scholarly journals Hazelnut Pollen Phenotyping Using Label-Free Impedance Flow Cytometry

2020 ◽  
Vol 11 ◽  
Author(s):  
Lorenzo Ascari ◽  
Valerio Cristofori ◽  
Federico Macrì ◽  
Roberto Botta ◽  
Cristian Silvestri ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Pollen Viability ◽  
Central Italy ◽  
Chromosome Translocation ◽  
Pollen Sterility ◽  
Tree Canopy ◽  
Label Free ◽  
Lab On Chip ◽  
Pollen Hydration ◽  
Chip Technology

Impedance flow cytometry (IFC) is a versatile lab-on-chip technology which enables fast and label-free analysis of pollen grains in various plant species, promising new research possibilities in agriculture and plant breeding. Hazelnut is a monoecious, anemophilous species, exhibiting sporophytic self-incompatibility. Its pollen is dispersed by wind in midwinter when temperatures are still low and relative humidity is usually high. Previous research found that hazelnut can be characterized by high degrees of pollen sterility following a reciprocal chromosome translocation occurring in some cultivated genotypes. In this study, IFC was used for the first time to characterize hazelnut pollen biology. IFC was validated via dye exclusion in microscopy and employed to (i) follow pollen hydration over time to define the best pre-hydration treatment for pollen viability evaluation; (ii) test hazelnut pollen viability and sterility on 33 cultivars grown in a collection field located in central Italy, and two wild hazelnuts. The accessions were also characterized by their amount and distribution of catkins in the tree canopy. Pollen sterility rate greatly varied among hazelnut accessions, with one main group of highly sterile cultivars and a second group, comprising wild genotypes and the remaining cultivars, producing good quality pollen. The results support the hypothesis of recurring reciprocal translocation events in Corylus avellana cultivars, leading to the observed gametic semi-sterility. The measured hazelnut pollen viability was also strongly influenced by pollen hydration (Radj2 = 0.83, P ≤ 0.0001) and reached its maximum at around 6 h of pre-hydration in humid chambers. Viable and dead pollen were best discriminated at around the same time of pollen pre-hydration, suggesting that high humidity levels are required for hazelnut pollen to maintain its functionality. Altogether, our results detail the value of impedance flow cytometry for high throughput phenotyping of hazelnut pollen. Further research is required to clarify the causes of pollen sterility in hazelnut, to confirm the role of reciprocal chromosome translocations and to investigate its effects on plant productivity.

scholarly journals MICRO-SCALE TECHNOLOGIES FOR CELL MECHANICS: EXPERIMENTAL AND MODELLING APPROACHES

2018 ◽  
Author(s):  
Federica Caselli ◽  
Nicola A. Nodargi ◽  
Paolo Bisegna
Keyword(s):  
Cell Mechanics ◽  
Cell Biology ◽  
Single Cell Level ◽  
Mechanical Function ◽  
Label Free ◽  
Cell Level ◽  
Lab On Chip ◽  
Non Invasive ◽  
Chip Technology ◽  
On Chip

Cell mechanics is a discipline that bridges cell biology with mechanics. Emerging microscale technologies are opening new venues in the field, due to their costeffectiveness, relatively easy fabrication, and high throughput. Two examples of those technologies are discussed here: microfluidic impedance cytometry and erythrocyte electrodeformation. The former is a lab-on-chip technology offering a simple, non-invasive, label-free method for counting, identifying and monitoring cellular biophysical and mechanical function at the single-cell level. The latter is a useful complement to the former, enabling cell deformation under the influence of an applied electric field.

Label-Free Electrochemical Detection of DNA Hybridization Related to Anthrax Lethal Factor by using Carbon Nanotube Modified Sensors

2019 ◽  
Vol 15 (4) ◽  
pp. 502-510 ◽  
Author(s):  
Hakan Karadeniz ◽  
Arzum Erdem
Keyword(s):  
Detection Limit ◽  
Dna Hybridization ◽  
Lethal Factor ◽  
Modified Electrodes ◽  
Label Free ◽  
Carbon Paste ◽  
Chip Technology ◽  
Anthrax Lethal Factor ◽  
Hybridization Time ◽  
Electrochemical Monitoring

Background: Anthrax Lethal Factor (ANT) is the dominant virulence factor produced by B. anthracis and is the major cause of death of infected animals. In this paper, pencil graphite electrodes GE were modified with single-walled and multi-walled carbon nanotubes (CNTs) for the detection of hybridization related to the ANT DNA for the first time in the literature. Methods: The electrochemical monitoring of label-free DNA hybridization related to ANT DNA was explored using both SCNT and MCNT modified PGEs with differential pulse voltammetry (DPV). The performance characteristics of ANT-DNA hybridization on disposable GEs were explored by measuring the guanine signal in terms of optimum analytical conditions; the concentration of SCNT and MCNT, the concentrations of probe and target, and also the hybridization time. Under the optimum conditions, the selectivity of probe modified electrodes was tested and the detection limit was calculated. Results: The selectivity of ANT probes immobilized onto MCNT-GEs was tested in the presence of hybridization of probe with NC no response was observed and with MM, smaller responses were observed in comparison to full-match DNA hybridization case. Even though there are unwanted substituents in the mixture samples containing both the target and NC in the ratio 1:1 and both the target and MM in the ratio 1:1, it has been found that ANT probe immobilized CNT modified graphite sensor can also select its target by resulting with 20.9% decreased response in comparison to the one measured in the case of full-match DNA hybridization case Therefore, it was concluded that the detection of direct DNA hybridization was performed by using MCNT-GEs with an acceptable selectivity. Conclusion: Disposable SCNT/MCNT modified GEs bring some important advantages to our assay including easy use, cost-effectiveness and giving a response in a shorter time compared to unmodified PGE, carbon paste electrode and glassy carbon electrode developed for electrochemical monitoring of DNA hybridization. Consequently, the detection of DNA hybridization related to the ANT DNA by MCNT modified sensors was performed by using lower CNT, probe and target concentrations, in a shorter hybridization time and resulting in a lower detection limit according to the SCNT modified sensors. In conclusion, MCNT modified sensors can yield the possibilities leading to the development of nucleic acid sensors platforms for the improvement of fast and cost-effective detection systems with respect to DNA chip technology.

scholarly journals Multi-pronged approach to human mesenchymal stromal cells senescence quantification with a focus on label-free methods

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Weichao Zhai ◽  
Jerome Tan ◽  
Tobias Russell ◽  
Sixun Chen ◽  
Dennis McGonagle ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Preclinical Model ◽  
Label Free ◽  
Current Standard ◽  
Differentiation Potential ◽  
Human Mesenchymal Stromal Cells ◽  
Endogenous Fluorophores

AbstractHuman mesenchymal stromal cells (hMSCs) have demonstrated, in various preclinical settings, consistent ability in promoting tissue healing and improving outcomes in animal disease models. However, translation from the preclinical model into clinical practice has proven to be considerably more difficult. One key challenge being the inability to perform in situ assessment of the hMSCs in continuous culture, where the accumulation of the senescent cells impairs the culture’s viability, differentiation potential and ultimately leads to reduced therapeutic efficacies. Histochemical $$\upbeta $$ β -galactosidase staining is the current standard for measuring hMSC senescence, but this method is destructive and not label-free. In this study, we have investigated alternatives in quantification of hMSCs senescence, which included flow cytometry methods that are based on a combination of cell size measurements and fluorescence detection of SA-$$\upbeta $$ β -galactosidase activity using the fluorogenic substrate, C$${_{12}}$$ 12 FDG; and autofluorescence methods that measure fluorescence output from endogenous fluorophores including lipopigments. For identification of senescent cells in the hMSC batches produced, the non-destructive and label-free methods could be a better way forward as they involve minimum manipulations of the cells of interest, increasing the final output of the therapeutic-grade hMSC cultures. In this work, we have grown hMSC cultures over a period of 7 months and compared early and senescent hMSC passages using the advanced flow cytometry and autofluorescence methods, which were benchmarked with the current standard in $$\upbeta $$ β -galactosidase staining. Both the advanced methods demonstrated statistically significant values, (r = 0.76, p $$\le $$ ≤ 0.001 for the fluorogenic C$${_{12}}$$ 12 FDG method, and r = 0.72, p $$\le $$ ≤ 0.05 for the forward scatter method), and good fold difference ranges (1.120–4.436 for total autofluorescence mean and 1.082–6.362 for lipopigment autofluorescence mean) between early and senescent passage hMSCs. Our autofluroescence imaging and spectra decomposition platform offers additional benefit in label-free characterisation of senescent hMSC cells and could be further developed for adoption for future in situ cellular senescence evaluation by the cell manufacturers.

Deep Learning-enabled Holographic Imaging Flow-Cytometry for Label-Free Detection of Giardia Lamblia in Water Samples

2021 ◽  
Author(s):  
Zoltán Göröcs ◽  
David Baum ◽  
Fang Song ◽  
Kevin de Haan ◽  
Hatice Ceylan Koydemir ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Deep Learning ◽  
Water Samples ◽  
Giardia Lamblia ◽  
Label Free ◽  
Holographic Imaging ◽  
Imaging Flow Cytometry ◽  
Label Free Detection

scholarly journals Simultaneous acoustic and photoacoustic microfluidic flow cytometry for label-free analysis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Vaskar Gnyawali ◽  
Eric M. Strohm ◽  
Jun-Zhi Wang ◽  
Scott S. H. Tsai ◽  
Michael C. Kolios
Keyword(s):  
Flow Cytometry ◽  
Label Free ◽  
Microfluidic Flow

scholarly journals Methods and applications of label-free cell-based systems

2016 ◽  
Vol 1 (1) ◽  
Author(s):  
Joachim Wiest
Keyword(s):  
Environmental Monitoring ◽  
Morphological Changes ◽  
Free Cell ◽  
Living Cells ◽  
Label Free ◽  
Controlled System ◽  
Chip Technology ◽  
Cell Monitoring ◽  
A Cell ◽  
On Chip

Label-free monitoring of living cells is used in various applications such as drug development, toxicology, regenerative medicine or environmental monitoring. The most prominent methods for monitoring the extracellular acidification, oxygen consumption, electrophysiological activity and morphological changes of living cells are described. Furthermore, the intelligent mobile lab (IMOLA) – a computer controlled system integrating cell monitoring and automated cell cultivation – is described as an example of a cell-based system for microphysiometry. Results from experiments in the field of environmental monitoring using algae are presented. An outlook toward the development of an organ-on-chip technology is given.

scholarly journals Label-free chemical imaging flow cytometry by high-speed multicolor stimulated Raman scattering

2019 ◽  
Vol 116 (32) ◽  
pp. 15842-15848 ◽  
Author(s):  
Yuta Suzuki ◽  
Koya Kobayashi ◽  
Yoshifumi Wakisaka ◽  
Dinghuan Deng ◽  
Shunji Tanaka ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Raman Scattering ◽  
Cancer Biology ◽  
High Speed ◽  
Stimulated Raman Scattering ◽  
Chemical Imaging ◽  
Fluorescent Labeling ◽  
Label Free ◽  
Imaging Flow Cytometry ◽  
Stimulated Raman

Combining the strength of flow cytometry with fluorescence imaging and digital image analysis, imaging flow cytometry is a powerful tool in diverse fields including cancer biology, immunology, drug discovery, microbiology, and metabolic engineering. It enables measurements and statistical analyses of chemical, structural, and morphological phenotypes of numerous living cells to provide systematic insights into biological processes. However, its utility is constrained by its requirement of fluorescent labeling for phenotyping. Here we present label-free chemical imaging flow cytometry to overcome the issue. It builds on a pulse pair-resolved wavelength-switchable Stokes laser for the fastest-to-date multicolor stimulated Raman scattering (SRS) microscopy of fast-flowing cells on a 3D acoustic focusing microfluidic chip, enabling an unprecedented throughput of up to ∼140 cells/s. To show its broad utility, we use the SRS imaging flow cytometry with the aid of deep learning to study the metabolic heterogeneity of microalgal cells and perform marker-free cancer detection in blood.

A low-cost, label-free DNA detection method in lab-on-chip format based on electrohydrodynamic instabilities, with application to long-range PCR

Lab on a Chip ◽  
2012 ◽  
Vol 12 (22) ◽  
pp. 4738 ◽  
Author(s):  
Mohamed Lemine Youba Diakité ◽  
Jerôme Champ ◽  
Stephanie Descroix ◽  
Laurent Malaquin ◽  
François Amblard ◽  
...  
Keyword(s):  
Long Range ◽  
Detection Method ◽  
Dna Detection ◽  
Low Cost ◽  
Label Free ◽  
Lab On Chip ◽  
Free Dna ◽  
Chip Format ◽  
Long Range Pcr ◽  
On Chip

scholarly journals Label-free impedance flow cytometry for nanotoxicity screening

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Melanie Ostermann ◽  
Alexander Sauter ◽  
Ying Xue ◽  
Eivind Birkeland ◽  
Julia Schoelermann ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Human Health ◽  
Trypan Blue ◽  
Label Free ◽  
Trigger Level ◽  
Lymphoma Cells ◽  
Human Lymphoma ◽  
Buffer Composition ◽  
Cost Efficient

AbstractThe development of reliable and cost-efficient methods to assess the toxicity of nanomaterials (NMs) is critical for the proper identification of their impact on human health and for ensuring a safe progress of nanotechnology. In this study, we investigated the reliability and applicability of label-free impedance flow cytometry (IFC) for in vitro nanotoxicity screening, which avoids time-consuming labelling steps and minimizes possible NM-induced interferences. U937 human lymphoma cells were exposed for 24 h to eight different nanomaterials at five concentrations (2, 10, 20, 50, and 100 μg/mL). The NMs’ effect on viability was measured using IFC and the results were compared to those obtained by trypan blue (TB) dye exclusion and conventional flow cytometry (FC). To discriminate viable from necrotic cells, the IFC measurement settings regarding signal trigger level and frequency, as well as the buffer composition, were optimised. A clear discrimination between viable and necrotic cells was obtained at 6 MHz in a sucrose-based measurement buffer. Nanomaterial-induced interferences were not detected for IFC. The IFC and TB assay results were in accordance for all NMs. The IFC was found to be robust, reliable and less prone to interferences due to the advantage of being label-free.

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