These are human skin cells (HaCaT keratinocytes) expressing fluorescently tagged keratin.
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Drums thundered at earsplitting volumes and children shrieked in delight as the lions chased them through the fluorescently lit streets.
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When I'm packed into a musty, fluorescently lit rail car during rush hour, I want to be anywhere else, and fast.
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Standing in the white-tiled, blood-swept aisle of Butcher Row in Leeds Market, one thing is fluorescently clear: Yorkshire loves pies.
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She took me to a dungeon of a room, the kind of fluorescently lit place where you might be sent for school detention.
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After the lobby and a short elevator ride, a bare, fluorescently lit hall led to an office door with a sign advertising weekly office doughnuts.
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His current solo show incorporates about a dozen spray painted canvases and two watercolors on paper, varying in size, both visually intoxicating and fluorescently frenetic.
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After being heavily bombed during World War II, Pantelleria Town, with its fluorescently lit groceries and dingy cafes, is considered a less picturesque part of the island.
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In a narrow, fluorescently lit hallway papered with old show fliers, they fueled themselves with beer, coffee and Red Bull while psyching each other up for the "last dance" and speculating about the future.
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See, most modern sequencing technologies work by taking a sample of your DNA, chopping it up into millions of short snippets, and then using fluorescently-tagged nucleotides to produce reads—the list of As, Ts, Cs, and Gs that correspond to each snippet.
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Signs hanging among the fluorescently lit shelves, which resemble library stacks if the stacks went on so far that you couldn't see the opposite end, tell workers they have 10 minutes to pick up their first item when they start their shift.
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I saw at least two female butchers in overalls and aprons fingering huge cuts of red-pink marbled meat, not to mention a host of female tellers sitting in their little fluorescently-lit cash cabins, totting up payments and counting out change.
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But instead of waiting for the antibodies to destroy the cancer cells, Green's 2.0 treatment—called FLALANT, for fluorescently-labeled bodies and laser-activated nanotherapy—uses the antibodies' ability to find tumors and lesions throughout the body as a delivery vehicle for her nanoparticles.
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This record feels good when you listen to it in a dark, sweaty club next to your crush or when walking along a busy fluorescently lit street on a hot summer night as the sky turns from cotton candy pink to inky dark blue.
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The Federal Department of Oxygen (DOO) was a jewel of a building in the center of Washington D.C. Through a curtain of smog, it shined with newness, a line of fluorescently green plastic shrubs following the cut of its foundation and a rise of Greek steps leading toward the entrance.
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ZIF-8 luminescence is highly sensitive to Cu2+, and Cd2+ ions as well as acetone. ZIF nanoparticles can also sense fluorescently tagged single stranded pieces of DNA.
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Sartorius Stedium has developed several reagents for the Virus Counter since acquiring it in 2016. These reagents include Combodye, a duel stained reagent for enveloped viruses and Virotag, specific fluorescently labeled, high-affinity reagents.
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Derivatives conjugated with fluorophores are sold widely. Because of its ability to selectively bind filamentous actin (F-actin) and not actin monomers (G-actin), fluorescently labeled phalloidin is more effective than antibodies against actin.
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CO-FISH, or strand-specific fluorescence in situ hybridization, facilitates strand-specific targeting of DNA with fluorescently-tagged probes. It exploits the uniform orientation of major satellites relative to the direction of telomeres, thus allowing strands to be unambiguously designated as "Watson" or "Crick" strands. Using unidirectional probes that recognize major satellite regions, coupled to fluorescently labelled dyes, individual strands can be bound. To ensure that only the template strand is labelled, the newly formed strands must be degraded by BrdU incorporation and photolysis.
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Katanin has proved necessary in this task. An experiment using time-lapse digital imaging of fluorescently labeled tubulin demonstrated that axon growth cones pause, and microtubules fragment, at sites of branching during neural development. Dent, E., Callaway, J., Gyorgyi, S., Baas, P. & Kalil, K. (1999) Reorganization and Movement of Microtubules in Axonal Growth Cones and Developing Interstitial Branches. A similar experiment using fluorescently labeled tubulin observed local microtubule fragmentation in newt lung cell lamellipodia during developmental migration, in which the fragments run perpendicular to the advancing cell membrane to aid exploration.
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To detect those human antibodies, the array is covered with a solution of a fluorescently labeled secondary antibody. This secondary antibody binds to the patient antibody already on the array from the diluted serum sample, and since this secondary antibody is fluorescently labeled, it is detectable using fluorescence microscopy. After the microarray is washed to remove unbound secondary antibody, and dried via centrifugation, it is scanned using fluorescence microscopy, and the pattern of peptide spots with bound antibodies versus those without antibodies becomes visible. This pattern is called the immunosignature.
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Microscopy is often also used in conjunction with biochemical staining techniques, and can be made exquisitely specific when used in combination with antibody based techniques. For example, the use of antibodies made artificially fluorescent (fluorescently labeled antibodies) can be directed to bind to and identify a specific antigens present on a pathogen. A fluorescence microscope is then used to detect fluorescently labeled antibodies bound to internalized antigens within clinical samples or cultured cells. This technique is especially useful in the diagnosis of viral diseases, where the light microscope is incapable of identifying a virus directly.
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This allows for the nanometer height measurements. FLIC microscope is well suited to measuring the topography of a membrane that contains fluorescent probes e.g. an artificial lipid bilayer, or a living cell membrane or the structure of fluorescently labeled proteins on a surface.
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Fluorescent signal strength depends on many factors such as probe labeling efficiency, the type of probe, and the type of dye. Fluorescently tagged antibodies or streptavidin are bound to the dye molecule. These secondary components are selected so that they have a strong signal.
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A recent study found that this resistance is due to expression of multidrug resistance protein 1 (MDR1). Fluorescently labeled eribulin has been used to study the pharmacokinetics and pharmacodynamics at single cell level in vivo. A new synthetic route to the drug was published in 2009.
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The overall assay consisted of RT-PCR amplification of influenza RNA, subsequent runoff transcription using the PCR product as template, and hybridization of fluorescently-labeled fragmented RNA to the microarray surface. The overall pattern of fluorescence intensities were utilized to type and subtype the influenza virus(es) present.
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CHPs can be used for visualizing many different types of collagen bands in SDS-PAGE gels. Collagen is denatured by heating in the presence of SDS prior to loading the gel. The collagen bands are visualized through CHP-collagen hybridization when the gels are stained by fluorescently- labeled CHPs.
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Colocalization is used in real-time single-molecule fluorescence microscopy to detect interactions between fluorescently labeled molecular species. In this case, one species (e.g. a DNA molecule) is typically immobilized on the imaging surface, and the other species (e.g. a DNA-binding protein) is supplied to the solution.
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Terminal restriction fragment length polymorphism (T-RFLP) is a method that uses fluorescently-labeled DNA fragments to produce a community fingerprint. This section presents a brief explanation of T-RFLP in the specific context of community fingerprinting. For a more detailed explanation, refer to the T-RFLP article.
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To perform T-RFLP (Figure 1), one must select a target gene (e.g. the 16S rRNA gene) to amplify by PCR. At least one primer used in the PCR reaction is fluorescently labeled at the 5´ end. After PCR amplification, each copied DNA segment carries the fluorescent label.
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Primer extension is able to genotype most SNPs under very similar reaction conditions making it also highly flexible. The primer extension method is used in a number of assay formats. These formats use a wide range of detection techniques that include MALDI-TOF Mass spectrometry (see Sequenom) and ELISA-like methods. Generally, there are two main approaches which use the incorporation of either fluorescently labeled dideoxynucleotides (ddNTP) or fluorescently labeled deoxynucleotides (dNTP). With ddNTPs, probes hybridize to the target DNA immediately upstream of SNP nucleotide, and a single, ddNTP complementary to the SNP allele is added to the 3’ end of the probe (the missing 3'-hydroxyl in didioxynucleotide prevents further nucleotides from being added).
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DNA Microarrays. Within the organisms, genes are transcribed and spliced (in eukaryotes) to produce mature mRNA transcripts (red). The mRNA is extracted from the organism and reverse transcriptase is used to copy the mRNA into stable ds-cDNA (blue). In microarrays, the ds-cDNA is fragmented and fluorescently labelled (orange).
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Antibody epitope mapping is used to find the specificity of an antibody. The epitope (antibody binding site of antigens) is expressed on the bacterial cell surface by expressing a region of the gene encoding the antigen. Flow cytometry with fluorescently-labelled antibodies is used to detect the amount of antibody binding to epitope.
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Fluorescent image of a cell in telophase. Multiple dyes were imaged and are shown in different colours. Fluorescent microscopy allows the direct visualization of molecules at the subcellular level, in both live and fixed cells. Molecules of interest are marked with either green fluorescent protein (GFP), another fluorescent protein, or a fluorescently-labeled antibody.
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Since the mid-20th century chemical fluorescent stains, such as DAPI which binds to DNA, have been used to label specific structures within the cell. More recent developments include immunofluorescence, which uses fluorescently labelled antibodies to recognise specific proteins within a sample, and fluorescent proteins like GFP which a live cell can express making it fluorescent.
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This technique offers several advantages over traditional sequencing methods such as Sanger sequencing. Sanger sequencing requires two reactions, one for the forward primer and another for the reverse primer. Unlike Illumina, Sanger sequencing uses fluorescently labeled dideoxynucleoside triphosphates (ddNTPs) to determine the sequence of the DNA fragment. ddNTPs are missing the 3' OH group and terminates DNA synthesis permanently.
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Amplicon melting using a fluorescently-labeled primer has been described (Gundry et al., 2003) but is less practical than using ds-specific dyes due to the cost of the fluorogenic primer. Scanning of larger amplicons is based on the same principles as outlined above. However, melting temperature and the overall shape of the melting curve become informative.
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One example of artificial ncRNA functionalization is incorporating RNA domains recognized by specific antibodies to the sgRNA. CRISPR-Display can target the sgRNA with a particular epitope sequence to various loci, and fluorescently tagged antibodies can be used to image the locus, showing its localization in the nucleus, and possible interactions with other tagged proteins or genomic loci.
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Fluorescent biomaterials are a possible way of using external factors to observe a pathway more visibly. The method involves fluorescently labeling peptide molecules that would alter an organism's natural pathway. When this peptide is inserted into the organism's cell, it can induce a different reaction. This method can be used, for example to treat a patient and then visibly see the treatment's outcome.
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This combined fluorescent protein is, in general, non-toxic to the organism and rarely interferes with the function of the protein under study. Genetically modified cells or organisms directly express the fluorescently tagged proteins, which enables the study of the function of the original protein in vivo. Growth of protein crystals results in both protein and salt crystals. Both are colorless and microscopic.
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UroVysion is a fluorescence in situ hybridization assay that was developed for the detection of bladder cancer in urine specimens. It consists of fluorescently labeled DNA probes to the pericentromeric regions of chromosomes 3 (red), 7 (green), and 17 (aqua) and to the 9p21 band (gold) location of the P16 tumor suppressor gene.Adv Anat Pathol. 2008 Sep;15(5):279-86.
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Comparative genomic hybridization (CGH), derived from FISH, is used to compare variations in copy number between a biological sample and a reference. CGH was originally developed to observe chromosomal aberrations in tumour cells. This method uses two genomes, a sample and a control, which are labeled fluorescently to distinguish them. In CGH, DNA is isolated from a tumour sample and biotin is attached.
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Array comparative genomic hybridization (aCGH) allows CGH to be performed without cell culture and isolation. Instead, it is performed on glass slides containing small DNA fragments. Removing the cell culture and isolation step dramatically simplifies and expedites the process. Using similar principles to CGH, the sample DNA is isolated and fluorescently labelled, then co-hybridized to single stranded probes to generate signals.
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The resulting PCR products attached to the beads are then covalently bound to a glass slide. Primers hybridize to the P1 adapter sequence within the library template. A set of four fluorescently labelled di-base probes compete for ligation to the sequencing primer. Specificity of the di-base probe is achieved by interrogating every 1st and 2nd base in each ligation reaction.
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With regard to the overall method, FISH can be performed using direct labelling—fluorochromes are attached to the probes—or indirect labelling—the probes are labelled with biotin or digoxigenin which are then detected using fluorescently-labelled streptavidin or antibodies, respectively. CISH is performed using indirect labelling in which antibodies or streptavidin are conjugated to enzymes such as HRP or alkaline phosphatase (AP).
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The first method consists of using fluorescent dyes that are retained nonspecifically in between the double strands. The second method involves probes that code for specific sequences and are fluorescently labeled. Detection of DNA using these methods can only be seen after the hybridization of probes with its complementary DNA takes place. An interesting technique combination is real-time PCR and reverse transcription.
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The manuscript describing the work was rejected multiple times before its eventual publication as an article (together with a shorter jointly-written "letter") within the journal Nature. A series of experiments, first turning a mouse embryo green by fluorescently tagging STAP cells, then videotaping the transformation of T-cells into pluripotent cells, finally convinced skeptics that the results were real.
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Developing P. hawaiensis embryos are clear, allowing for both detailed microscopic analyses in situ and the use of fluorescently tagged tracer molecules in live embryos. Early cleavage is holoblastic (total), allowing the fates of individual early cells to be explored through experimental manipulation. The transcriptome of P. hawaiensis was generated using the pyrosequencing technique. This resource is freely available for download.
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The guiding principle behind flow cytometry is that cells or subcellular particles are tagged with fluorescent probes are passed through a laser beam and sorted by the strength of fluorescence emitted by cells contained in the droplets. MHC tetramer staining by flow cytometry identifies and isolates specific T cells based on the binding specificity of their cell surface receptors with fluorescently- tagged MHC-peptide complexes.
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In fluorescence-detection size exclusion chromatography the protein of interest is fluorescently tagged (e.g., with GFP) and run through a gel filtration column on an FPLC system equipped with a fluorescence detector. The resulting chromatogram allows the researcher to estimate the dispersity and expression level of the tagged protein in the current buffer. Since only fluorescence is measured, only the tagged protein is seen in the chromatogram.
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The resulting bead-DNA complexes are then analyzed using flow cytometry. This technique is able to capture allele and mutation frequencies due to coupling with ddPCR. However, unlike with ddPCR, a larger number of DNA sequences can be interrogated due to the flexibility of using fluorescently bound probes. Another advantage of this system is that the DNA isolated can also be used for downstream sequencing.
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The enzymes bind to the DNA and cut the phosphate backbone, allowing the DNA to be unwound. Topotecan unsilences the paternal UBE2A allele by reducing the transcription of an antisense transcript. Topotecan inhibits topoisomerase I restoring UBE3A levels to wild-type range in cultured mince neurons. Transgenic mice with a fluorescently tagged UBE3A were used to test the effectiveness of unsilencing the paternal copy.
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IFT20 subunit of the particle is localized to the Golgi complex in addition to the basal body and cilia where all previous IFT particle proteins had been found. In living cells, fluorescently tagged IFT20 is highly dynamic and moves between the Golgi complex and the cilium as well as along ciliary microtubules. IFT20 has been shown to interact with SPEF2 in the testis, and plays a role in sperm motility.
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Such an ester with an acid and NHS, sometimes called succinate ester, is stable enough to be purified and stored at low temperatures in the absence of water and, as such, is commercially available. NHS esters are commonly used for protein modification (e.g. an NHS ester of fluorescein is commercially available, and can be added to a protein to obtain a fluorescently labeled protein in one simple reaction and purification step).
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Immunofluorescence of Hep2 Intermediate Filaments Later, Osborn and Weber pioneered fluorescent antibody staining of cellular substructures, a major technique called indirect immunofluorescence microscopy. In developing the method, they tagged microtubules with specific antibodies, then used fluorescently-tagged secondary antibodies (antibodies to the first set of antibodies) to light up the locations of the microtubules in cells. When they began their work in Germany, the cytoskeleton was not heavily researched.
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Antibodies, or another type of binder molecule, are fixed onto a solid support and a fluorescently labeled protein mixture is added. Signal intensities are used to identify proteins. Antibody microarrays are extremely versatile – they can be used to analyze the amount of protein in a mixture, different protein isoforms, posttranslational modifications, and the biochemical activity of proteins. In addition, these microarrays are highly sensitive – they can detect single molecules of protein.
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In processes that involve following a labelled molecule as it is incorporated in some larger polymer, such markers can be used to follow the dynamics of growth/shrinkage of the polymer, as well as its movement. Commonly used fiducial markers are fluorescently labelled monomers of bio-polymers. The task of measuring and quantifying what happens to these is borrowed from methods in physics and computational imaging like Speckle imaging.
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These chain-terminating nucleotides lack a 3'-OH group required for the formation of a phosphodiester bond between two nucleotides, causing DNA polymerase to cease extension of DNA when a ddNTP is incorporated. The ddNTPs may be radioactively or fluorescently labelled for detection in DNA sequencers. Typically, these machines can sequence up to 96 DNA samples in a single batch (run) in up to 48 runs a day.
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However, depending on the numerical aperture used, the experiment will yield good lateral resolution (x-y) or good vertical resolution (z), but not both. A high N.A. (~1.0) gives good lateral resolution which is best if the goal is to determine long range topography. Low N.A. (~0.001), on the other hand, provides accurate z-height measurement to determine the height of a fluorescently labeled molecule in a system.
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A multispectral image of tissue from a mouse intestine, showing how autofluoresce can obscure several fluorescence signals. Autofluorescence can be problematic in fluorescence microscopy. Light-emitting stains (such as fluorescently labelled antibodies) are applied to samples to enable vizualisation of specific structures. Autofluorescence interferes with detection of specific fluorescent signals, especially when the signals of interest are very dim — it causes structures other than those of interest to become visible.
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In these techniques, a sample containing a fluorescently-tagged protein of interest is plunge-frozen and first imaged in a light microscope equipped with a special stage to allow the sample to be kept at sub- crystallization temperatures (< −150 °C). The location of the fluorescent signal is identified and the sample is transferred to the CryoTEM, where the same location is then imaged at high resolution by CryoET.
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Thermophoresis depends on the interface between molecule and solvent. Under constant buffer conditions, thermophoresis probes the size, charge and solvation entropy of the molecules. The thermophoresis of a fluorescently labeled molecule A typically differs significantly from the thermophoresis of a molecule-target complex AT due to size, charge and solvation entropy differences. This difference in the molecule's thermophoresis is used to quantify the binding in titration experiments under constant buffer conditions.
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The fluorescently labeled probe is excited by light and the emission of the excitation is then detected by a photosensor such as a CCD camera equipped with appropriate emission filters which captures a digital image of the western blot and allows further data analysis such as molecular weight analysis and a quantitative western blot analysis. Fluorescence is considered to be one of the best methods for quantification but is less sensitive than chemiluminescence.
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To utilize FRET for phosphorylation studies, fluorescent proteins are coupled to both a phosphoamino acid binding domain and a peptide that can by phosphorylated. Upon phosphorylation or dephosphorylation of a substrate peptide, a conformational change occurs that results in a change in fluorescence. FRET has also been used in tandem with Fluorescence Lifetime Imaging Microscopy (FLIM) or fluorescently conjugated antibodies and flow cytometry to provide quantitative results with excellent temporal and spatial resolution.
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Bacterial display can be used to find peptides which bind to specific cells e.g. breast cancer cells or stem cells. Displayed proteins are fluorescently tagged with GFP, so binding interactions between peptides and target cells can be seen by flow cytometry. Control samples are required in order to measure fluorescence levels in the absence of displayed peptides. Samples are also required which don’t contain displayed peptides, but contain mammalian cells and bacterial cells (including the scaffold).
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Some technologies that analyze the sequences can use cluster amplification of adapter-ligated ChIP DNA fragments on a solid flow cell substrate to create clusters of approximately 1000 clonal copies each. The resulting high density array of template clusters on the flow cell surface is sequenced by a Genome analyzing program. Each template cluster undergoes sequencing-by-synthesis in parallel using novel fluorescently labelled reversible terminator nucleotides. Templates are sequenced base-by-base during each read.
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Microscale thermophoresis (MST) measures the size, charge and hydration entropy of molecules/substrates at equilibrium. The thermophoretic movement of a fluorescently labeled substrate changes significantly as it is modified by an enzyme. This enzymatic activity can be measured with high time resolution in real time. The material consumption of the all optical MST method is very low, only 5 µl sample volume and 10nM enzyme concentration are needed to measure the enzymatic rate constants for activity and inhibition.
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Once the DNA template has been completely synthesized, the fragments are separated by capillary electrophoresis. At the bottom of the capillary tube a laser excites the fluorescently labeled ddNTPs and a camera captures the color emitted. Due to the automated nature of Illumina dye sequencing it is possible to sequence multiple strands at once and gain actual sequencing data quickly. With Sanger sequencing, only one strand is able to be sequenced at a time and is relatively slow.
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These include several protein kinases, ATPases, myosin, and other nucleotide binding proteins. Over the past twenty years, there have been hundreds of papers describing TNP-ATP’s use and applications. Many applications involving this fluorescently labeled nucleotide have helped to clarify structure-function relationships of many ATP-requiring proteins and enzymes. There have also been a growing number of papers that display TNP-ATP use as a means of assessing the ATP-binding capacity of various mutant proteins.
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In literature, this powerful technique of flow cytometry and CFSE has been used to find the efficiency of T-cells in killing the target cells in cancer such as leukemia. In order to visualize the target cell death, both rapid and slow, scientists have used CFSE labelling with antibody staining of certain kinds of cells and fluorescently labelled microbeads. This also gave information regarding the proliferation of the target cells upon the treatment of certain cytokines.
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Many of the competitive single-molecule sequencing methods rely on the incorporation of fluorescently labeled nucleotides. In next-generation sequencing, the fluorescence signal of clusters can be easily detected. However, when the same concept is applied to single-molecule sequencing, the largest complication results from the high error rates. Because it is difficult to detect single labeled molecules, these platforms suffer from low signal-to-noise ratios, often resulting in misdetection or non-detection of fluorescent signals.
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Dye-primer sequencing facilitates reading in an optical system for faster and more economical analysis and automation. The later development by Leroy Hood and coworkers of fluorescently labeled ddNTPs and primers set the stage for automated, high- throughput DNA sequencing.Sequence ladder by radioactive sequencing compared to fluorescent peaks Chain-termination methods have greatly simplified DNA sequencing. For example, chain-termination-based kits are commercially available that contain the reagents needed for sequencing, pre-aliquoted and ready to use.
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Then an oligonucleotide complementary to the suspected pathogen's genetic code is synthesized and chemically tagged with a fluorescent probe. The tissue sample is chemically treated in order to make the cell membranes permeable to the fluorescently tagged oligonucleotide. The fluorescent tag is then added and only binds to the complementary DNA of the suspected pathogen. If the pathogen is present in the tissue sample, then the pathogen's cells will fluoresce after treatment with the tagged oligonucleotide.
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A silicon wafer is typically used as the reflective surface in a FLIC experiment. An oxide layer is then thermally grown on top of the silicon wafer to act as a spacer. On top of the oxide is placed the fluorescently labeled specimen, such as a lipid membrane, a cell or membrane bound proteins. With the sample system built, all that is needed is an epifluorescence microscope and a CCD camera to make quantitative intensity measurements.
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Jablonski diagram of FRET Fluorescence Resonance Energy Transfer (FRET) utilizes energy transferred between the donor and the acceptor molecules that are in close proximity. FRET uses a fluorescently labeled ligand, as with FP. Energy transfer within FRET begins by exciting the donor. The dipole-dipole interaction between the donor and the acceptor molecule transfers the energy from the donor to the acceptor molecule. If the ligand is bound to the receptor-antibody complex, then the acceptor will emit light.
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In a study that used fluorescently labelled bacteria in fishponds to observe protistan bacterivory, ciliate grazing accounted for 56% of total protistan grazing and Halteria, along with two other ciliate genera, Pelagohalteria and Rimostrombidium were responsible approximately 71% of the total ciliate bacterivory.Šimek, K., Jürgens, K., Nedoma, J., Comerma, M., & Armengol, J. (2000). Ecological role and bacterial grazing of Halteria spp.: small freshwater oligotrichs as dominant pelagic ciliate bacterivores. Aquatic Microbial Ecology, 22(1), 43-56.
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The result of digestion is a set of restriction fragments of different lengths, each of which is fluorescently labeled at one end. These are known as "terminal fragments" because they are labeled at the end where the PCR primer attached. (The unlabeled ends are not recorded in the final analysis.) Next, the fragments are separated by size through either gel or capillary electrophoresis. Laser detection captures the size and fluorescence-intensity patterns of the terminal fragments.
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Due to variable non-coding regions, the output for RISA is a gel with different banding patterns, and output for ARISA is an electropherogram with different peaks (similar to T-RFLP). The brightness of the fluorescently labeled primers correlates to how prevalent that bacterial type is in the community. The banding pattern on the gel can be interpreted as a community-specific profile. Each DNA band or peak indicates at least one representative of that organism.
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Additionally, Lippincott-Schwartz's laboratory demonstrated that Golgi enzymes constitutively recycle back to the endoplasmic reticulum and that such recycling plays a central role in the maintenance, biogenesis, and inheritance of the Golgi apparatus in mammalian cells. Within Lippincott-Schwartz lab, current projects include several cell biological areas. For example, protein transport and cytoskeleton interaction, organelle assembly and disassembly, and cell polarity generation. There are also projects analyzing the dynamics of proteins that have been fluorescently labeled.
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Common methods include PCR, multiplex ligation-dependent probe amplification (MLPA), and chromosomal microarray (CMA). Cytogenetic/FISH analysis attaches fluorescently labels DNA markers to a denatured chromosome and is then examined under fluorescent lighting, which reveals mutations caused by translocations or inversions involving 7p21. Occasionally, individuals with SCS have a chromosome translocation, inversion, or ring chromosome 7 involving 7p21 resulting in atypical findings, such as, increased developmental delay. Individuals with SCS, typically have normal brain functioning and rarely have mental impairments.
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As a result, multiple amplification events can be initiated, producing multiple RCA products ("Multiprimed RCA"). # Amplification product detection and visualization, which is most commonly conducted through fluorescent detection, with fluorophore-conjugated dNTP, fluorophore-tethered complementary or fluorescently-labeled molecular beacons. In addition to the fluorescent approaches, gel electrophoresis is also widely used for the detection of RCA product. RCA produces a linear amplification of DNA, as each circular template grows at a given speed for a certain amount of time.
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The effects of neighboring bases and secondary structure to detect the frequency of frameshift mutations has been investigated in depth using fluorescence. Fluorescently tagged DNA, by means of base analogues, permits one to study the local changes of a DNA sequence. Studies on the effects of the length of the primer strand reveal that an equilibrium mixture of four hybridization conformations was observed when template bases looped-out as a bulge, i.e. a structure flanked on both sides by duplex DNA.
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This allowed his laboratory to fluorescently trace synapses with markers to yield high-quality, vivid images of brain activity. These 'brainbow’'line images have been examined around the world to determine specific neuronal pathways within the mosaic of neuron tangles in the brain. Additionally, over his career, Sanes has been a part of hundreds of published papers involving the study of synapses from molecular and embryological perspectives. Most recently, Sanes lab is studying the function of neuronal circuits specifically in the retina.
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Cells can produce antisense RNA molecules naturally, called microRNAs, which interact with complementary mRNA molecules and inhibit their expression. The concept has also been exploited as a molecular biology technique, by artificially introducing a transgene coding for antisense RNA in order to block the expression of a gene of interest. Radioactively or fluorescently labelled antisense RNA can be used to show the level of transcription of genes in various cell types. Some alternative antisense structural types have been experimentally applied as antisense therapy.
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Amsterdam: Elsevier/Academic Press, p. 165. Microtubule in vitro assays for motor proteins such as dynein and kinesin are researched by fluorescently tagging a microtubule and fixing either the microtubule or motor proteins to a microscope slide then visualizing the slide with video-enhanced microscopy to record the travel of the microtubule motor proteins. This allows the movement of the motor proteins along the microtubule or the microtubule moving across the motor proteins. Consequently, some microtubule processes can be determined by kymograph.
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This is often called "whole-chromosome painting." If every possible probe is used, every chromosome, (the whole genome) would be marked fluorescently, which would not be particularly useful for determining features of individual sequences. However, it is possible to create a mixture of smaller probes that are specific to a particular region (locus) of DNA; these mixtures are used to detect deletion mutations. When combined with a specific color, a locus-specific probe mixture is used to detect very specific translocations.
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These multinucleated cells appear very similar to virally induced syncytia. HERV-W's main gene expression is ERVWE-1 which is a highly fusogenic env glycoprotein also called syncytin-1 because it induces the formation of syncytia (multinucleated cells). Scientists began searching for ways that syncytin was involved in placental cytotrophoblast fusion and differentiation. Using monoclonal fluorescently labeled antibodies the Frendo Lab was able to visualize the Env-W expression at the apical membrane of the synctiotrophoblast in first- trimester placentas.
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It has been shown that integration of linkage maps can aid de novo assemblies with long range, chromosome scale recombination data, without which, assemblies can be subject to macro ordering errors. Optical mapping is the process of immobilizing the DNA on a slide and digesting it with restriction enzymes. The fragment ends are then fluorescently tagged and stitched back together. For the last two decades, optical mapping has been prohibitively expensive, but recent advances in technology have reduced cost significantly.
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The microbeads are then arrayed in a flow cell for sequencing and quantification. The sequence signatures are deciphered by the parallel identification of four bases by hybridization to fluorescently labeled encoders (Figure 5). Each of the encoders has a unique label which is detected after hybridization by taking an image of the microbead array. The next step is to cleave and remove that set of four bases and reveal the next four bases for a new round of hybridization to encoders and image acquisition.
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Finally, the thermocycling of the PCR reaction continues, starting the third portion of the KASP method. A fluorescently labeled primer is present in the master mix where it is quenched due to hybridization with its complementary part that has a quencher at the end. The fluorescent-labelled primer complements the tail sequence of the allele- specific forward primer, allowing for elongation to occur. This occurs multiple times throughout the thermocycling settings and the fluorescent signalling becomes stronger as more fluorescent primers are used in the amplification process.
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Microarrays consist of short nucleotide oligomers, known as "probes", which are typically arrayed in a grid on a glass slide. Transcript abundance is determined by hybridisation of fluorescently labelled transcripts to these probes. The fluorescence intensity at each probe location on the array indicates the transcript abundance for that probe sequence. Microarrays require some genomic knowledge from the organism of interest, for example, in the form of an annotated genome sequence, or a library of ESTs that can be used to generate the probes for the array.
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VChR1 from the colonial alga Volvox carteri absorbs maximally at 535 nm and had been used to stimulate cells with yellow light (580 nm), although photocurrents generated by VChR1 are typically very small. However, VChR1-ChR2 hybrids have been developed using directed evolution that display maximal excitation at 560 nm, and 50% of peak absorbance at wavelengths over 600 nm. Using fluorescently labeled ChR2, light-stimulated axons and synapses can be identified. This is useful to study the molecular events during the induction of synaptic plasticity.
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Fluorescent dyes, with no maturation time, offer higher photo stability and brightness in comparison to fluorescent proteins. In terms of brightness, luminosity is dependent on the fluorophores’ extinction coefficient or ability to absorb light, and its quantum efficiency or effectiveness at transforming absorbed light into fluorescently emitting luminescence. The dyes themselves are not very fluorescent, but when they bind to proteins, they become more easily detectable. One example, NanoOrange, binds to the coating and hydrophobic regions of a protein while being immune to reducing agents.
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Other methods for elucidating the cellular location of proteins requires the use of known compartmental markers for regions such as the ER, the Golgi, lysosomes or vacuoles, mitochondria, chloroplasts, plasma membrane, etc. With the use of fluorescently tagged versions of these markers or of antibodies to known markers, it becomes much simpler to identify the localization of a protein of interest. For example, indirect immunofluorescence will allow for fluorescence colocalization and demonstration of location. Fluorescent dyes are used to label cellular compartments for a similar purpose.
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The method has subsequently been improved dramatically and if performed correctly should only identify cells in the last phase of apoptosis. New methods incorporate the dUTPs modified by fluorophores or haptens, including biotin or bromine, which can be detected directly in the case of a fluorescently-modified nucleotide (i.e., fluorescein-dUTP), or indirectly with streptavidin or antibodies, if biotin- dUTP or BrdUTP are used, respectively. The most sensitive of them is the method utlilizing incorporation of BrdUTP by TdT followed by immunocytochemical detection of BrdU.
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Additionally, for a number of applications it is important to be able to acquire images in different colors at different exposure times. For example, to visualize exocytosis in TIRFM, very fast acquisition is necessary. However, to image a fluorescently labeled stationary organelle in the cell, low excitation is necessary to avoid photobleaching and as a result the acquisition has to be relatively slow. In this regard, the above implementation offers great flexibility, since different cameras can be used to acquire images in different channels.
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Microfluidic Sanger sequencing is a lab-on-a-chip application for DNA sequencing, in which the Sanger sequencing steps (thermal cycling, sample purification, and capillary electrophoresis) are integrated on a wafer-scale chip using nanoliter-scale sample volumes. This technology generates long and accurate sequence reads, while obviating many of the significant shortcomings of the conventional Sanger method (e.g. high consumption of expensive reagents, reliance on expensive equipment, personnel-intensive manipulations, etc.) by integrating and automating the Sanger sequencing steps. In its modern inception, high-throughput genome sequencing involves fragmenting the genome into small single-stranded pieces, followed by amplification of the fragments by Polymerase Chain Reaction (PCR). Adopting the Sanger method, each DNA fragment is irreversibly terminated with the incorporation of a fluorescently labeled dideoxy chain-terminating nucleotide, thereby producing a DNA “ladder” of fragments that each differ in length by one base and bear a base-specific fluorescent label at the terminal base. Amplified base ladders are then separated by Capillary Array Electrophoresis (CAE) with automated, in situ “finish-line” detection of the fluorescently labeled ssDNA fragments, which provides an ordered sequence of the fragments.
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Plates are subsequently probed with fluorescently labeled antibodies against a viral antigen, and fluorescence microscopy is used to count and quantify the number of foci. The FFA method typically yields results in less time than plaque or fifty-percent-tissue-culture-infective-dose (TCID50) assays, but it can be more expensive in terms of required reagents and equipment. Assay completion time is also dependent on the size of area that the user is counting. A larger area will require more time but can provide a more accurate representation of the sample.
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With transgenic lines that fluorescently label zebrafish neutrophils, the cells can be tracked by epifluorescence or confocal microscopy during the course of an inflammatory response. Through this method, specific subpopulations of neutrophils can be tracked and their origin and fate during the induction and resolution of inflammation is observed. Another advantage for using zebrafish to study neutrophil swarming is that adaptive immunity for this organism does not develop until around 4 weeks of age. This allows for the study of neutrophil movement and other host immune responses independent of adaptive immune responses.
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At the end of clonal amplification, all of the reverse strands are washed off the flow cell, leaving only forward strands. A primer attaches to the forward strands adapter primer binding site, and a polymerase adds a fluorescently tagged dNTP to the DNA strand. Only one base is able to be added per round due to the fluorophore acting as a blocking group; however, the blocking group is reversible. Using the four-color chemistry, each of the four bases has a unique emission, and after each round, the machine records which base was added.
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Thus, such a method provides more information about the dynamics and stages of biological events and processes. The method takes advantage of the easily detectable fluorescent proteins fused to a protein of interest, which can then be followed inside a cell using a fluorescence microscope. The cell may then be treated by a perturbation of interest (e.g. a drug, expression of a misfolded protein), and various properties of the fluorescently tagged protein can be assayed using time-lapse microscopy: #Changes of the fluorescence level indicates changes of expression levels (i.e.
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The microemulsion droplets are then broken to release the magnetic beads, which have the amplified copies of DNA attached. The beads are magnetically purified and base pair-specific fluorescent probes are attached. This helps distinguish between wild-type and mutant DNA fragments, as one fluorescent probe binds specifically to the wild-type DNA and the other to specific mutant DNA. Each fluorescently labeled bead is analyzed in a flow cytometer, resulting in a separation of mutant from wild- type DNA as well as the ratio of mutant to wild-type DNA present in a sample.
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QR has many uses as a fluorescent probe. The use of QR as a probe is relatively safe, inexpensive, and a sensitive method compared with other fluorescence probes like ethidium bromide or dimeric cyanine dyes. QR is also an ideal fluorescent probe because substrates of interest, such as antibodies, can be detected within a 0.3nM detection limit without the use of radiolabeled or fluorescently labeled oligonucleotides, which are the DNA components. In other words, quinaldine red is preferred tag since its binding increases the fluorescence without extra tags being needed.
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Abbott Laboratories' ID Now nucleic acid test uses isothermal amplification technology. The assay amplifies a unique region of the virus's RdRp gene; the resulting copies are then detected with "fluorescently-labeled molecular beacons".ID NOW COVID-19, Instruction for Use, FDA The test kit uses the company's "toaster-size" ID Now device, which is widely deployed in the US. The device can be used in laboratories or in point of care settings, and provides results in 13 minutes or less. Primerdesign offers its Genesig Real-Time PCR Coronavirus (COVID‑19).
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Dynamic morphometrics technology involves new methods of labeling, imaging, and quantifying dendritogenesis. The transparent, externally developing vertebrate embryos of Xenopus laevis and zebrafish allow direct imaging of the organism in the critical stages of development while keeping the embryos intact. Individual brain neurons can be fluorescently labeled using single cell electroporation while leaving the rest of the brain unaltered. Also, two- photon microscopy allows in vivo time-lapse imaging to create high-resolution, 3D images of neurons deep within the living brain, again with minimal damage to the brain.
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It samples cerebrospinal fluid (CSF) and so it is applicable to scrapie, chronic wasting disease (CWD), bovine spongiform encephalopathy (BSE) and sporadic Creutzfeldt–Jakob disease, amongst others. The RT-QuIC assay uses as reagents normally folded prions, fluorescently labelled so that they indicate when they are misfolded; samples suspected of containing misfolded prions are added and misfolded reagents can be detected by thioflavin T visible spectrum fluorescence detection. The Centers for Disease Control and Prevention includes a positive RT-QuIC result in its diagnostic criteria for the probable diagnosis of sCJD.
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While motility measurements are critical for identifying the presence of hyperactive motility, additional methods have been developed to identify the occurrence of the acrosome reaction. A simple method uses Coomassie brilliant blue G250 to stain cells, providing visual evidence of intact or reacted acrosomes. More advanced techniques employ fluorescent or electron microscopy methods. Fluorescein-conjugated Peanut agglutinin (FITC-PNA) or Pisum sativum agglutinin (FITC-PSA) can be used to fluorescently tag the acrosome of sperm cells, which can be then used to assess the status of the acrosome using a fluorescent microscope.
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Hybridization of the target to the probe The core principle behind microarrays is hybridization between two DNA strands, the property of complementary nucleic acid sequences to specifically pair with each other by forming hydrogen bonds between complementary nucleotide base pairs. A high number of complementary base pairs in a nucleotide sequence means tighter non- covalent bonding between the two strands. After washing off non-specific bonding sequences, only strongly paired strands will remain hybridized. Fluorescently labeled target sequences that bind to a probe sequence generate a signal that depends on the hybridization conditions (such as temperature), and washing after hybridization.
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Because the forward primer used for probe amplification is fluorescently labeled, each amplicon generates a fluorescent peak which can be detected by a capillary sequencer. Comparing the peak pattern obtained on a given sample with that obtained on various reference samples, the relative quantity of each amplicon can be determined. This ratio is a measure for the ratio in which the target sequence is present in the sample DNA. Various techniques including DGGE (Denaturing Gradient Gel Electrophoresis), DHPLC (Denaturing High Performance Liquid Chromatography), and SSCA (Single Strand Conformation Analysis) effectively identify SNPs and small insertions and deletions.
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These random segments are inserted into a plasmid or bacteriophage vector, which is in turn implanted into Escherichia coli bacteria. Colonies are then developed, and screened with fluorescently–labelled oligonucleotide sequences that will hybridize to a microsatellite repeat, if present on the DNA segment. If positive clones can be obtained from this procedure, the DNA is sequenced and PCR primers are chosen from sequences flanking such regions to determine a specific locus. This process involves significant trial and error on the part of researchers, as microsatellite repeat sequences must be predicted and primers that are randomly isolated may not display significant polymorphism.
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Strategies involving combing recently replicated DNA typically involve incorporating modified nucleotides (such as BrdU, bromodeoxyuridine) into the nascent DNA, then fluorescently detecting it. As replication forks spread bidirectionally from origins of replication at (approximately) equal speeds, then origin position can be inferred. Replacing the modified nucleotide pool with a different type of modified nucleotide after a certain amount of time allows development of a time-resolved picture of the firing of sites, and the kinetics of replication forks. Pause sites can be identified, merged replication forks resolved, and the frequency of origin firings in different time periods to be studied.
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So when the immune cells of mice reacted with fluorescently labeled antibodies specific to effector cells, the mature cells were differentiated from the newly forming stem cells. His work has contributed to the understanding of how a single hematopoietic stem cell can give rise to specialized blood cells. Weissman is also a leading expert in the field of cancer stem cell biology, where his work sheds light on the understanding of the pathogenesis of multiple human malignancies. He is also known for transgenic research in which human brain cells are grown in the brains of mice.
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Differential comparison in cDNA microarray cDNA microarrays are often used for large-scale screening and expression studies. In cDNA microarrays, mRNA from cells are collected and converted into cDNA by reverse transcription. Subsequently, cDNA molecules (each corresponding to one gene) are immobilized as ~100 µm diameter spots on a membrane, glass, or silicon chip by metallic pins. For detection, fluorescently-labelled single strand cDNA from cells hybridize to the molecules on the microarray and a differential comparison between a treated sample (labelled red, for example) and an untreated sample (labelled in another color such as green) is used for analysis.
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This is detected by the incorporation of the fluorescently labeled dNTPs onto the end of the probe. If the target DNA does not contain an allele complementary to the probe's 3’ base, the target DNA will produce a mismatch at the 3’ end of the probe and DNA polymerase will not be able to extend from the 3' end of the probe. The benefit of the second approach is that several labeled dNTPs may get incorporated into the growing strand, allowing for increased signal. However, DNA polymerase in some rare cases, can extend from mismatched 3’ probes giving a false positive result.
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The two other, rather new but more reliable approaches are either by detecting areas of different probe mobility on an individual image basis or by physical modeling of fluorescence loss from moving bodies. Loss of fluorescence is defined by the mobile fraction, or the fraction of fluorophores capable of recovering into a photobleached area, of the fluorescently labeled protein. Incomplete loss of fluorescence indicates that there are fluorophores that do not move or travel to the bleached area. This allows for definition of the immobile fraction, or the fraction of fluorophores incapable of recovering into a photobleached area, of fluorescent-labeled proteins.
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In order to simultaneously manipulate and image samples that exhibit fluorescence, optical tweezers can be built alongside a fluorescence microscope. Such instruments are particularly useful when it comes to studying single or small numbers of biological molecules that have been fluorescently labelled, or in applications in which fluorescence is used to track and visualize objects that are to be trapped. This approach has been extended for simultaneous sensing and imaging of dynamic protein complexes using long and strong tethers generated by a highly efficient multi- step enzymatic approach and applied to investigations of disaggregation machines in action.
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Spectral karyogram of a human female Multicolor FISH and the older spectral karyotyping are molecular cytogenetic techniques used to simultaneously visualize all the pairs of chromosomes in an organism in different colors. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. Because there are a limited number of spectrally distinct fluorophores, a combinatorial labeling method is used to generate many different colors. Fluorophore combinations are captured and analyzed by a fluorescence microscope using up to 7 narrow-banded fluorescence filters or, in the case of spectral karyotyping, by using an interferometer attached to a fluorescence microscope.
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A DNA microarray Microarrays measure the amount of mRNA in a sample that corresponds to a given gene or probe DNA sequence. Probe sequences are immobilized on a solid surface and allowed to hybridize with fluorescently labeled “target” mRNA. The intensity of fluorescence of a spot is proportional to the amount of target sequence that has hybridized to that spot, and therefore to the abundance of that mRNA sequence in the sample. Microarrays allow for identification of candidate genes involved in a given process based on variation between transcript levels for different conditions and shared expression patterns with genes of known function.
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Previous experiments involving the analysis of cell mechanics had depended on fluorescently labeled phalloidin and actin GFP fusion proteins obtained from utrophin in Xenopus laevis and ABP120 in Dictyostelium discoideum. However, due to their large protein size, markers such as phalloidin and GFP fusion proteins are limited to cells that can be transfected and tend to compete with their orthologous protein. These localization markers, often referred to as dyes, affect cellular mechanical properties and F-actin structures, thus making these markers unreliable. An alternative to these markers is Life Act- TagGFP2, which is a much smaller protein and does not affect cell mechanics.
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The thermophoretic movement of the fluorescently labelled molecule is measured by monitoring the fluorescence distribution F inside a capillary. The microscopic temperature gradient is generated by an IR-Laser, which is focused into the capillary and is strongly absorbed by water. The temperature of the aqueous solution in the laser spot is raised by ΔT=1-10 K. Before the IR-Laser is switched on a homogeneous fluorescence distribution Fcold is observed inside the capillary. When the IR-Laser is switched on, two effects, occur on the same time-scale, contributing to the new fluorescence distribution Fhot.
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Much current research (in the early 21st century) on optical microscope techniques is focused on development of superresolution analysis of fluorescently labelled samples. Structured illumination can improve resolution by around two to four times and techniques like stimulated emission depletion (STED) microscopy are approaching the resolution of electron microscopes. This occurs because the diffraction limit is occurred from light or excitation, which makes the resolution must be doubled to become super saturated. Stefan Hell was awarded the 2014 Nobel Prize in Chemistry for the development of the STED technique, along with Eric Betzig and William Moerner who adapted fluorescence microscopy for single-molecule visualization.
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Another method of heterologous protein fusion is fusion with fimbriae/flagella, which are filamentous protrusions on the cell surface. There are many fimbriae on mainly Gram-negative bacteria, so displaying proteins on fimbriae is advantageous over some other surface proteins which are less numerous. A disadvantage of using fimbriae is that there is a relatively small insert size limit of 10-30 amino acids. Flow Cytometer Instrument Once the heterologous protein has been fused with the bacterial cell surface protein, it is exposed to either an enzyme, a cell (expressing a target protein) or an antibody (usually fluorescently tagged), depending on the application of the experiment.
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The fixation of the fluorophore molecule provides a fluorescent signal that indicates whether there is an A, C, G, or T at the query position on the genomic DNA tag. After four-colour imaging, the anchor primer/nonamer complexes are stripped off and a new cycle is begun by replacing the anchor primer. A new mixture of the fluorescently tagged nonamers is introduced, for which the query position is shifted one base further into the genomic DNA tag. 5' Cy5‐NNNNNNNTN 5' Cy3‐NNNNNNNAN 5' TexasRed‐NNNNNNNCN 5' 6FAM‐NNNNNNNGN Seven bases from the 5’ to 3’ direction and six bases from the 3’ end could be queried in this fashion.
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New York: Pantheon Books. Modern humans who spend most of their time indoors, in dimly or fluorescently lit buildings may be at risk of development of myopia. People, and children especially, who spend more time doing physical exercise and outdoor play have lower rates of myopia, suggesting the increased magnitude and complexity of the visual stimuli encountered during these types of activities decrease myopic progression. There is preliminary evidence that the protective effect of outdoor activities on the development of myopia is due, at least in part, to the effect of long hours of exposure to daylight on the production and the release of retinal dopamine.
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Fluorescent ddNTP molecules The classical chain-termination method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, normal deoxynucleotidetriphosphates (dNTPs), and modified di-deoxynucleotidetriphosphates (ddNTPs), the latter of which terminate DNA strand elongation. These chain-terminating nucleotides lack a 3'-OH group required for the formation of a phosphodiester bond between two nucleotides, causing DNA polymerase to cease extension of DNA when a modified ddNTP is incorporated. The ddNTPs may be radioactively or fluorescently labelled for detection in automated sequencing machines. The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase.
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PLCγ1 activity is necessary for mediating GBM cell migration in the absence of PTPmu, thus it seems likely that PTPmu dephosphorylation of PLCγ1 prevents PLCγ1-mediated migration. Cleavage of cell adhesion molecules, like PTPmu, has also been linked to the deregulation of contact inhibition of growth observed in cancer cells. Visualization of the shed extracellular fragment of PTPmu has been proposed to be an effective means of delineating the borders of a GBM tumor ‘’in vivo.’’ Fluorescently tagged PTPmu peptides that bind homophilically to the shed PTPmu extracellular domains are capable of crossing the blood–brain barrier and identifying tumor margins in rodent models of GBM.
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Fluorescence guided surgery (FGS), (also called 'Fluorescence image-guided surgery', or in the specific case of tumor resection, 'fluorescence guided resection') is a medical imaging technique used to detect fluorescently labelled structures during surgery. Similarly to standard image-guided surgery, FGS has the purpose of guiding the surgical procedure and providing the surgeon of real time visualization of the operating field. When compared to other medical imaging modalities, FGS is cheaper and superior in terms of resolution and number of molecules detectable. As a drawback, penetration depth is usually very poor (100 μm) in the visible wavelengths, but it can reach up to 1–2 cm when excitation wavelengths in the near infrared are used.
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Denny then began to exploring the idea of memory traces, cells in the brain where memories are stored, and tried to tag these memory traces in the dentate gyrus and CA3 regions of the hippocampus. She was able to develop a genetic tool that enabled her to fluorescently tag neurons that were activated in specific memories. Since her tool enabled long term labelling of memory associated neurons, she is able to observe memory traces overtime and reported that memory traces become more faint over time even though learned behaviors persist. Her findings highlights the possibility that these memory traces are being redistributed to areas other than the hippocampus for even longer-term storage.
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Helicos was co-founded in 2003 by life science entrepreneur Stanley Lapidus, Stephen Quake, and Noubar Afeyan with investments from Atlas Venture, Flagship Ventures, Highland Capital Partners, MPM Capital, and Versant Ventures. Helicos's technology images the extension of individual DNA molecules using a defined primer and individual fluorescently labeled nucleotides, which contain a "Virtual Terminator" preventing incorporation of multiple nucleotides per cycle. The "Virtual Terminator" technology was developed by Dr. Suhaib Siddiqi, while at Helicos Biosciences. In the August 2009 issue of Nature Biotechnology, Dr. Stephen Quake, a professor of bioengineering at Stanford University and a co-founder of Helicos BioSciences, sequenced his own genome, using Single Molecule Sequencing for under $50,000 in reagents.
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Trophallaxis - the mouth-to-mouth transfer of liquid food - is a main mechanism of food dissemination in ant colonies. In C. fellah, the colony trophallactic network has been quantified by combining unique marking of individuals with fluorescently labelled food. This procedure refined our understanding of trophallaxis, revealing that transfer flow can switch direction during a trophallaxis event, that foragers receive (as well as unload) food, that foragers often leave the nest after offloading only a small amount of the food in their crop, and that non- foragers also offload considerable amounts of food. Further, the vast majority of trophallaxis events were short in duration, possibly functioning to maintain the colony odour rather than disseminate food.
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The Allen Institute for Cell Science was modeled on the Allen Institute for Brain Science and was launched to capture a global view of human cells, developing gene-edited, fluorescently tagged human induced pluripotent stem cells that form the backbone of an openly available library of digital microscopy images and computational models to predict cellular organization. The tagged cell lines are available for others in the scientific community to use, and have been used in research on kidney disease and cardiomyocyte function, among others. Ongoing projects at the institute include studies of cardiomyocyte differentiation and mitosis. Cell biology resources from the institute have been used in high school and college biology education, including at Washington State University.
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Reverse transcription first generates a DNA template from the mRNA; this single-stranded template is called cDNA. The cDNA template is then amplified in the quantitative step, during which the fluorescence emitted by labeled hybridization probes or intercalating dyes changes as the DNA amplification process progresses. With a carefully constructed standard curve, qPCR can produce an absolute measurement of the number of copies of original mRNA, typically in units of copies per nanolitre of homogenized tissue or copies per cell. qPCR is very sensitive (detection of a single mRNA molecule is theoretically possible), but can be expensive depending on the type of reporter used; fluorescently labeled oligonucleotide probes are more expensive than non-specific intercalating fluorescent dyes.
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Later, blood samples from various primates: hominoids, Old World monkeys, New World monkeys and prosimians were probed using a fluorescently labeled HERV-W element derived from the gorilla fosmid library. Fluorescence in situ hybridization (FISH) revealed HERV-W elements in all the primate blood samples except the tupaia. With this information and the divergence values of the 5’ and 3’ LTRs the construction of a phylogenetic tree was possible. This data implies that the HERV-W genome integrated into its host's germ-line around 63 million years ago, expanded in the era of Old and New World monkeys and then evolved independently. Since its integration the 5’ and 3’ LTR have followed independent evolution in each species.
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A surface may absorb part of the light ray, resulting in a loss of intensity of the reflected and/or refracted light. It might also reflect all or part of the light ray, in one or more directions. If the surface has any transparent or translucent properties, it refracts a portion of the light beam into itself in a different direction while absorbing some (or all) of the spectrum (and possibly altering the color). Less commonly, a surface may absorb some portion of the light and fluorescently re-emit the light at a longer wavelength color in a random direction, though this is rare enough that it can be discounted from most rendering applications.
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Real-time PCR or quantitative PCR (qPCR), is becoming a well-established method to quickly amplify and simultaneously quantify targeted AM fungal DNA from biological samples (plant roots or soils). Fairly recent developments in qPCR markers allow researchers to explore the relative abundance of AM fungal species within roots in greenhouse experiments as well as in the field to identify local AM fungal communities. qPCR markers for arbuscular mycorrhizal fungi will consist of AM specific primers and fluorescently labeled hydrolysis probes. These AM specific primers (discussed above) can be chosen by the researcher and this decision is typically guided by the question at hand, resources available, and willingness to troubleshoot in the lab.
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Direct FA stained mouse brain impression smear reveals the presence of the bacterium Chlamydia psittaci. 400X. A direct fluorescent antibody (DFA or dFA), also known as "direct immunofluorescence", is an antibody that has been tagged in a direct fluorescent antibody test. Its name derives from the fact that it directly tests the presence of an antigen with the tagged antibody, unlike western blotting, which uses an indirect method of detection, where the primary antibody binds the target antigen, with a secondary antibody directed against the primary, and a tag attached to the secondary antibody. Commercial DFA testing kits are available, which contain fluorescently labelled antibodies, designed to specifically target unique antigens present in the bacteria or virus, but not present in mammals (Eukaryotes).
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Applications: There has additionally been interest in expressing these artificial structures in engineered living bacterial cells, most likely using the transcribed RNA for the assembly, although it is unknown whether these complex structures are able to efficiently fold or assemble in the cell's cytoplasm. If successful, this could enable directed evolution of nucleic acid nanostructures. Scientists at Oxford University reported the self-assembly of four short strands of synthetic DNA into a cage which can enter cells and survive for at least 48 hours. The fluorescently labeled DNA tetrahedra were found to remain intact in the laboratory cultured human kidney cells despite the attack by cellular enzymes after two days. This experiment showed the potential of drug delivery inside the living cells using the DNA ‘cage’.
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Figure 4. Single nucleotide extension reaction The chips also provides a good platform for performing PCR directly on the chip (in individual gel pads) as it is easy to isolate each gel pad from its neighbour unlike typical microarray chips which face serious problems in doing the same task For the analysis of hybridization results obtained with fluorescently labelled target molecules fluorescence microscopes are employed. The instrument is equipped with controlled- temperature sample table to vary the temperature in the chip-containing reaction chamber during the course of the experiment. A cooled charge-coupled device (CCD) camera is used to record the light signals from the chip, which are then sent to the computer program for quantitative evaluation of the hybridization signals over the entire chip.
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Both assumptions listed above, however, are not always met. Often, several different bacteria in a population might give a single peak on the electropherogram due to the presence of a restriction site for the particular restriction enzyme used in the experiment at the same position. To overcome this problem and to increase the resolving power of this technique a single sample can be digested in parallel by several enzymes (often three) resulting in three T-RFLP profiles per sample each resolving some variants while missing others. Another modification which is sometimes used is to fluorescently label the reverse primer as well using a different dye, again resulting in two parallel profiles per sample each resolving a different number of variants.
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Unlike pyrosequencing, the DNA chains are extended one nucleotide at a time and image acquisition can be performed at a delayed moment, allowing for very large arrays of DNA colonies to be captured by sequential images taken from a single camera. Decoupling the enzymatic reaction and the image capture allows for optimal throughput and theoretically unlimited sequencing capacity; with an optimal configuration, the ultimate throughput of the instrument depends only on the A/D conversion rate of the camera. The camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3' blocker is chemically removed from the DNA, allowing the next cycle. An alternative approach, ion semiconductor sequencing, is based on standard DNA replication chemistry.
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A recent design used piezoelectric inkjet printing to imprint paper with the enzyme acetylcholinesterase (AChE) and the substrate indophenyl acetate (IPA), and this paper-based microfluidic device was used to detect organophosphate pesticides (AChE inhibitors) via a decrease in blue-purple color. This device is distinguished by its use of bioactive paper instead of compartments with pre-stored reagents, and it was demonstrated to have good long-term stability, making it ideal for field use. A more recent paper-based microfluidic design utilized a sensor, consisting of fluorescently labeled single-stranded DNA (ssDNA) coupled with graphene oxide, on its surface to simultaneously detect heavy metals and antibiotics in food products. Heavy metals increased fluorescence intensity, whereas antibiotics decreased fluorescence intensity.
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The pair moved to Germany in 1975 when Weber was offered the position of Director of the Department of Biochemistry and Cell Biology at the Max Planck Institute for Biophysical Chemistry in Göttingen. There they pioneered another new technique: immunofluorescence microscopy. They and Elias Lazarides had previously found that they could tag the subunit proteins of microtubules, microfilaments, intermediate filaments and other cellular structures with specific antibodies and then tag these antibodies with a second fluorescently labelled antibody as described in a series of papers such as "Actin antibody: the specific visualization of actin filaments in non-muscle cells". The fluorescent signal could be easily visualized using a fluorescence microscope and this allowed the rapid examination of the localization of molecules in cells and in tissues.
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The ELISA template, commonly used for performing immunoassays and other enzyme-based biochemical assays, has been adapted for use with the DMF platform for the detection of analytes such as IgE and IgG. In one example, a series of bioassays were conducted to establish the quantification capabilities of DMF devices, including an ELISA-based immunoassay for the detection of IgE. Superparamagnetic nanoparticles were immobilized with anti-IgE antibodies and fluorescently labeled aptamers to quantify IgE using an ELISA template. Similarly, for the detection of IgG, IgG can be immobilized onto a DMF chip, conjugated with horseradish-peroxidase (HRP)-labeled IgG, and then quantified through measurement of the color change associated with product formation of the reaction between HRP and tetramethylbenzidine.
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1983, 1984 NASA Lewis Research Center Summer Faculty Fellow. 1985, 1986, 1987: Senior Visiting Scientist, Burroughs Wellcome Co.See also Burroughs Wellcome Fund 1987: East Carolina University Sigma XI Helms Award for outstanding Research 1999: East Carolina University Distinguished Research Professor of Chemistry Award, 5-Year Achievement Research/Creative Activity AwardEast Carolina University Past Award Recipients 2001: East Carolina University College of Arts and Sciences Distinguished Professor of Chemistry, Lifetime Achievement Award 2003: Eastern Analytical Symposium Award for Achievement in Chemometrics. 2010: Applied Spectroscopy William F. Meggers Award for outstanding paper appearing in Applied Spectroscopy.Patrick J. Cutler, David M. Haaland, and Paul J. Gemperline for 'Systematic Method for the Kinetic Modeling of Temporally Resolved Hyperspectral Microscope Images of Fluorescently Labeled Cells.
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In molecular biology, a hybridization probe is a fragment of DNA or RNA of variable length (usually 100–10000 bases long) which can be radioactively or fluorescently labeled. It can then be used in DNA or RNA samples to detect the presence of nucleotide substances (the RNA target) that are complementary to the sequence in the probe. The probe thereby hybridizes to single-stranded nucleic acid (DNA or RNA) whose base sequence allows probe–target base pairing due to complementarity between the probe and target. The labeled probe is first denatured (by heating or under alkaline conditions such as exposure to sodium hydroxide) into single stranded DNA (ssDNA) and then hybridized to the target ssDNA (Southern blotting) or RNA (northern blotting) immobilized on a membrane or in situ.
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Seven Magic Mountains (2016–2021) Moonrise East (2008), Basel, San Francisco The Wise (2014), 10 meters h, 84 tons Liverpool Mountain installed outside Tate Liverpool in October 2018 Ugo Rondinone (born 1964) is a New York-based, Swiss-born mixed-media artist noted for a range of contemporary paintings and sculptures. Rondinone is widely known for his temporary, large-scale land art sculpture, Seven Magic Mountains (2016–2021), with its seven fluorescently- painted totems of large, car-size stones stacked high.Graham Bowley (April 9, 2015), Splash of color in the desert New York Times The installation's organizers, Art Production Fund (APF) and the Nevada Museum of Art, estimated that 16 million people would see the work—from its location just south of Las Vegas, alongside Interstate 15, the primary Los Angeles – Las Vegas interstate.
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He showed these complexes were highly mobile and clustered before entry into cells, and measured their lateral diffusion coefficients.Schlessinger, J., Shechter, Y., Cuatrecasas, P., Willingham, M., and Pastan, I.: Quantitative determination of the lateral diffusion coefficients of the hormone-receptor complexes of insulin and epidermal growth factor on the plasma membrane of cultured fibroblasts. Proc. Natl. Acad. Sci. USA 75: 5353-5357, 1978Schlessinger, J., Shechter, Y., Willingham, M.C., and Pastan, I.: Direct visualization of the binding, aggregation and internalization of insulin and epidermal growth factor on fibroblastic cells. Proc. Natl. Acad. Sci. USA 75: 2659-2663, 1978 In collaboration with Mark Willingham, he developed and used video intensified microscopy to visualize fluorescently labeled insulin and EGF forming clusters on the surface of living cells prior to entry through the endocytic pathway.
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Cells infected by rotavirus (top) and uninfected cells (bottom) The focus forming assay (FFA) is a variation of the plaque assay, but instead of relying on cell lysis in order to detect plaque formation, the FFA employs immunostaining techniques using fluorescently labeled antibodies specific for a viral antigen to detect infected host cells and infectious virus particles before an actual plaque is formed. The FFA is particularly useful for quantifying classes of viruses that do not lyse the cell membranes, as these viruses would not be amenable to the plaque assay. Like the plaque assay, host cell monolayers are infected with various dilutions of the virus sample and allowed to incubate for a relatively brief incubation period (e.g., 24–72 hours) under a semisolid overlay medium that restricts the spread of infectious virus, creating localized clusters (foci) of infected cells.
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Principle of FRAP A) The bilayer is uniformly labeled with a fluorescent tag B) This label is selectively photobleached by a small (~30 micrometre) fast light pulse C) The intensity within this bleached area is monitored as the bleached dye diffuses out and new dye diffuses in D) Eventually uniform intensity is restored Fluorescence recovery after photobleaching (FRAP) is a method for determining the kinetics of diffusion through tissue or cells. It is capable of quantifying the two dimensional lateral diffusion of a molecularly thin film containing fluorescently labeled probes, or to examine single cells. This technique is very useful in biological studies of cell membrane diffusion and protein binding. In addition, surface deposition of a fluorescing phospholipid bilayer (or monolayer) allows the characterization of hydrophilic (or hydrophobic) surfaces in terms of surface structure and free energy.
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FISH images allow both the identification of mega-telomeric chromosomes and the visualization of chromosome structure, GC-rich DNA regions, and, depending on the experiment, co-localization with genetic regions or genes. Slot blot method of analysis can be utilized to determine the total amount of telomeric sequence per genome (inclusive of interstitial and terminal arrays) Molecular techniques for quantifying telomeric sequences include pulse-field gel electrophoresis (PFGE), slot blot, horizontal gel electrophoresis, and Contour-clamped homogeneous electric field pulse field gel electrophoresis (CHEF-PFGE). In these techniques, purified genomic DNA is isolated and digested with restriction enzymes, such as HaeIII, HinfI, AluI, Sau3AI, EcoRI, EcoRV, PstI, SstI, BamHI, HindIII or BglII, and quantified by fluorometry. The digestion of DNA into smaller fragments by restriction enzymes, separation of variable-sized DNA fragments via electrophoresis, and labeling of fragments containing telomeric DNA using a specific radio- or fluorescently-labeled probe are the essential steps completed within many molecular techniques.
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A form of MHC multimer developed and trademarked by the Danish biotechnology company, Immudex in 2002. Dextramer reagents are fluorescently labeled with FITC, PE or APC, and contain MHC molecules attached to a dextran backbone, which are used to detect antigen-specific T-cells in fluid cells and solid tissue samples using flow cytometry. These T-cells contain T-cell receptors (TCR) that recognize a specific MHC-peptide complex displayed on the surface of antigen presenting cells allowing for detection, isolation, and quantification of these specific T-cell populations due to an improved signal-to-noise ratio not present in prior generations of multimers. Dextramers have been developed with a larger number of MHC-peptides for various human, mouse, and rhesus macaque genes involved in diseases including but not limited to: cancer, HIV, Epstein-Barr virus (EBV), cytomegalovirus (CMV), LCMV, human papillomavirus (HPV), BK polyomavirus, HTLV, hepatitis, mycobacterium, and graft-versus-host disease.
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In the early 1990s, Brown began developing a new technology to enable systematic investigation of the behavior and properties of whole genomes—called DNA microarrays. "I had a mental image of a DNA microarray, even including the red and green fluorescent spots, a few years before I'd figured out the details of making them," Brown told The Scientist. Brown and his colleagues created a robotic dispenser that could deposit minute quantities of tens of thousands of individual genes onto a single glass slide, a “DNA microarray” or "gene chip." By flooding the slide with fluorescently labeled genetic material derived from a living sample, a researcher could see which genes were being expressed in cells. Shortly after their first description of DNA microarrays, the Brown laboratory published a “how-to” manual on the Web that helped these robotic devices become standard equipment in life science labs throughout the world, in an effort led by Joe DeRisi, Michael Eisen, Ash Alizadeh, and others.
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Next, multiple copies of the same transgenic construct were inserted into the genome of the target species, resulting in the random expression of different XFP ratios and subsequently causing different cells to exhibit a variety of colorful hues. Brainbow was originally created as an improvement over more traditional neuroimaging techniques, such as Golgi staining and dye injection, both of which presented severe limitations to researchers in their ability to visualize the intricate architecture of neural circuitry in the brain. While older techniques were only able to stain cells with a constricted range of colors, often utilizing bi- and tri-color transgenic mice to unveil limited information in regards to neuronal structures, Brainbow is much more flexible in that it has the capacity to fluorescently label individual neurons with up to approximately 100 different hues so that scientists can identify and even differentiate between dendritic and axonal processes. By revealing such detailed information about neuronal connectivity and patterns, sometimes even in vivo, scientists are often able to infer information regarding neuronal interactions and their subsequent impact upon behavior and function.
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