Spectrophotometry and fluorimetry of cellular compartments and Intracellular processes

2000 ◽  
Author(s):  
C. Lindsay Bashford
Keyword(s):  
Experimental Design ◽  
Optical Spectroscopy ◽  
Single Cells ◽  
Full Range ◽  
Experimental System ◽  
Two Dimensions ◽  
Optical Measurements ◽  
Spectroscopic Techniques ◽  
Experimental Protocols ◽  
Cellular Compartments

Optical spectroscopy, Spectrophotometry and fluorimetry can be used to monitor processes occurring in living cells provided that suitable chromophores are present which ‘report’ on the events in which they participate. The advantages of optical techniques are manifold. Firstly they can be fast—with appropriate apparatus events in the pico- and nano-second domains can be studied by fluorescence spectroscopy. Secondly they are continuous—instant feedback from the experimental system can guide the most complex of experimental protocols, and allow the experimenter to adjust system parameters as necessary. Thirdly they are convenient, and most laboratories have access to equipment that can provide quantitative analysis of optical signals; examples include conventional spectrophotometers/fluorimeters, dedicated instruments (e.g. for fluorescence lifetime and polarization measurements), cameras, microscopes and plate readers. Significantly detectors from one apparatus can often be used on others to open up new experimental protocols. Fortunately the principles underlying the use of such a diverse array of optical devices are straightforward and universal—they apply just as much to laboratory ‘work-horse’ instruments as they do to the most specialized, laser-illuminated fluorescence microscope. The availability of fast laboratory computers with large storage capacities means that most modern spectrometers are microprocessor controlled and digitization of signals opens up the full range of possibilities of data accumulation, storage, analysis and interpretation. The main problem with optical measurements is not the acquisition but rather the interpretation of the data obtained. Straightforward analysis of the results depends on the clarity of the experimental design and the appropriate choice of chromophore. This chapter describes some of the problems that can be addressed by spectroscopic techniques and attempts to give guidance on good experimental design. Optical spectroscopy requires either spectrophotometers, to measure absorbance, fluorimeters, to measure fluorescence, or microscopes, which can measure fluorescence or absorbance of single cells or small groups of cells. Fluorimeters and spectrophotometers usually require solutions or suspensions of material in conventional cuvettes; microscopes provide two-dimensional images from smears, slices or surfaces. Other devices that record signals resolved in two-dimensions include gel scanners and microplate readers.

scholarly journals Spectroscopic Techniques versus Pitot Tube for the Measurement of Flow Velocity in Narrow Ducts

Sensors ◽  
2020 ◽  
Vol 20 (24) ◽  
pp. 7349
Author(s):  
Francesco D’Amato ◽  
Silvia Viciani ◽  
Alessio Montori ◽  
Marco Barucci ◽  
Carmen Morreale ◽  
...  
Keyword(s):  
Flow Field ◽  
Flow Velocity ◽  
Pitot Tube ◽  
Optical Measurements ◽  
Trace Gas ◽  
Dual Function ◽  
Spectroscopic Techniques ◽  
Gas Composition ◽  
Flow Conditions ◽  
Theoretical Simulation

In order to assess the limits and applicability of Pitot tubes for the measurement of flow velocity in narrow ducts, e.g., biomass burning plants, an optical, dual function device was implemented. This sensor, based on spectroscopic techniques, targets a trace gas, injected inside the stack either in bursts, or continuously, so performing transit time or dilution measurements. A comparison of the two optical techniques with respect to Pitot readings was carried out in different flow conditions (speed, temperature, gas composition). The results of the two optical measurements are in agreement with each other and fit quite well the theoretical simulation of the flow field, while the results of the Pitot measurements show a remarkable dependence on position and inclination of the Pitot tube with respect to the duct axis. The implications for the metrology of small combustors’ emissions are outlined.

scholarly journals μCB-seq: Microfluidic cell barcoding and sequencing for high-resolution imaging and sequencing of single cells

2020 ◽  
Author(s):  
Tyler N. Chen ◽  
Anushka Gupta ◽  
Mansi Zalavadia ◽  
Aaron M. Streets
Keyword(s):  
High Resolution ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Protein Localization ◽  
Single Cells ◽  
Optical Measurements ◽  
Detection Sensitivity ◽  
High Resolution Imaging ◽  
Cell Analysis ◽  
Resolution Imaging

AbstractSingle-cell RNA sequencing (scRNA-seq) enables the investigation of complex biological processes in multicellular organisms with high resolution. However, many phenotypic features that are critical to understanding the functional role of cells in a heterogeneous tissue or organ are not directly encoded in the genome and therefore cannot be profiled with scRNA-seq. Quantitative optical microscopy has long been a powerful approach for characterizing diverse cellular phenotypes including cell morphology, protein localization, and chemical composition. Combining scRNA-seq with optical imaging has the potential to provide comprehensive single-cell analysis, allowing for functional integration of gene expression profiling and cell-state characterization. However, it is difficult to track single cells through both measurements; therefore, coupling current scRNA-seq protocols with optical measurements remains a challenge. Here, we report Microfluidic Cell Barcoding and Sequencing (μCB-seq), a microfluidic platform that combines high-resolution imaging and sequencing of single cells. μCB-seq is enabled by a novel fabrication method that preloads primers with known barcode sequences inside addressable reaction chambers of a microfluidic device. In addition to enabling multi-modal single-cell analysis, μCB-seq improves gene detection sensitivity, providing a scalable and accurate method for information-rich characterization of single cells.

scholarly journals Simultaneous Optical Measurements of Axial and Tangential Steady-State Blade Deflections

1999 ◽  
Author(s):  
Anatole P. Kurkov ◽  
Harbans S. Dhadwal
Keyword(s):  
Wind Tunnel Test ◽  
Design Feature ◽  
Aircraft Engine ◽  
Optical Probe ◽  
Two Dimensions ◽  
Optical Measurements ◽  
Axial Position ◽  
Aerodynamic Loading ◽  
Blade Edge ◽  
Alternate Solution

Currently, the majority of fiber-optic blade instrumentation is being designed and manufactured by aircraft-engine companies for their own use. The most commonly employed probe for optical blade deflection measurements is the spot probe. One of its characteristics is that the incident spot on a blade is not fixed relative to the blade, but changes depending on the blade deformation associated with centrifugal and aerodynamic loading. While there are geometrically more complicated optical probe designs in use by different engine companies, this paper offers an alternate solution derived from a probe-mount design feature that allows one to change the probe axial position until the incident spot contacts either a leading or a trailing edge. By tracing the axial position of either blade edge one is essentially extending the deflection measurement to two dimensions, axial and tangential. The blade deflection measurements were obtained during a wind tunnel test of a fan prototype.

scholarly journals A computational method to aid the design and analysis of single cell RNA-seq experiments for cell type identification

2018 ◽  
Author(s):  
Douglas Abrams ◽  
Parveen Kumar ◽  
R. Krishna Murthy Karuturi ◽  
Joshy George
Keyword(s):  
Experimental Design ◽  
Single Cell ◽  
Single Cells ◽  
Cell Types ◽  
Cell Number ◽  
Fold Change ◽  
Computational Method ◽  
Marker Genes ◽  
Cell Type ◽  
Estimate Sample Size

AbstractBackgroundThe advent of single cell RNA sequencing (scRNA-seq) enabled researchers to study transcriptomic activity within individual cells and identify inherent cell types in the sample. Although numerous computational tools have been developed to analyze single cell transcriptomes, there are no published studies and analytical packages available to guide experimental design and to devise suitable analysis procedure for cell type identification.ResultsWe have developed an empirical methodology to address this important gap in single cell experimental design and analysis into an easy-to-use tool called SCEED (Single Cell Empirical Experimental Design and analysis). With SCEED, user can choose a variety of combinations of tools for analysis, conduct performance analysis of analytical procedures and choose the best procedure, and estimate sample size (number of cells to be profiled) required for a given analytical procedure at varying levels of cell type rarity and other experimental parameters. Using SCEED, we examined 3 single cell algorithms using 48 simulated single cell datasets that were generated for varying number of cell types and their proportions, number of genes expressed per cell, number of marker genes and their fold change, and number of single cells successfully profiled in the experiment.ConclusionsBased on our study, we found that when marker genes are expressed at fold change of 4 or more than the rest of the genes, either Seurat or Simlr algorithm can be used to analyze single cell dataset for any number of single cells isolated (minimum 1000 single cells were tested). However, when marker genes are expected to be only up to fC 2 upregulated, choice of the single cell algorithm is dependent on the number of single cells isolated and proportion of rare cell type to be identified. In conclusion, our work allows the assessment of various single cell methods and also aids in examining the single cell experimental design.

Clonal analysis of expression of epithelial antigens in cultures of normal human breast

1986 ◽  
Vol 80 (1) ◽  
pp. 91-101
Author(s):  
P.A. Edwards ◽  
I.M. Brooks ◽  
H.J. Bunnage ◽  
A.V. Foster ◽  
M.L. Ellison ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Epithelial Cells ◽  
Phase Contrast ◽  
Single Cells ◽  
Full Range ◽  
Human Breast ◽  
Intact Tissue ◽  
Normal Human Breast ◽  
Normal Human ◽  
Luminal Epithelial Cells

Cells from normal human breast epithelium were cloned in monolayer culture and the clones were stained with monoclonal antibodies. Tissue was from reduction mammoplasty operations. Cloning efficiencies were 5–30%. Two types of clone were identified: 10 to 30% were of relatively spread cells whose boundaries were often difficult to see by phase-contrast microscopy but where they were visible they appeared as dark lines. The edges of the clones usually appeared to be under tension. These clones were stained by two monoclonal antibodies, LICR-LON-M8 and M24, that stain luminal epithelial cells in the intact tissue, but not myoepithelial or stromal cells. Within a clone the cells showed a full range of antigenic phenotypes. This was confirmed for clones grown from single cells that had been isolated manually. The second type of clone was more compact with little evidence of tension at the edges, and cell boundaries were clearly visible and bright under phase contrast. These clones were not stained by antibodies M8 or M24. Both types of clone stained with a third monoclonal antibody that is specific for luminal epithelial cells in the intact tissue, LICR-LON-M18, but the distribution of staining was different in the different types of clone. The simplest interpretation of the two types of clone is that luminal epithelial cells give rise to the spread type of clone while the myoepithelial cells give rise to the more abundant and vigorous compact clones. Alternatively, the compact clones may be from luminal epithelial cells that have lost differentiated characteristics.

scholarly journals The Design and Implementation of Adsorptive Removal of Cu(II) from Leachate Using ANFIS

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Nurdan Gamze Turan ◽  
Okan Ozgonenel
Keyword(s):  
Experimental Design ◽  
Contact Time ◽  
Fixed Bed ◽  
Experimental System ◽  
Factorial Experimental Design ◽  
Adsorbent Dosage ◽  
Initial Ph ◽  
Full Factorial Experimental Design ◽  
Full Factorial ◽  
Aqueous Leachate

Clinoptilolite was investigated for the removal of Cu(II) ions from industrial leachate. Adaptive neural fuzzy interface system (ANFIS) was used for modeling the batch experimental system and predicting the optimal input values, that is, initial pH, adsorbent dosage, and contact time. Experiments were studied under laboratory batch and fixed bed conditions. The outcomes of suggested ANFIS modeling were then compared to a full factorial experimental design (23), which was utilized to assess the effect of three factors on the adsorption of Cu(II) ions in aqueous leachate of industrial waste. It was observed that the optimized parameters are almost close to each other. The highest removal efficiency was found as about 93.65% at pH 6, adsorbent dosage 11.4 g/L, and contact time 33 min for batch conditions of 23experimental design and about 90.43% at pH 5, adsorbent dosage 15 g/L and contact time 35 min for batch conditions of ANFIS. The results show that clinoptilolite is an efficient sorbent and ANFIS, which is easy to implement and is able to model the batch experimental system.

Experimental Study on Skin Friction Reduction With Micro-Blowing

Volume 4 ◽  
2004 ◽  
Author(s):  
Song Liu ◽  
Hongmin Li ◽  
Minel J. Braun
Keyword(s):  
Boundary Layer ◽  
Shear Stress ◽  
Skin Friction ◽  
Full Range ◽  
Experimental System ◽  
Main Flow ◽  
Friction Reduction ◽  
Long Distance ◽  
Full Field ◽  
Set Up

Reducing skin friction, such as friction on a car hood or a plane wing, can significantly reduce the drag force and decrease specific fuel consumption. Many techniques and methods have been tried. The Micro-blowing Technique (MBT) is an innovative way to reduce skin friction. Suggested by early research in boundary layer injection in 1950s, MBT was actually brought to effective use in 1994 by Hwang [1]. The basic idea is that by blowing fluid, same as or different from the mainstream flow, at an angle with that of the main flow, a decrease in the velocity gradient at the wall can be achieved, and thus the shear stress on the surface is reduced. Although the experimental data on boundary layer with micro blowing show a significant friction reduction, the mechanism of MBT is still not well understood and thus its full range of application is not yet established. In this paper, we further the understanding of the MBT mechanism. An experimental system is set up to visualize the flow structure on a plate with and without micro blowing in a tunnel. A long distance microscope is combined with a Full Field Flow Tracking visualization method in order to elucidate the nature of the flow interaction and mixing between the blowing flow and the main flow. The flow above the porous plates is visualized and velocities both in the blowing layer immediately adjacent to the plate and in the main flow are quantified using the PIV procedure. The flow and shear stress analysis shows that MTB has significantly different effects on a flow with a boundary layer and fully developed internal flows.

Microwave-assisted synthesis of zinc 5-(4-carboxyphenyl)-10,15,20-triphenylporphyrin and zinc 5-(4-carboxyphenyl)-10,15,20-triphenylchlorin

2015 ◽  
Vol 19 (04) ◽  
pp. 595-600 ◽  
Author(s):  
Rima Chouikrat ◽  
Aymeric Champion ◽  
Régis Vanderesse ◽  
Céline Frochot ◽  
Albert Moussaron
Keyword(s):  
Reaction Times ◽  
Microwave Assisted Synthesis ◽  
Spectroscopic Techniques ◽  
H Nmr ◽  
Microwave Assisted ◽  
Experimental Protocols ◽  
Time Of Reaction

The microwave-assisted synthesis of zinc 5-(4-carboxyphenyl)-10,15,20-triphenylporphyrin and zinc 5-(4-carboxyphenyl)-10,15,20-triphenylchlrorin are described and compared to classic conditions of synthesis in terms of time of reaction and yields obtained. The new experimental protocols are easy to implement required small amounts of reagents and solvents and lead to short reaction times. All compounds have been characterized by 1 H NMR, MS and spectroscopic techniques.

scholarly journals Interest Groups in Multi-Level Contexts: European Integration as Cross-Cutting Issue in Party-Interest Group Contacts

2020 ◽  
Vol 8 (1) ◽  
pp. 61-71 ◽  
Author(s):  
Joost Berkhout ◽  
Marcel Hanegraaff ◽  
Patrick Statsch
Keyword(s):  
Interest Groups ◽  
European Integration ◽  
Interest Group ◽  
Full Range ◽  
Two Dimensions ◽  
European Policy ◽  
Group Leaders ◽  
Domain Specific ◽  
European Public ◽  
Multi Level

Policy-specific actor-constellations consisting of party- and group-representatives commonly drive the effective establishment of new policy programmes or changes in existing policies. In the EU multi-level system, the creation of such constellations is complicated because it practically requires consensus on two dimensions: the European public policy at stake and the issue of European integration. This means that, for interest groups with interests in particular policy domains, and with limited interest in the actual issue of European integration, non-Eurosceptic parties must be their main ally in their policy battles. We hypothesise that interest groups with relevant European domain-specific interests will ally with non-Eurosceptic parties, whereas interest groups whose interests are hardly affected by the European policy process will have party-political allies across the full range of positions on European integration. We assess this argument on the basis of an elite-survey of interest group leaders and study group-party dyads in several European countries (i.e., Belgium, Lithuania, Italy, Netherlands, Poland, and Slovenia) in a large number of policy domains. Our dependent variable is the group-party dyad and the main independent variables are the European policy interests of the group and the level of Euroscepticism of the party. We broadly find support for our hypotheses. The findings of our study speak to the debate concerning the implications of the politicisation of European integration and, more specifically, the way in which party-political polarisation of Europe may divide domestic interest group systems and potentially drive group and party systems apart.

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