lapse migration data. Our quantitative approach identified a previously unappreciated heterogeneity of non-migratory cells and predicted the influence of 2D-packing on migration.

scholarly journals Robust Optical Flow Algorithm for General, Label-free Cell Segmentation

2020 ◽  
Author(s):  
Michael C. Robitaille ◽  
Jeff M. Byers ◽  
Joseph A. Christodoulides ◽  
Marc P. Raphael
Keyword(s):  
Optical Flow ◽  
Cell Biology ◽  
Cell Movement ◽  
Time Lapse ◽  
Cell Types ◽  
Free Cell ◽  
Cell Segmentation ◽  
Label Free ◽  
Segmentation Approach

ABSTRACTCell segmentation is crucial to the field of cell biology, as the accurate extraction of cell morphology, migration, and ultimately behavior from time-lapse live cell imagery are of paramount importance to elucidate and understand basic cellular processes. Here, we introduce a novel segmentation approach centered around optical flow and show that it achieves robust segmentation by validating it on multiple cell types, phenotypes, optical modalities, and in-vitro environments without the need of labels. By leveraging cell movement in time-lapse imagery as a means to distinguish cells from their background and augmenting the output with machine vision operations, our algorithm reduces the number of adjustable parameters needed for optimization to two. The code is packaged within a MATLAB executable file, offering an accessible means for general cell segmentation typically unavailable in most cell biology laboratories.

scholarly journals Detection of Cell Aggregation and Altered Cell Viability by Automated Label-Free Video Microscopy

2014 ◽  
Vol 20 (3) ◽  
pp. 372-381 ◽  
Author(s):  
Obaid Aftab ◽  
Mårten Fryknäs ◽  
Ulf Hammerling ◽  
Rolf Larsson ◽  
Mats G. Gustafsson
Keyword(s):  
Cell Viability ◽  
Phase Contrast ◽  
Growth Factor Receptor ◽  
Cell Aggregation ◽  
Well Being ◽  
Time Lapse ◽  
Video Microscopy ◽  
Label Free ◽  
Monitoring Method

Automated phase-contrast video microscopy now makes it feasible to monitor a high-throughput (HT) screening experiment in a 384-well microtiter plate format by collecting one time-lapse video per well. Being a very cost-effective and label-free monitoring method, its potential as an alternative to cell viability assays was evaluated. Three simple morphology feature extraction and comparison algorithms were developed and implemented for analysis of differentially time-evolving morphologies (DTEMs) monitored in phase-contrast microscopy videos. The most promising layout, pixel histogram hierarchy comparison (PHHC), was able to detect several compounds that did not induce any significant change in cell viability, but made the cell population appear as spheroidal cell aggregates. According to recent reports, all these compounds seem to be involved in inhibition of platelet-derived growth factor receptor (PDGFR) signaling. Thus, automated quantification of DTEM (AQDTEM) holds strong promise as an alternative or complement to viability assays in HT in vitro screening of chemical compounds.

Discrimination of normal and malignant mouse ovarian surface epithelial cells in vitro using Raman microspectroscopy

2015 ◽  
Vol 7 (22) ◽  
pp. 9520-9528 ◽  
Author(s):  
S. Borel ◽  
E. A. Prikryl ◽  
N. H. Vuong ◽  
J. Jonkman ◽  
B. Vanderhyden ◽  
...  
Keyword(s):  
Statistical Analysis ◽  
Multivariate Statistical Analysis ◽  
Raman Microspectroscopy ◽  
Live Cells ◽  
Label Free ◽  
Multivariate Statistical ◽  
Ovarian Surface Epithelial ◽  
Free Classification

Raman microspectroscopy in conjunction with multivariate statistical analysis is a powerful technique for label-free classification of live cells based on their molecular composition, which can be correlated to variations in protein, DNA/RNA, and lipid macromolecules.

scholarly journals Imaging cell biology in live animals: Ready for prime time

2013 ◽  
Vol 201 (7) ◽  
pp. 969-979 ◽  
Author(s):  
Roberto Weigert ◽  
Natalie Porat-Shliom ◽  
Panomwat Amornphimoltham
Keyword(s):  
Cell Biology ◽  
Cancer Biology ◽  
In Vitro Model ◽  
Time Lapse ◽  
Living Cells ◽  
Model Systems ◽  
Prime Time ◽  
Vitro Model

Time-lapse fluorescence microscopy is one of the main tools used to image subcellular structures in living cells. Yet for decades it has been applied primarily to in vitro model systems. Thanks to the most recent advancements in intravital microscopy, this approach has finally been extended to live rodents. This represents a major breakthrough that will provide unprecedented new opportunities to study mammalian cell biology in vivo and has already provided new insight in the fields of neurobiology, immunology, and cancer biology.

scholarly journals Organoids: the third dimension of human brain development and disease

2021 ◽  
Author(s):  
Georgia Kouroupi ◽  
Kanella Prodromidou ◽  
Florentia Papastefanaki ◽  
Era Taoufik ◽  
Rebecca Matsas
Keyword(s):  
Stem Cells ◽  
Human Brain ◽  
Brain Development ◽  
Cell Biology ◽  
Embryonic Stem ◽  
Two Dimensional ◽  
The Third ◽  
Human Brain Development ◽  
Third Dimension

Stem cell technologies have opened up new avenues in the study of human biology and disease. Especially, the advent of human embryonic stem cells followed by reprograming technologies for generation of induced pluripotent stem cells have instigated studies for modeling human brain development and disease by providing a means to simulate a human tissue with otherwise limited or no accessibility to researchers. Brain development is a complex process achieved in a remarkably controlled spatial and temporal manner through coordinated cellular and molecular events. In vitro models aim to mimic these processes and recapitulate brain organogenesis. Initially, two-dimensional neural cultures presented an innovative landmark for investigating human neuronal and, more recently, glial biology as well as for modeling brain neurodevelopmental and neurodegenerative diseases. The establishment of three-dimensional cultures in the form of brain organoids was an equally important milestone in the field. Brain organoids mimic more closely the in vivo tissue composition and architecture and are more physiologically relevant than monolayer cultures. They therefore represent a more realistic cellular environment for modeling the cell biology and pathology of the nervous system. Here we highlight the journey to recapitulate human brain development and disease in-a-dish, starting from two-dimensional in vitro systems up to the third dimension provided by brain organoids. We discuss the potential of these approaches for modeling human brain development and evolution and their promise for understanding and treating brain disease.

scholarly journals Design of Biologically Active Binary Protein 2D Materials

2020 ◽  
Author(s):  
Ariel J. Ben-Sasson ◽  
Joseph Watson ◽  
William Sheffler ◽  
Matthew Camp Johnson ◽  
Alice Bittleston ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Biology ◽  
Computational Design ◽  
Building Blocks ◽  
Computational Method ◽  
Biologically Active ◽  
Cell Surface Receptor ◽  
Two Dimensional ◽  
Synthetic Cell

AbstractProteins that assemble into ordered two-dimensional arrays such as S-layers1,2 and designed analogues3–5 have intrigued bioengineers,6,7 but with the exception of a single lattice formed through non-rigid template streptavidin linkers,8 they are constituted from just one protein component. For modulating assembly dynamics and incorporating more complex functionality, materials composed of two components would have considerable advantages.9–12 Here we describe a computational method to generate de-novo binary 2D non-covalent co-assemblies by designing rigid asymmetric interfaces between two distinct protein dihedral building-blocks. The designed array components are soluble at mM concentrations, but when combined at nM concentrations, rapidly assemble into nearly-crystalline micrometer-scale p6m arrays nearly identical to the computational design model in vitro and in cells without the need of a two-dimensional support. Because the material is designed from the ground up, the components can be readily functionalized, and their symmetry reconfigured, enabling formation of ligand arrays with distinguishable surfaces to drive extensive receptor clustering, downstream protein recruitment, and signaling. Using quantitative microscopy we show that arrays assembled on living cells have component stoichiometry and likely structure similar to arrays formed in vitro, suggesting that our material can impose order onto fundamentally disordered substrates like cell membranes. We find further that in sharp contrast to previously characterized cell surface receptor binding assemblies such as antibodies and nanocages, which are rapidly endocytosed, large arrays assembled at the cell surface suppress endocytosis in a tunable manner, with potential therapeutic relevance for extending receptor engagement and immune evasion. Our work paves the way towards synthetic cell biology, where a new generation of multi-protein macroscale materials is designed to modulate cell responses and reshape synthetic and living systems.One Sentence SummaryCo-assembling binary 2D protein crystals enables robust formation of complex large scale ordered biologically active materials

scholarly journals Two-Dimensional and Three-Dimensional Cell Culture and Their Applications

2021 ◽  
Author(s):  
Sangeeta Ballav ◽  
Ankita Jaywant Deshmukh ◽  
Shafina Siddiqui ◽  
Jyotirmoi Aich ◽  
Soumya Basu
Keyword(s):  
Cell Culture ◽  
Cell Biology ◽  
Low Cost ◽  
Three Dimensional ◽  
3D Culture ◽  
Two Dimensional ◽  
Living Body ◽  
Preclinical Trials ◽  
Dimensional Cell

Cell culture is one of the most important and commonly used in vitro tools to comprehend various aspects of cells or tissues of a living body such as cell biology, tissue morphology, mechanism of diseases, cell signaling, drug action, cancer research and also finds its great importance in preclinical trials of various drugs. There are two major types of cell cultures that are most commonly used- two-dimensional (2D) and three-dimensional culture (3D). The former has been used since the 1900s, owing to its simplicity and low-cost maintenance as it forms a monolayer, while the latter being the advanced version and currently most worked upon. This chapter intends to provide the true meaning and significance to both cultures. It starts by making a clear distinction between the two and proceeds further to discuss their different applications in vitro. The significance of 2D culture is projected through different assays and therapeutic treatment to understand cell motility and treatment of diseases, whereas 3D culture includes different models and spheroid structures consisting of multiple layers of cells, and puts a light on its use in drug discovery and development. The chapter is concluded with a detailed account of the production of therapeutic proteins by the use of cells.

Control of cortical interneuron migration by neurotrophins and PI3-kinase signaling

Development ◽  
2002 ◽  
Vol 129 (13) ◽  
pp. 3147-3160 ◽  
Author(s):  
Franck Polleux ◽  
Kristin L. Whitford ◽  
Paul A. Dijkhuizen ◽  
Tania Vitalis ◽  
Anirvan Ghosh
Keyword(s):  
Marginal Zone ◽  
Time Lapse ◽  
Pi3 Kinase ◽  
Cortical Plate ◽  
Kinase Activation ◽  
Migration Assay ◽  
Cortical Interneuron ◽  
Tangential Migration ◽  
Rapid Activation

During telencephalic development, cells from the medial ganglionic eminence (MGE) are thought to migrate to the neocortex to give rise to a majority of cortical GABAergic interneurons. By combining time-lapse video-microscopy, immunofluorescence and pharmacological perturbations in a new in vitro migration assay, we find that MGE-derived cells migrate through the entire extent of the cortex and into the CA fields of the hippocampus, but avoid the dentate gyrus. Migrating neurons initially travel within the marginal zone and intermediate zone, and can enter the cortical plate from either location. Tangential migration is strongly stimulated by BDNF and NT4 and attenuated by the Trk-family inhibitor, K252a, suggesting that migration is regulated by TrkB signaling. Furthermore, TrkB-null mice show a significant decrease in the number of calbindin-positive neurons migrating tangentially in the embryonic cortex. BDNF and NT4 cause rapid activation of PI3-kinase in MGE cells, and inhibition of PI3-kinase (but not of MAP kinase or PLCγ) dramatically attenuates tangential migration. These observations suggest that TrkB signaling, via PI3-kinase activation, plays an important role in controlling interneuron migration in the developing cerebral cortex.

scholarly journals Label-free three-dimensional analyses of live cells with deep-learning-based segmentation exploiting refractive index distributions

2021 ◽  
Author(s):  
Jinho Choi ◽  
Hye-Jin Kim ◽  
Gyuhyeon Sim ◽  
Sumin Lee ◽  
Wei Sun Park ◽  
...  
Keyword(s):  
Cell Biology ◽  
Lipid Droplets ◽  
Three Dimensional ◽  
Time Lapse ◽  
Cell Types ◽  
Live Cells ◽  
Label Free ◽  
Subcellular Organelles ◽  
Long Time ◽  
Three Dimensional Segmentation

Visualisations and analyses of cellular and subcellular organelles in biological cells is crucial for the study of cell biology. However, existing imaging methods require the use of exogenous labelling agents, which prevents the long-time assessments of live cells in their native states. Here we propose and experimentally demonstrate three-dimensional segmentation of subcellular organelles in unlabelled live cells, exploiting a 3D U-Net-based architecture. We present the high-precision three-dimensional segmentation of cell membrane, nucleus membrane, nucleoli, and lipid droplets of various cell types. Time-lapse analyses of dynamics of activated immune cells are also analysed using label-free segmentation.

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