scholarly journals Fibronectin-Based Nanomechanical Biosensors to Map 3D Strains in Live Cells and Tissues

2020 ◽  
Author(s):  
Daniel J. Shiwarski ◽  
Joshua W. Tashman ◽  
Alkiviadis Tsamis ◽  
Jacqueline M. Bliley ◽  
Malachi A. Blundon ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
Single Cells ◽  
Time Lapse ◽  
Confocal Imaging ◽  
Square Lattice ◽  
Live Cells ◽  
3D Analysis ◽  
Wide Range ◽  
And Function ◽  
Analysis Platform

AbstractMechanical forces are integral to a wide range of cellular processes including migration, differentiation and tissue morphogenesis; however, it has proved challenging to directly measure strain at high spatial resolution and with minimal tissue perturbation. Here, we fabricated, calibrated, and tested a fibronectin (FN)-based nanomechanical biosensor (NMBS) that can be applied to cells and tissues to measure the magnitude, direction, and dynamics of strain from subcellular to tissue length-scales. The NMBS is a fluorescently-labeled, ultrathin square lattice FN mesh with spatial resolution tailored by adjusting the width and spacing of the lattice fibers from 2-100 µm. Time-lapse 3D confocal imaging of the NMBS demonstrated strain tracking in 2D and 3D following mechanical deformation of known materials and was validated with finite element modeling. Imaging and 3D analysis of the NMBS applied to single cells, cell monolayers, and Drosophila ovarioles demonstrated the ability to dynamically track microscopic tensile and compressive strains in various biological applications with minimal tissue perturbation. This fabrication and analysis platform serves as a novel tool for studying cells, tissues, and more complex systems where forces guide structure and function.

scholarly journals Fibronectin-based nanomechanical biosensors to map 3D surface strains in live cells and tissue

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Daniel J. Shiwarski ◽  
Joshua W. Tashman ◽  
Alkiviadis Tsamis ◽  
Jaci M. Bliley ◽  
Malachi A. Blundon ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
Single Cells ◽  
Time Lapse ◽  
Confocal Imaging ◽  
Surface Strain ◽  
Cellular Migration ◽  
Live Cells ◽  
3D Surface ◽  
Modeling Analysis ◽  
And Function

AbstractMechanical forces are integral to cellular migration, differentiation and tissue morphogenesis; however, it has proved challenging to directly measure strain at high spatial resolution with minimal perturbation in living sytems. Here, we fabricate, calibrate, and test a fibronectin (FN)-based nanomechanical biosensor (NMBS) that can be applied to the surface of cells and tissues to measure the magnitude, direction, and strain dynamics from subcellular to tissue length-scales. The NMBS is a fluorescently-labeled, ultra-thin FN lattice-mesh with spatial resolution tailored by adjusting the width and spacing of the lattice from 2–100 µm. Time-lapse 3D confocal imaging of the NMBS demonstrates 2D and 3D surface strain tracking during mechanical deformation of known materials and is validated with finite element modeling. Analysis of the NMBS applied to single cells, cell monolayers, and Drosophila ovarioles highlights the NMBS’s ability to dynamically track microscopic tensile and compressive strains across diverse biological systems where forces guide structure and function.

scholarly journals In vivo single cell analysis reveals Gata2 dynamics in cells transitioning to hematopoietic fate

2017 ◽  
Vol 215 (1) ◽  
pp. 233-248 ◽  
Author(s):  
Christina Eich ◽  
Jochen Arlt ◽  
Chris S. Vink ◽  
Parham Solaimani Kartalaei ◽  
Polynikis Kaimakis ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Fate ◽  
Regulatory Networks ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Time Lapse ◽  
Hematopoietic Stem ◽  
And Function ◽  
In Cells

Cell fate is established through coordinated gene expression programs in individual cells. Regulatory networks that include the Gata2 transcription factor play central roles in hematopoietic fate establishment. Although Gata2 is essential to the embryonic development and function of hematopoietic stem cells that form the adult hierarchy, little is known about the in vivo expression dynamics of Gata2 in single cells. Here, we examine Gata2 expression in single aortic cells as they establish hematopoietic fate in Gata2Venus mouse embryos. Time-lapse imaging reveals rapid pulsatile level changes in Gata2 reporter expression in cells undergoing endothelial-to-hematopoietic transition. Moreover, Gata2 reporter pulsatile expression is dramatically altered in Gata2+/− aortic cells, which undergo fewer transitions and are reduced in hematopoietic potential. Our novel finding of dynamic pulsatile expression of Gata2 suggests a highly unstable genetic state in single cells concomitant with their transition to hematopoietic fate. This reinforces the notion that threshold levels of Gata2 influence fate establishment and has implications for transcription factor–related hematologic dysfunctions.

scholarly journals A new method for post-translationally labeling proteins in live cells for fluorescence imaging and tracking

2017 ◽  
Vol 30 (12) ◽  
pp. 771-780 ◽  
Author(s):  
M Hinrichsen ◽  
M Lenz ◽  
J M Edwards ◽  
O K Miller ◽  
S G J Mochrie ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Single Cells ◽  
Covalent Bond ◽  
Subcellular Location ◽  
Turnover Time ◽  
Target Protein ◽  
Live Cells ◽  
General Applicability ◽  
Novel Approach ◽  
And Function

AbstractWe present a novel method to fluorescently label proteins, post-translationally, within live Saccharomycescerevisiae. The premise underlying this work is that fluorescent protein (FP) tags are less disruptive to normal processing and function when they are attached post-translationally, because target proteins are allowed to fold properly and reach their final subcellular location before being labeled. We accomplish this post-translational labeling by expressing the target protein fused to a short peptide tag (SpyTag), which is then covalently labeled in situ by controlled expression of an open isopeptide domain (SpyoIPD, a more stable derivative of the SpyCatcher protein) fused to an FP. The formation of a covalent bond between SpyTag and SpyoIPD attaches the FP to the target protein. We demonstrate the general applicability of this strategy by labeling several yeast proteins. Importantly, we show that labeling the membrane protein Pma1 in this manner avoids the mislocalization and growth impairment that occur when Pma1 is genetically fused to an FP. We also demonstrate that this strategy enables a novel approach to spatiotemporal tracking in single cells and we develop a Bayesian analysis to determine the protein’s turnover time from such data.

scholarly journals Autofluorescence in freshly isolated adult human liver sinusoidal cells

2021 ◽  
Vol 65 (4) ◽  
Author(s):  
Anett Kristin Larsen ◽  
Jaione Simón-Santamaría ◽  
Kjetil Elvevold ◽  
Bo Göran Ericzon ◽  
Kim Erlend Mortensen ◽  
...  
Keyword(s):  
Human Liver ◽  
Cultured Cells ◽  
Confocal Imaging ◽  
Live Cells ◽  
Sinusoidal Cells ◽  
Imaging Flow Cytometry ◽  
Wide Range ◽  
Liver Sinusoidal Cells ◽  
Liver Endothelial Cells ◽  
Selection Of

Autofluorescent granules of various sizes were observed in primary human liver endothelial cells (LSECs) upon laser irradiation using a wide range of wavelengths. Autofluorescence was detected in LAMP-1 positive vesicles, suggesting lysosomal location. Confocal imaging of freshly prepared cultures and imaging flow cytometry of non-cultured cells revealed fluorescence in all channels used. Treatment with a lipofuscin autofluorescence quencher reduced autofluorescence, most efficiently in the near UV-area. These results, combined with the knowledge of the very active blood clearance function of LSECs support the notion that lysosomally located autofluorescent material reflected accumulation of lipofuscin in the intact liver. These results illustrate the importance of careful selection of fluorophores, especially when labelling of live cells where the quencher is not compatible.

scholarly journals Microscopy quantification of microbial birth and death dynamics

2018 ◽  
Author(s):  
Samuel F. M. Hart ◽  
David Skelding ◽  
Adam J. Waite ◽  
Justin Burton ◽  
Li Xie ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Time Lapse ◽  
Microbial Evolution ◽  
Cell Counting ◽  
Live Cells ◽  
Nutrient Concentrations ◽  
Death Rates ◽  
Time Lapse Microscopy ◽  
Wide Range ◽  
Total Fluorescence

AbstractMicrobes live in dynamic environments where nutrient concentrations fluctuate. Quantifying fitness (birth and death) in a wide range of environments is critical for understanding microbial evolution as well as ecological interactions where one species alters the fitness of another. Here, using high-throughput time-lapse microscopy, we have quantified howSaccharomyces cerevisiaemutants incapable of synthesizing an essential metabolite grow or die in various concentrations of the required metabolite. We establish that cells normally expressing fluorescent proteins lose fluorescence upon death and that the total fluorescence in an imaging frame is proportional to the number of live cells even when cells form multiple layers. We validate our microscopy approach of measuring birth and death rates using flow cytometry, cell counting, and chemostat culturing. For lysine-requiring cells, very low concentrations of lysine are not detectably consumed and do not support cell birth, but delay the onset of death phase and reduce the death rate. In contrast, in low hypoxanthine, hypoxanthine-requiring cells can produce new cells, yet also die faster than in the absence of hypoxanthine. For both strains, birth rates under various metabolite concentrations are better described by the sigmoidal-shaped Moser model than the well-known Monod model, while death rates depend on the metabolite concentration and can vary with time. Our work reveals how time-lapse microscopy can be used to discover non-intuitive microbial dynamics and to quantify growth rates in many environments.

scholarly journals Preventing Photomorbidity in Long-Term Multi-color Fluorescence Imaging of Saccharomyces cerevisiae and S. pombe

2020 ◽  
Vol 10 (12) ◽  
pp. 4373-4385
Author(s):  
Gregor W. Schmidt ◽  
Andreas P. Cuny ◽  
Fabian Rudolf
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Time Lapse ◽  
Light Dose ◽  
Live Cells ◽  
Excitation Light ◽  
Minimal Impact ◽  
Imaging Conditions

Time-lapse imaging of live cells using multiple fluorescent reporters is an essential tool to study molecular processes in single cells. However, exposure to even moderate doses of visible excitation light can disturb cellular physiology and alter the quantitative behavior of the cells under study. Here, we set out to develop guidelines to avoid the confounding effects of excitation light in multi-color long-term imaging. We use widefield fluorescence microscopy to measure the effect of the administered excitation light on growth rate (here called photomorbidity) in yeast. We find that photomorbidity is determined by the cumulative light dose at each wavelength, but independent of the way excitation light is applied. Importantly, photomorbidity possesses a threshold light dose below which no effect is detectable (NOEL). We found, that the suitability of fluorescent proteins for live-cell imaging at the respective excitation light NOEL is equally determined by the cellular autofluorescence and the fluorescent protein brightness. Last, we show that photomorbidity of multiple wavelengths is additive and imaging conditions absent of photomorbidity can be predicted. Our findings enable researchers to find imaging conditions with minimal impact on physiology and can provide framework for how to approach photomorbidity in other organisms.

scholarly journals Generating kinetic environments to study dynamic cellular processes in single cells

2019 ◽  
Author(s):  
Alexander Thiemicke ◽  
Hossein Jashnsaz ◽  
Guoliang Li ◽  
Gregor Neuert
Keyword(s):  
Cell Culture ◽  
Single Molecule ◽  
Cellular Response ◽  
Single Cells ◽  
Mammalian Cell Culture ◽  
Time Lapse ◽  
Culture Conditions ◽  
Wide Range ◽  
Quantitative Manner ◽  
Fate Decision

AbstractCells of any organism are consistently exposed to changes over time in their environment. The kinetics by which these changes occur are critical for the cellular response and fate decision. It is therefore important to control the temporal changes of extracellular stimuli precisely to understand biological mechanisms in a quantitative manner. Most current cell culture and biochemical studies focus on instant changes in the environment and therefore neglect the importance of kinetic environments. To address these shortcomings, we developed two experimental methodologies to precisely control the environment of single cells. These methodologies are compatible with standard biochemistry, molecular, cell and quantitative biology assays. We demonstrate applicability by obtaining time series and time point measurements in both live and fixed cells. We demonstrate the feasibility of the methodology in yeast and mammalian cell culture in combination with widely used assays such as flow cytometry, time-lapse microscopy and single-molecule RNA Fluorescent in-situ Hybridization. Our experimental methodologies are easy to implement in most laboratory settings and allows the study of kinetic environments in a wide range of assays and different cell culture conditions.

High spatiotemporal resolution and low photo-toxicity fluorescence imaging in live cells and in vivo

2019 ◽  
Vol 47 (6) ◽  
pp. 1635-1650 ◽  
Author(s):  
Xiaohong Peng ◽  
Xiaoshuai Huang ◽  
Ke Du ◽  
Huisheng Liu ◽  
Liangyi Chen
Keyword(s):  
Fluorescence Microscopy ◽  
Spatial Resolution ◽  
Cell Biology ◽  
Critical Role ◽  
Super Resolution ◽  
Light Sheet ◽  
Live Cells ◽  
Spatiotemporal Resolution ◽  
And Function

Taking advantage of high contrast and molecular specificity, fluorescence microscopy has played a critical role in the visualization of subcellular structures and function, enabling unprecedented exploration from cell biology to neuroscience in living animals. To record and quantitatively analyse complex and dynamic biological processes in real time, fluorescence microscopes must be capable of rapid, targeted access deep within samples at high spatial resolutions, using techniques including super-resolution fluorescence microscopy, light sheet fluorescence microscopy, and multiple photon microscopy. In recent years, tremendous breakthroughs have improved the performance of these fluorescence microscopies in spatial resolution, imaging speed, and penetration. Here, we will review recent advancements of these microscopies in terms of the trade-off among spatial resolution, sampling speed and penetration depth and provide a view of their possible applications.

scholarly journals Cellular crowding influences extrusion and proliferation to facilitate epithelial tissue repair

2019 ◽  
Vol 30 (16) ◽  
pp. 1890-1899 ◽  
Author(s):  
Jovany J. Franco ◽  
Youmna Atieh ◽  
Chase D. Bryan ◽  
Kristen M. Kwan ◽  
George T. Eisenhoffer
Keyword(s):  
Tissue Repair ◽  
Wound Closure ◽  
Time Lapse ◽  
Cell Number ◽  
Confocal Imaging ◽  
Epithelial Tissue ◽  
Directed Cell Migration ◽  
Epithelial Wound Healing ◽  
Cellular Behaviors ◽  
And Function

Epithelial wound healing requires a complex orchestration of cellular rearrangements and movements to restore tissue architecture and function after injury. While it is well known that mechanical forces can affect tissue morphogenesis and patterning, how the biophysical cues generated after injury influence cellular behaviors during tissue repair is not well understood. Using time-lapse confocal imaging of epithelial tissues in living zebrafish larvae, we provide evidence that localized increases in cellular crowding during wound closure promote the extrusion of nonapoptotic cells via mechanically regulated stretch-activated ion channels (SACs). Directed cell migration toward the injury site promoted rapid changes in cell number and generated shifts in tension at cellular interfaces over long spatial distances. Perturbation of SAC activity resulted in failed extrusion and increased proliferation in crowded areas of the tissue. Together, we conclude that localized cell number plays a key role in dictating cellular behaviors that facilitate wound closure and tissue repair.

scholarly journals Depth-resolved mid-infrared photothermal imaging of living cells and organisms with submicrometer spatial resolution

2016 ◽  
Vol 2 (9) ◽  
pp. e1600521 ◽  
Author(s):  
Delong Zhang ◽  
Chen Li ◽  
Chi Zhang ◽  
Mikhail N. Slipchenko ◽  
Gregory Eakins ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
Single Cells ◽  
Chemical Imaging ◽  
Detection Sensitivity ◽  
Living Systems ◽  
Live Cells ◽  
Photothermal Effect ◽  
Label Free ◽  
Infrared Microscopy ◽  
Mid Infrared

Chemical contrast has long been sought for label-free visualization of biomolecules and materials in complex living systems. Although infrared spectroscopic imaging has come a long way in this direction, it is thus far only applicable to dried tissues because of the strong infrared absorption by water. It also suffers from low spatial resolution due to long wavelengths and lacks optical sectioning capabilities. We overcome these limitations through sensing vibrational absorption–induced photothermal effect by a visible laser beam. Our mid-infrared photothermal (MIP) approach reached 10 μM detection sensitivity and submicrometer lateral spatial resolution. This performance has exceeded the diffraction limit of infrared microscopy and allowed label-free three-dimensional chemical imaging of live cells and organisms. Distributions of endogenous lipid and exogenous drug inside single cells were visualized. We further demonstrated in vivo MIP imaging of lipids and proteins inCaenorhabditis elegans. The reported MIP imaging technology promises broad applications from monitoring metabolic activities to high-resolution mapping of drug molecules in living systems, which are beyond the reach of current infrared microscopy.

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