scholarly journals Hyperspectral imaging as a tool for assessing coral health utilising natural fluorescence

2019 ◽  
Author(s):  
Jonathan Teague ◽  
Jack Willans ◽  
Michael Allen ◽  
Thomas Scott ◽  
John Day
Keyword(s):  
Hyperspectral Imaging ◽  
Coral Bleaching ◽  
Cell Biology ◽  
Fluorescent Proteins ◽  
Emission Spectra ◽  
Pocillopora Damicornis ◽  
Remotely Operated Vehicle ◽  
Underwater Visual Census ◽  
Research Fields ◽  
Natural Fluorescence

Fluorescent proteins are a crucial visualisation tool in a myriad of research fields including cell biology, microbiology and medicine. Fluorescence is a result of the absorption of electromagnetic radiation at one wavelength and its reemission at a longer wavelength. Coral communities exhibit a natural fluorescence which can be used to distinguish between diseased and healthy specimens, however, current methods, such as the underwater visual census, are expensive and time-consuming constituting many manned dive hours. We propose the use of a remotely operated vehicle mounted with a novel hyperspectral fluorescence imaging (HyFI) “payload” for more rapid surveying and data collection. We have tested our system in a laboratory environment on common coral species including Seriatopora spp., Montipora verrucosa, Montipora spp., Montipora capricornis, Echinopora lamellose, Euphyllia ancora, Pocillopora damicornis and Montipora confusa. With the aid of hyperspectral imaging, the coral specimens’ emission wavelengths can be accurately assessed by capturing the emission spectra of the corals when excited with light emitting diodes (395–405 and 440 nm). Fluorescence can also provide an indicator of coral bleaching as shown in our bleaching experiment where we observe fluorescence reduction alongside coral bleaching.

scholarly journals Multicolor fluorescent imaging by space-constrained computational hyperspectral imaging

2018 ◽  
Author(s):  
Yina Wang ◽  
Bin Yang ◽  
Siyu Feng ◽  
Veronica Pessino ◽  
Bo Huang
Keyword(s):  
Experimental Data ◽  
Hyperspectral Imaging ◽  
Fluorescent Proteins ◽  
Emission Spectra ◽  
Fluorescent Imaging ◽  
Cellular Structures ◽  
Imaging Method ◽  
Color Imaging ◽  
Powerful Technique ◽  
Under Sampling

AbstractHyperspectral imaging is a powerful technique to simultaneously study multiple fluorophore labels with overlapping emissions. Here we present a computational hyperspectral imaging method, which uses the sample spatial fluorescence information as a reconstruction constraint. Our method addresses both the under-sampling issue of compressive hyperspectral imaging and the low throughput issue of scanning hyperspectral imaging. With simulated and experimental data, we have demonstrated the superior reconstruction precision of our method in two and three-color imaging. We have experimentally validated this method in differentiating cellular structures labeled with two red-colored fluorescent proteins, tdTomato and mCherry, which have highly overlapping emission spectra. Our method has the advantage of totally free wavelength choice and can also be combined with conventional filter-based sequential multi-color imaging to further expand the choices of probes.

Illuminating subcellular structures and dynamics in plants: a fluorescent protein toolboxThis review is one of a selection of papers published in the Special Issue on Plant Cell Biology.

2006 ◽  
Vol 84 (4) ◽  
pp. 515-522 ◽  
Author(s):  
Preetinder K. Dhanoa ◽  
Alison M. Sinclair ◽  
Robert T. Mullen ◽  
Jaideep Mathur
Keyword(s):  
Plant Cell ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Live Imaging ◽  
Living Cells ◽  
Special Issue ◽  
Exciting Possibility ◽  
Selection Of

The discovery and development of multicoloured fluorescent proteins has led to the exciting possibility of observing a remarkable array of subcellular structures and dynamics in living cells. This minireview highlights a number of the more common fluorescent protein probes in plants and is a testimonial to the fact that the plant cell has not lagged behind during the live-imaging revolution and is ready for even more in-depth exploration.

scholarly journals Differential equation methods for simulation of GFP kinetics in non–steady state experiments

2018 ◽  
Vol 29 (6) ◽  
pp. 763-771 ◽  
Author(s):  
Robert D. Phair
Keyword(s):  
Differential Equation ◽  
Steady State ◽  
Fluorescence Microscopy ◽  
Cell Biology ◽  
Fluorescent Proteins ◽  
Quantitative Information ◽  
Tracer Kinetics ◽  
Mathematical Framework ◽  
Experimental Protocols ◽  
Tracer Kinetic

Genetically encoded fluorescent proteins, combined with fluorescence microscopy, are widely used in cell biology to collect kinetic data on intracellular trafficking. Methods for extraction of quantitative information from these data are based on the mathematics of diffusion and tracer kinetics. Current methods, although useful and powerful, depend on the assumption that the cellular system being studied is in a steady state, that is, the assumption that all the molecular concentrations and fluxes are constant for the duration of the experiment. Here, we derive new tracer kinetic analytical methods for non–steady state biological systems by constructing mechanistic nonlinear differential equation models of the underlying cell biological processes and linking them to a separate set of differential equations governing the kinetics of the fluorescent tracer. Linking the two sets of equations is based on a new application of the fundamental tracer principle of indistinguishability and, unlike current methods, supports correct dependence of tracer kinetics on cellular dynamics. This approach thus provides a general mathematical framework for applications of GFP fluorescence microscopy (including photobleaching [FRAP, FLIP] and photoactivation to frequently encountered experimental protocols involving physiological or pharmacological perturbations (e.g., growth factors, neurotransmitters, acute knockouts, inhibitors, hormones, cytokines, and metabolites) that initiate mechanistically informative intracellular transients. When a new steady state is achieved, these methods automatically reduce to classical steady state tracer kinetic analysis.

scholarly journals Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Alejandro Sarrion-Perdigones ◽  
Lyra Chang ◽  
Yezabel Gonzalez ◽  
Tatiana Gallego-Flores ◽  
Damian W. Young ◽  
...  
Keyword(s):  
Transcriptional Response ◽  
Emission Spectra ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Research Fields ◽  
Experimental Errors ◽  
Cellular Pathways ◽  
Multiple Signaling ◽  
Simultaneous Assessment ◽  
Spectra Deconvolution

AbstractSensitive simultaneous assessment of multiple signaling pathways within the same cells requires orthogonal reporters that can assay over large dynamic ranges. Luciferases are such genetically encoded candidates due to their sensitivity, versatility, and cost-effectiveness. We expand luciferase multiplexing in post-lysis endpoint luciferase assays from two to six. Light emissions are distinguished by a combination of distinct substrates and emission spectra deconvolution. All six luciferase reporter units are stitched together into one plasmid facilitating delivery of all reporter units through a process we termed solotransfection, minimizing experimental errors. We engineer a multiplex hextuple luciferase assay to probe pathway fluxes through five transcriptional response elements against a control constitutive promoter. We can monitor effects of siRNA, ligand, and chemical compound treatments on their target pathways along with the four other probed cellular pathways. We demonstrate the effectiveness and adaptiveness of multiplex luciferase assaying, and its broad application across different research fields.

scholarly journals Intracellular Imaging with Genetically Encoded RNA-based Molecular Sensors

Nanomaterials ◽  
2019 ◽  
Vol 9 (2) ◽  
pp. 233 ◽  
Author(s):  
Zhining Sun ◽  
Tony Nguyen ◽  
Kathleen McAuliffe ◽  
Mingxu You
Keyword(s):  
Cell Biology ◽  
Design Principle ◽  
Fluorescent Proteins ◽  
Rna Recognition ◽  
Molecular Sensors ◽  
Intracellular Imaging ◽  
Biological Targets ◽  
Target Analytes ◽  
Wide Range ◽  
Reporting Systems

Genetically encodable sensors have been widely used in the detection of intracellular molecules ranging from metal ions and metabolites to nucleic acids and proteins. These biosensors are capable of monitoring in real-time the cellular levels, locations, and cell-to-cell variations of the target compounds in living systems. Traditionally, the majority of these sensors have been developed based on fluorescent proteins. As an exciting alternative, genetically encoded RNA-based molecular sensors (GERMS) have emerged over the past few years for the intracellular imaging and detection of various biological targets. In view of their ability for the general detection of a wide range of target analytes, and the modular and simple design principle, GERMS are becoming a popular choice for intracellular analysis. In this review, we summarize different design principles of GERMS based on various RNA recognition modules, transducer modules, and reporting systems. Some recent advances in the application of GERMS for intracellular imaging are also discussed. With further improvement in biostability, sensitivity, and robustness, GERMS can potentially be widely used in cell biology and biotechnology.

scholarly journals Comparative Analysis of Bacteriophytochrome Agp2 and Its Engineered Photoactivatable NIR Fluorescent Proteins PAiRFP1 and PAiRFP2

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1286 ◽  
Author(s):  
Faez Iqbal Khan ◽  
Fakhrul Hassan ◽  
Razique Anwer ◽  
Feng Juan ◽  
Dakun Lai
Keyword(s):  
Comparative Analysis ◽  
Structural Changes ◽  
Near Infrared ◽  
Fluorescent Proteins ◽  
Irradiation Time ◽  
Fluorescence Emission ◽  
Emission Spectra ◽  
Md Simulations ◽  
Fluorescence Emission Spectra ◽  
Molecular Stability

Two photoactivatable near infrared fluorescent proteins (NIR FPs) named “PAiRFP1” and “PAiRFP2” are formed by directed molecular evolution from Agp2, a bathy bacteriophytochrome of Agrobacterium tumefaciens C58. There are 15 and 24 amino acid substitutions in the structure of PAiRFP1 and PAiRFP2, respectively. A comprehensive molecular exploration of these bacteriophytochrome photoreceptors (BphPs) are required to understand the structure dynamics. In this study, the NIR fluorescence emission spectra for PAiRFP1 were recorded upon repeated excitation and the fluorescence intensity of PAiRFP1 tends to increase as the irradiation time was prolonged. We also predicted that mutations Q168L, V244F, and A480V in Agp2 will enhance the molecular stability and flexibility. During molecular dynamics (MD) simulations, the average root mean square deviations of Agp2, PAiRFP1, and PAiRFP2 were found to be 0.40, 0.49, and 0.48 nm, respectively. The structure of PAiRFP1 and PAiRFP2 were more deviated than Agp2 from its native conformation and the hydrophobic regions that were buried in PAiRFP1 and PAiRFP2 core exposed to solvent molecules. The eigenvalues and the trace of covariance matrix were found to be high for PAiRFP1 (597.90 nm2) and PAiRFP2 (726.74 nm2) when compared with Agp2 (535.79 nm2). It was also found that PAiRFP1 has more sharp Gibbs free energy global minima than Agp2 and PAiRFP2. This comparative analysis will help to gain deeper understanding on the structural changes during the evolution of photoactivatable NIR FPs. Further work can be carried out by combining PCR-based directed mutagenesis and spectroscopic methods to provide strategies for the rational designing of these PAiRFPs.

scholarly journals Genetically encoded fluorescent tags

2017 ◽  
Vol 28 (7) ◽  
pp. 848-857 ◽  
Author(s):  
Kurt Thorn
Keyword(s):  
Flow Cytometry ◽  
Nucleic Acid ◽  
Light Microscopy ◽  
Cell Biology ◽  
Fluorescent Proteins ◽  
Protein Sequences ◽  
Biological Properties ◽  
Protein Abundance ◽  
Fluorescent Tags ◽  
Exogenous Fluorophores

Genetically encoded fluorescent tags are protein sequences that can be fused to a protein of interest to render it fluorescent. These tags have revolutionized cell biology by allowing nearly any protein to be imaged by light microscopy at submicrometer spatial resolution and subsecond time resolution in a live cell or organism. They can also be used to measure protein abundance in thousands to millions of cells using flow cytometry. Here I provide an introduction to the different genetic tags available, including both intrinsically fluorescent proteins and proteins that derive their fluorescence from binding of either endogenous or exogenous fluorophores. I discuss their optical and biological properties and guidelines for choosing appropriate tags for an experiment. Tools for tagging nucleic acid sequences and reporter molecules that detect the presence of different biomolecules are also briefly discussed.

scholarly journals From imaging to understanding: Frontiers in Live Cell Imaging, Bethesda, MD, April 19–21, 2006

2006 ◽  
Vol 174 (4) ◽  
pp. 481-484 ◽  
Author(s):  
Yu-li Wang ◽  
Klaus M. Hahn ◽  
Robert F. Murphy ◽  
Alan F. Horwitz
Keyword(s):  
Cell Biology ◽  
Live Cell Imaging ◽  
Recent Progress ◽  
Cell Imaging ◽  
Live Cell ◽  
The Other ◽  
Biological Applications ◽  
Research Fields ◽  
Recent Meeting ◽  
The One

A recent meeting entitled Frontiers in Live Cell Imaging was attended by more than 400 cell biologists, physicists, chemists, mathematicians, and engineers. Unlike typical special topics meetings, which bring together investigators in a defined field primarily to review recent progress, the purpose of this meeting was to promote cross-disciplinary interactions by introducing emerging methods on the one hand and important biological applications on the other. The goal was to turn live cell imaging from a “technique” used in cell biology into a new exploratory science that combines a number of research fields.

FRET Studies Between CdTe Capped by Small-Molecule Ligands and Fluorescent Protein

2014 ◽  
Vol 13 (05n06) ◽  
pp. 1460013
Author(s):  
Yue Zhang ◽  
Dejian Zhou ◽  
Junhui He
Keyword(s):  
Fluorescent Protein ◽  
Organic Dyes ◽  
Fluorescent Proteins ◽  
Semiconductor Nanocrystals ◽  
Resonance Energy ◽  
Emission Spectra ◽  
Binding Constants ◽  
Dipole Interaction ◽  
Water Soluble ◽  
Donor Acceptor

Water-soluble luminescent semiconductor nanocrystals also known as quantum dots (QDs) that have prominent photostability, wide absorption cross sections and tunable narrow emission, have been shown as promising probes in immunoassays. QDs are often used as donors in fluorescence resonance energy transfer (FRET) based sensors using organic dyes or fluorescent proteins as acceptors. Here, the FRET between a QD donor and fluorescent protein acceptors has been studied. The fluorescent protein (FP)mCherry appended with a hexa-histidine-tag could effectively self-assemble onto CdTe to produce small donor-acceptor distances and hence highly efficient FRET (efficiency > 80%) at relatively low FP: CdTe copy numbers (ca.1). Using the Förster dipole–dipole interaction formula, the Förster radius (R0) and respective donor-acceptor distances for the CdTe -FP FRET systems have been calculated. The binding constants (Kd) of the QD-FP systems have also been evaluated by the emission spectra.

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