scholarly journals Fluorescent Markers: Proteins and Nanocrystals

2021 ◽  
Author(s):  
Anielle Christine Almeida Silva ◽  
Jerusa Maria de Oliveira ◽  
Kelen Talita Romão da Silva ◽  
Francisco Rubens Alves dos Santos ◽  
João Paulo Santos de Carvalho ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Biological Systems ◽  
Biological Molecules ◽  
Fluorescent Reporter ◽  
Fluorescent Markers ◽  
Drosophila Model ◽  
Reporter Proteins ◽  
Red Luminescence

This book chapter will comment on fluorescent reporter proteins and nanocrystals’ applicability as fluorescent markers. Fluorescent reporter proteins in the Drosophila model system offer a degree of specificity that allows monitoring cellular and biochemical phenomena in vivo, such as autophagy, mitophagy, and changes in the redox state of cells. Titanium dioxide (TiO2) nanocrystals (NCs) have several biological applications and emit in the ultraviolet, with doping of europium ions can be visualized in the red luminescence. Therefore, it is possible to monitor nanocrystals in biological systems using different emission channels. CdSe/CdS magic-sized quantum dots (MSQDs) show high luminescence stability in biological systems and can be bioconjugated with biological molecules. Therefore, this chapter will show exciting results of the group using fluorescent proteins and nanocrystals in biological systems.

scholarly journals Collection of Recombinant Rotaviruses Expressing Fluorescent Reporter Proteins

2019 ◽  
Vol 8 (27) ◽  
Author(s):  
Asha A. Philip ◽  
Brittany E. Herrin ◽  
Maximiliano L. Garcia ◽  
Andrew T. Abad ◽  
Sarah P. Katen ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Proteins ◽  
Genome Segment ◽  
Live Cell ◽  
Fluorescent Reporter ◽  
Fluorescent Markers ◽  
Reporter Proteins

ABSTRACT A collection of recombinant rotaviruses that express the fluorescent markers UnaG, mKate, mRuby, TagBFP, CFP, or YFP as separate proteins was generated. Genes for the fluorescent proteins were inserted into genome segment 7 without compromising expression of the protein NSP3. These recombinant rotaviruses are valuable for analyzing rotavirus biology by fluorescence-based live-cell imaging.

Cellular Uptake of Carbon Nanotubes Conjugated to Semiconductor Quantum Dots for Breast Cancer Imaging and Treatment Applications

2010 ◽  
Author(s):  
Kristen A. Zimmermann ◽  
Jianfei Zhang ◽  
Harry Dorn ◽  
Christopher Rylander ◽  
Marissa Nichole Rylander
Keyword(s):  
Carbon Nanotubes ◽  
Quantum Dots ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Properties ◽  
Breast Cancer Imaging ◽  
Fluorescent Markers ◽  
Green Fluorescent

Carbon nanotubes (CNTs) are attractive materials for early detection, treatment, and imaging of cancer malignancies; however, they are limited by their inability to be monitored in vitro and in vivo [1]. Unlabeled CNTs are difficult to distinguish using elemental analysis because they are composed entirely of carbon, which is also characteristic of cellular membranes. Although some single walled nanotubes (SWNT) have been found to exhibit fluorescent properties, not all particles in a single batch fluoresce [2]. Additionally, these emissions may be too weak to be detected using conventional imaging modalities [3]. Incorporating fluorescent markers, such as fluorescent proteins or quantum dots, allows the non-fluorescent particles to be visualized. Previously, fluorophores, such as green fluorescent protein (GFP) or red fluorescent protein (RFP), have been used to visualize and track cells or other particles in biological environments, but their low quantum yield and tendency to photobleach generate limitations for their use in such applications.

scholarly journals A Bright Future for Fluorescence Imaging of Fungi in Living Hosts

2019 ◽  
Vol 5 (2) ◽  
pp. 29 ◽  
Author(s):  
Ambre Chapuis ◽  
Elizabeth Ballou ◽  
Donna MacCallum
Keyword(s):  
Mouse Models ◽  
Fluorescent Proteins ◽  
Imaging Techniques ◽  
Post Mortem ◽  
New Techniques ◽  
Ethical Concerns ◽  
Non Invasive ◽  
Reporter Proteins ◽  
Invasive Techniques

Traditional in vivo investigation of fungal infection and new antifungal therapies in mouse models is usually carried out using post mortem methodologies. However, biomedical imaging techniques focusing on non-invasive techniques using bioluminescent and fluorescent proteins have become valuable tools. These new techniques address ethical concerns as they allow reduction in the number of animals required to evaluate new antifungal therapies. They also allow better understanding of the growth and spread of the pathogen during infection. In this review, we concentrate on imaging technologies using different fungal reporter proteins. We discuss the advantages and limitations of these different reporters and compare the efficacy of bioluminescent and fluorescent proteins for fungal research.

scholarly journals Flavin Mononucleotide-Based Fluorescent Reporter Proteins Outperform Green Fluorescent Protein-Like Proteins as Quantitative In Vivo Real-Time Reporters

2010 ◽  
Vol 76 (17) ◽  
pp. 5990-5994 ◽  
Author(s):  
Thomas Drepper ◽  
Robert Huber ◽  
Achim Heck ◽  
Franco Circolone ◽  
Anne-Kathrin Hillmer ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Real Time ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Flavin Mononucleotide ◽  
Fluorescence Signal ◽  
Reporter Protein ◽  
Reporter Proteins ◽  
Green Fluorescent

ABSTRACT Fluorescent proteins of the green fluorescent protein (GFP) family are commonly used as reporter proteins for quantitative analysis of complex biological processes in living microorganisms. Here we demonstrate that the fluorescence signal intensity of GFP-like proteins is affected under oxygen limitation and therefore does not reflect the amount of reporter protein in Escherichia coli batch cultures. Instead, flavin mononucleotide (FMN)-binding fluorescent proteins (FbFPs) are suitable for quantitative real-time in vivo assays under these conditions.

scholarly journals A Fluorescent Split Aptamer for Visualizing RNA-RNA Assembly In Vivo

2017 ◽  
Author(s):  
Khalid K. Alam ◽  
Kwaku D. Tawiah ◽  
Matthew F. Lichte ◽  
David Porciani ◽  
Donald H. Burke
Keyword(s):  
Self Assembly ◽  
Living Cells ◽  
Rna Aptamers ◽  
Genetic Circuits ◽  
Fluorescent Reporter ◽  
Indirect Measures ◽  
Reporter Proteins ◽  
Circuit Function

AbstractRNA-RNA assembly governs key biological processes and is a powerful tool for engineering synthetic genetic circuits. Characterizing RNA assembly in living cells often involves monitoring fluorescent reporter proteins, which are at best indirect measures of underlying RNA-RNA hybridization events and are subject to additional temporal and load constraints associated with translation and activation of reporter proteins. In contrast, RNA aptamers that sequester small molecule dyes and activate their fluorescence are increasingly utilized in genetically-encoded strategies to report on RNA-level events. Split-aptamer systems have been rationally designed to generate signal upon hybridization of two or more discrete RNA transcripts, but none directly function when expressed in vivo. We reasoned that the improved physiological properties of the Broccoli aptamer enable construction of a split-aptamer system that could function in living cells. Here we present the Split-Broccoli system, in which self-assembly is nucleated by a thermostable, three-way junction RNA architecture and fluorescence activation requires both strands. Functional assembly of the system approximately follows second order kinetics in vitro and improves when cotranscribed, rather than when assembled from purified components. Split-Broccoli fluorescence is digital in vivo and retains functional modularity when fused to RNAs that regulate circuit function through RNA-RNA hybridization, as demonstrated with an RNA Toehold switch. Split-Broccoli represents the first functional split-aptamer system to operate in vivo. It offers a genetically-encoded and nondestructive platform to monitor and exploit RNA-RNA hybridization, whether as an all-RNA, stand-alone AND gate or as a tool for monitoring assembly of RNA-RNA hybrids.

scholarly journals FRAME-tags: genetically encoded fluorescent markers for multiplexed barcoding and time-resolved tracking of live cells

2021 ◽  
Author(s):  
Andrew V. Anzalone ◽  
Miguel Jimenez ◽  
Virginia W. Cornish
Keyword(s):  
Fitness Landscape ◽  
Fluorescent Proteins ◽  
Biological Systems ◽  
Emission Spectra ◽  
Live Cells ◽  
Time Resolved ◽  
Detection Techniques ◽  
Fluorescent Markers ◽  
Spectral Channels

Cellular barcodes offer critical tools for tracking cellular identity in biological systems. Although genetically encoded fluorescent barcodes are ideal for real-time tracking, their scalability is constrained by the broad, overlapping emission spectra characteristic of fluorescent proteins (FPs). Here, we describe a palette of genetically encoded fluorescent barcodes called FRAME- tags, which break this scalability barrier by encoding barcode identity as unique FP expression ratios. FRAME-tags use -1 programmed ribosomal frameshifting RNA motifs to precisely control the translational output of multiple FPs from a single mRNA, leading to extremely narrow and resolvable ratios of the corresponding cellular fluorescence distributions. With this platform, we constructed 20 resolvable FRAME-tags in yeast using just two FPs, and further demonstrated that 100 or more distinguishable FRAME-tags could be made by the addition of a third FP. We used FRAME-tags to map the dynamic fitness landscape of yeast co-cultures, and to characterize the expression pattern of 20 yeast promoters in multiplex across diverse conditions. FRAME-tags offer a valuable new tool for cellular barcoding that enables time- resolved characterization of complex biological systems using widely available fluorescence detection techniques and a minimal number of spectral channels.

scholarly journals FRaeppli, a multispectral imaging toolbox for cell tracing and dense tissue analysis in zebrafish

2022 ◽  
Author(s):  
Sara Caviglia ◽  
Iris A Unterweger ◽  
Akvile Gasiunaite ◽  
Alexandre E Vanoosthuyse ◽  
Francesco Cutrale ◽  
...  
Keyword(s):  
Multispectral Imaging ◽  
Fluorescent Proteins ◽  
Tissue Analysis ◽  
Internal Organs ◽  
Simultaneous Imaging ◽  
Fluorescent Markers ◽  
Mammalian Liver ◽  
Cell Shapes ◽  
Dense Tissue

Visualizing cell shapes, interactions and lineages of differentiating cells is instrumental for understanding organ development and repair. Across species, strategies for stochastic multicolour labelling have greatly facilitated tracking cells in in vivo and mapping neuronal connectivity. Nevertheless, integrating multi-fluorophore information into the context of developing tissues in zebrafish is challenging given their cytoplasmic localization and spectral incompatibility with commonly used fluorescent markers. Here, we developed FRaeppli (Fish-Raeppli) expressing bright membrane- or nuclear-targeted fluorescent proteins for efficient cell shape analysis and tracking. High spatiotemporal activation flexibility is provided by the Gal4/UAS system together with Cre/lox and/or PhiC31integrase. The distinct spectra of the FRaeppli fluorescent proteins allow simultaneous imaging with GFP and infrared subcellular reporters or tissue landmarks. By tailoring hyperspectral protocols for time-efficient acquisition, we demonstrate FRaeppli s suitability for live imaging of complex internal organs, like the liver. Combining FRaeppli with polarity markers revealed previously unknown canalicular topologies between differentiating hepatocytes, reminiscent of the mammalian liver, suggesting shared developmental mechanisms. The multispectral FRaeppli toolbox thus enables the comprehensive analysis of intricate cellular morphologies, topologies and tissue lineages at single-cell resolution in zebrafish.

scholarly journals Multi-spectral fluorescent reporter influenza viruses (Color-flu) as powerful tools for in vivo studies

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Satoshi Fukuyama ◽  
Hiroaki Katsura ◽  
Dongming Zhao ◽  
Makoto Ozawa ◽  
Tomomi Ando ◽  
...  
Keyword(s):  
Influenza A ◽  
Fluorescent Proteins ◽  
Virus Antigen ◽  
Influenza Viruses ◽  
In Vivo Studies ◽  
Live Cells ◽  
Influenza A Viruses ◽  
Fluorescent Reporter ◽  
Viral Spread

Abstract Seasonal influenza A viruses cause annual epidemics of respiratory disease; highly pathogenic avian H5N1 and the recently emerged H7N9 viruses cause severe infections in humans, often with fatal outcomes. Although numerous studies have addressed the pathogenicity of influenza viruses, influenza pathogenesis remains incompletely understood. Here we generate influenza viruses expressing fluorescent proteins of different colours (‘Color-flu’ viruses) to facilitate the study of viral infection in in vivo models. On adaptation to mice, stable expression of the fluorescent proteins in infected animals allows their detection by different types of microscopy and by flow cytometry. We use this system to analyse the progression of viral spread in mouse lungs, for live imaging of virus-infected cells, and for differential gene expression studies in virus antigen-positive and virus antigen-negative live cells in the lungs of Color-flu-infected mice. Collectively, Color-flu viruses are powerful tools to analyse virus infections at the cellular level in vivo to better understand influenza pathogenesis.

scholarly journals A series of dual-reporter vectors for ratiometric analysis of protein abundance in plants

2020 ◽  
Author(s):  
Aashima Khosla ◽  
Cecilia Rodriguez-Furlan ◽  
Suraj Kapoor ◽  
Jaimie M. Van Norman ◽  
David C. Nelson
Keyword(s):  
Signal To Noise Ratio ◽  
High Signal ◽  
Signal To Noise ◽  
Fluorescent Reporter ◽  
Dual Reporter ◽  
Reference Protein ◽  
Reporter Systems ◽  
Reporter Proteins ◽  
Single Transcript

ABSTRACTRatiometric reporter systems enable comparisons of the abundance of a protein of interest, or “target,” relative to a reference protein. Both proteins are encoded on a single transcript but are separated during translation. This arrangement bypasses the potential for discordant expression that can arise when the target and reference proteins are encoded by separate genes. We generated a set of 18 Gateway-compatible vectors termed pRATIO that combine a variety of promoters, fluorescent and bioluminescent reporters, and 2A “self-cleaving” peptides. These constructs are easily modified to produce additional combinations or introduce new reporter proteins. We found that mScarlet-I provides the best signal-to-noise ratio among several fluorescent reporter proteins during transient expression experiments in Nicotiana benthamiana. Firefly and Gaussia luciferase also produce high signal-to-noise in N. benthamiana. As proof of concept, we used this system to investigate whether degradation of the receptor KAI2 after karrikin treatment is influenced by its subcellular localization. KAI2 is normally found in the cytoplasm and the nucleus of plant cells. In N. benthamiana, karrikin-induced degradation of KAI2 was only observed when it was retained in the nucleus. These vectors are tools to easily monitor in vivo the abundance of a protein that is transiently expressed in plants, and will be particularly useful for investigating protein turnover in response to different stimuli.

scholarly journals Development of Pseudorabies Virus Strains Expressing Red Fluorescent Proteins: New Tools for Multisynaptic Labeling Applications

2003 ◽  
Vol 77 (18) ◽  
pp. 10106-10112 ◽  
Author(s):  
Bruce W. Banfield ◽  
Jessica D. Kaufman ◽  
Jessica A. Randall ◽  
Gary E. Pickard
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Retrograde Transport ◽  
Fluorescent Reporter ◽  
Recombinant Strains ◽  
Transsynaptic Tracing ◽  
Green Fluorescent ◽  
Virus Strains

ABSTRACT The transsynaptic retrograde transport of the pseudorabies virus Bartha (PRV-Bartha) strain has become an important neuroanatomical tract-tracing technique. Recently, dual viral transneuronal labeling has been introduced by employing recombinant strains of PRV-Bartha engineered to express different reporter proteins. Dual viral transsynaptic tracing has the potential of becoming an extremely powerful method for defining connections of single neurons to multiple neural circuits in the brain. However, the present use of recombinant strains of PRV expressing different reporters that are driven by different promoters, inserted in different regions of the viral genome, and detected by different methods limits the potential of these recombinant virus strains as useful reagents. We previously constructed and characterized PRV152, a PRV-Bartha derivative that expresses the enhanced green fluorescent protein. The development of a strain isogenic to PRV152 and differing only in the fluorescent reporter would have great utility for dual transsynaptic tracing. In this report, we describe the construction, characterization, and application of strain PRV614, a PRV-Bartha derivative expressing a novel monomeric red fluorescent protein, mRFP1. In contrast to viruses expressing DsRed and DsRed2, PRV614 displayed robust fluorescence both in cell culture and in vivo following transsynaptic transport through autonomic circuits afferent to the eye. Transneuronal retrograde dual PRV labeling has the potential to be a powerful addition to the neuroanatomical tools for investigation of neuronal circuits; the use of strain PRV614 in combination with strain PRV152 will eliminate many of the pitfalls associated with the presently used pairs of PRV recombinants.

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