Tandem Dye Research Articles

Role of the Anaphylatoxin Receptor C5aR2 in Angiotensin II-Induced Hypertension and Hypertensive End-Organ Damage.

Complement activation may facilitate hypertension through its effects on immune responses. The anaphylatoxin C5a, a major inflammatory effector, binds to the C5a receptors 1 and 2 (C5aR1, C5aR2). We have recently shown that C5aR1-/- mice have reduced hypertensive renal injury. The role of C5aR2 in hypertension is unknown. For examination of C5aR2 expression on infiltrating and resident renal cells a tandem dye Tomato-C5aR2 knock-in reporter mouse was used. Human C5aR2 expression was analyzed in a single-cell RNAseq data set from the kidneys of hypertensive patients. Finally, we examined the effect of angiotensin II-induced hypertension in C5aR2-deficient mice. Flow cytometric analysis of leukocytes isolated from kidneys of the reporter mice showed that dendritic cells are the major C5aR2-expressing population (34%) followed by monocyte/macrophages (30%) and neutrophils (14%). Using confocal microscopy C5aR2 was not detected in resident renal or cardiac cells. In the human kidney, C5aR2 was also mainly found in monocytes, macrophages, and dendritic cells with a significantly higher expression in hypertension (P < 0.05). Unilateral nephrectomy was performed followed by infusion of Ang II (0.75 ng/g/min) and a high salt diet in wildtype (n = 18) and C5aR2-deficient mice (n = 14). Blood pressure, renal injury (albuminuria, glomerular filtration rate, glomerular and tubulointerstitial injury, inflammation), and cardiac injury (cardiac fibrosis, heart weight, gene expression) did not differ between hypertensive wildtype and C5aR2-/- mice. In summary, C5aR2 is mainly expressed in myeloid cells in the kidney in mice and humans but its deficiency has no effect on Ang II-induced hypertensive injury.

Increasing Signal Intensity of Fluorescent Oligo-Labeled Antibodies to Enable Combination Multiplexing.

Full-spectrum flow cytometry has increased antibody-based multiplexing, yet further increases remain potentially impactful. We recently proposed how fluorescence multiplexing using spectral imaging and combinatorics (MuSIC) could do so using tandem dyes and an oligo-based antibody labeling method. In this work, we found that such labeled antibodies had significantly lower signal intensities than conventionally labeled antibodies in human cell experiments. To improve signal intensity, we tested moving the fluorophores from the original external (ext.) 5' or 3' end-labeled orientation to internal (int.) fluorophore modifications. Cell-free spectrophotometer measurements showed a ∼6-fold signal intensity increase of the new int. configuration compared to the previous ext. configuration. Time-resolved fluorescence and fluorescence correlation spectroscopy showed that the ∼3-fold brightness difference is due to static quenching most likely by the oligo or solution in the ext. configuration. Spectral flow cytometry experiments using peripheral blood mononuclear cells show int. MuSIC probe-labeled antibodies (i) retained increased signal intensity while having no significant difference in the estimated % of CD8+ lymphocytes and (ii) labeled with Atto488, Atto647, and Atto488/647 combinations can be demultiplexed in triple-stained samples. The antibody labeling approach is general and can be broadly applied to many biological and diagnostic applications where spectral detection is available.

Increasing Signal Intensity of Fluorescent Oligo-Labeled Antibodies to Enable Combination Multiplexing.

Full-spectrum flow cytometry has increased antibody-based multiplexing, yet further increases remain potentially impactful. We recently proposed how fluorescence Multiplexing using Spectral Imaging and Combinatorics (MuSIC) could do so using tandem dyes and an oligo-based antibody labeling method. In this work, we found that such labeled antibodies had significantly lower signal intensity than conventionally-labeled antibodies in human cell experiments. To improve signal intensity, we tested moving the fluorophores from the original external (ext.) 5' or 3' end-labeled orientation to internal (int.) fluorophore modifications. Cell-free spectrophotometer measurements showed a ~6-fold signal intensity increase of the new int. configuration compared to the previous ext. configuration. Time-resolved fluorescence and fluorescence correlation spectroscopy showed that ~3-fold brightness difference is due to static quenching most likely by the oligo or solution in the ext. configuration. Spectral flow cytometry experiments using peripheral blood mononuclear cells show int. MuSIC probe-labeled antibodies (i) retained increased signal intensity while having no significant difference in the estimated % of CD8+ lymphocytes and (ii) labeled with Atto488, Atto647, and Atto488/647 combinations can be demultiplexed in triple-stained samples. The antibody labeling approach is general and can be broadly applied to many biological and diagnostic applications where spectral detection is available.

Purified Pyropia yezoensis Pigment Extract-Based Tandem Dye Synthesis.

Red phycoerythrin (R-PE) is a highly valuable protein found in an edible seaweed, Pyropia yezoensis. It is used extensively in biotechnological applications due to its strong fluorescence and stability in diverse environments. However, the current methods for extracting and purifying R-PE are costly and unsustainable. The aim of the present study was to enhance the financial viability of the process by improving the extraction and purification of R-PE from dried P. yezoensis and to further enhance R-PE value by incorporating it into a tandem dye for molecular biology applications. A combination of ultrafiltration, ion exchange chromatography, and gel filtration yielded concentrated (1 mg·mL-1) R-PE at 99% purity. Using purified PE and Cyanine5 (Cy5), an organic tandem dye, phycoerythrin-Cy5 (PE-Cy5), was subsequently established. In comparison to a commercially available tandem dye, PE-Cy5 exhibited 202.3% stronger fluorescence, rendering it suitable for imaging and analyzes that require high sensitivity, enhanced signal-to-noise ratio, broad dynamic range, or shorter exposure times to minimize potential damage to samples. The techno-economic analysis confirmed the financial feasibility of the innovative technique for the extraction and purification of R-PE and PE-Cy5 production.

Conjugation of Fluorochromes to Monoclonal Antibodies.

Detection of cell surface molecules labeled by monoclonal or polyclonal antibodies conjugated to a fluorochrome is the most widely used application of flow cytometry. Here, we present protocols for tagging monoclonal antibodies with fluorescein, biotin, Texas Red, and phycobiliproteins. In addition, we provide a procedure for preparing a PE-Texas Red tandem conjugate dye that can then be used for antibody conjugation. These protocols enable investigators to label antibodies of their choice with multiple fluorochromes and permit more combinations of antibodies for multicolor flow applications. © 2023 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol 1: Labeling an antibody with fluorescein isothiocyanate (FITC) Basic Protocol 2: Labeling an antibody with long-armed biotin Basic Protocol 3: Labeling an antibody with Texas Red-X Basic Protocol 4: Labeling an antibody with a synthetic organic fluor kit Basic Protocol 5: Labeling an antibody with phycobiliproteins Basic Protocol 6: Conjugation of Texas Red to R-phycoerythrin to produce an energy transfer fluorochrome.

Photo-driven water splitting photoelectrochemical cells by tandem organic dye sensitized solar cells with I−/I3− as redox mediator

Dye-sensitized photoelectrochemical tandem cells have shown the promise for light driven hydrogen production from water owing to the low cost, wide absorption spectra in the visible region and ease to process of their constitutive photoelectrode materials. However, most photo-driven water splitting photoelectrochemical cells driven by organic dye sensitized solar cells exhibit unsatisfactory hydrogen evolution rate, primarily attributed to their poor light capturing ability and low photocurrent performance. Here we present the construction of a tandem system consisting of an organic blue-colored S5 sensitizer-based dye-sensitized photoelectrochemical cell (DSPEC) wired in series with three spectral-complemental dyes BTA-2, APP-3 and APP-1 sensitizers-based dye-sensitized solar cell (DSC), respectively. The two spectral-complemental chromophores were used in DSC and DSPEC to ensure that the full solar spectrum could be absorbed as much as possible. The results showed that the photocurrent of tandem device was closely related to the open-circuit voltage (Voc) of sensitized DSC, in which the tandem configuration consisting of S5 based DSPEC and BTA-2 based DSC gave the best photocurrent. On this basis, tandem device with the only light energy and no external applied electrical bias was further constructed of BTA-2 based 2-junction DSC and S5 based DSPEC and obtained a photocurrent of 500 µA cm−2 for hydrogen generation. Furthermore, I−/I3− was used as a redox couple between dye regeneration and O2 production on the surface of Pt-IrO2/WO3. The strategy opens up the application of pure organic dyes in DSC/DSPEC tandem device.

Simultaneous Polychromatic Immunofluorescent Staining of Tissue Sections and Consecutive Imaging of up to Seven Parameters by Standard Confocal Microscopy.

Confocal microscopy has been an important imaging tool for life scientists for over 20 years. Early techniques focused on indirect staining processes that involved staining with an unconjugated primary antibody, followed by incubation with a secondary fluorescent antibody that would reveal and amplify the signal of the primary antibody. With more and more directly conjugated fluorescent primary antibodies becoming commercially available, staining with multiple fluorescent primary antibodies is now more frequent. To date, staining with up to three primary antibodies and a nuclear dye is widely practiced. Here, we describe an important improvement to the standard polychromatic immunofluorescent staining protocol that allows the simultaneous detection of seven fluorescent parameters using a standard confocal laser scanning microscope with four laser lines and four photomultiplier tubes. By incorporating recently available tandem dyes that emit in the blue and violet regions of the visible light spectrum (Brilliant Blue and Brilliant Violet), we were able to differentiate several additional fluorochromes simultaneously. Due to the added complexity of 7-color immunofluorescent imaging, we developed a clear methodology to optimize antibody concentrations and simple guidelines on how to identify and correct non-specific signals. These are detailed in the following protocol. © 2019 by John Wiley & Sons, Inc. Basic Protocol: 7-Color immunofluorescent staining protocol using directly conjugated antibodies Support Protocol 1: Antibody titration protocol Support Protocol 2: Spillover optimization protocol.

Labeling Antibodies with Cy5-Phycoerythrin.

Conjugates of the FRET dye Cy5-phycoerythrin (Cy5PE) with antibodies are relatively straightforward to make. The protocol does require synthesis of the Cy5PE tandem dye. Phycoerythrin (PE) can be purchased from multiple vendors. This type of conjugate is useful for immunofluorescence studies involving protein targets with low expression levels. Although the entire conjugation can be performed in a single day, there is an overnight stopping point. When initially making Cy5PE derivatives, several different conjugates with varying ratios of Cy5 to PE should be made. These should be tested by conjugating to a well-characterized antibody. Absorbance spectra readings are a very worthwhile step to determine the quality of the Cy5PE label.

Tandem structured luminescent solar concentrator based on inorganic carbon quantum dots and organic dyes

In this study, we demonstrate the simultaneous use of carbon quantum dots and organic dyes as highly emissive luminescent material in high performance tandem luminescent solar concentrator (LSC). The top LSC layer is based on carbon quantum dots (CQDs), while the bottom one is based on organic dye. The role of förster resonance energy transfer (FRET) is demonstrated in organic dyes in a bottom LSC. Moreover, the CQDs layer which has the capability to harvest ultraviolet (UV) and near-UV photons acts as a protective layer to improve the photo-stability of the organic dyes contained in bottom waveguide. The electrical measurements showed that the optical conversion efficiency (ηopt) and power conversion efficiency (ηPCE) of CQDs based single LSC are 5.62% and 1.03% respectively. While, ηopt, and ηPCE the dye-LSC are 13.42% and 2.72% respectively. However, in the tandem LSC, overall ηopt and ηPCE are 16.32% and 3.2% respectively. Our results showed that tandem structured CQDs and dye based luminescent solar concentrators make the practical use of LSCs more feasible because of the unique properties such as good photostability and high efficiency.

Generation of endogenous pH-sensitive EGF receptor and its application in high-throughput screening for proteins involved in clathrin-mediated endocytosis.

Previously we used gene-editing to label endogenous EGF receptor (EGFR) with GFP and demonstrate that picomolar concentrations of EGFR ligand drive signaling and endocytosis of EGFR in tumors in vivo (Pinilla-Macua et al., 2017). We now use gene-editing to insert a fluorogen activating protein (FAP) in the EGFR extracellular domain. Binding of the tandem dye pair MG-Bis-SA to FAP-EGFR provides a ratiometric pH-sensitive model with dual fluorescence excitation and a single far-red emission. The excitation ratio of fluorescence intensities was demonstrated to faithfully report the fraction of FAP-EGFR located in acidic endosomal/lysosomal compartments. Coupling native FAP-EGFR expression with the high method sensitivity has allowed development of a high-throughput assay to measure the rates of clathrin-mediated FAP-EGFR endocytosis stimulated with physiological EGF concentrations. The assay was utilized to screen a phosphatase siRNA library. These studies highlight the utility of endogenous pH-sensitive FAP-receptor chimeras in high-throughput analysis of endocytosis.

Bioluminescence Imaging of Inflammation in Vivo Based on Bioluminescence and Fluorescence Resonance Energy Transfer Using Nanobubble Ultrasound Contrast Agent.

Inflammation is an immunological response involved in various inflammatory disorders ranging from neurodegenerative diseases to cancers. Luminol has been reported to detect myeloperoxidase (MPO) activity in an inflamed area through a light-emitting reaction. However, this method is limited by low tissue penetration and poor spatial resolution. Here, we fabricated a nanobubble (NB) doped with two tandem lipophilic dyes, red-shifting luminol-emitted blue light to near-infrared region through a process integrating bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET). This BRET-FRET process caused a 24-fold increase in detectable luminescence emission over luminol alone in an inflammation model induced by lipopolysaccharide. In addition, the echogenicity of the BRET-FRET NBs also enables perfused tissue microvasculature to be delineated by contrast-enhanced ultrasound imaging with high spatial resolution. Compared with commercially available ultrasound contrast agent, the BRET-FRET NBs exhibited comparable contrast-enhancing capability but much smaller size and higher concentration. This bioluminescence/ultrasound dual-modal contrast agent was then successfully applied for imaging of an animal model of breast cancer. Furthermore, biosafety experiments revealed that multi-injection of luminol and NBs did not induce any observable abnormality. By integrating the advantages of bioluminescence imaging and ultrasound imaging, this BRET-FRET system may have the potential to address a critical need of inflammation imaging.

Genetically Targeted Ratiometric and Activated pH Indicator Complexes (TRApHIC) for Receptor Trafficking.

Fluorescent protein-based pH sensors are useful tools for measuring protein trafficking through pH changes associated with endo- and exocytosis. However, commonly used pH-sensing probes are ubiquitously expressed with their protein of interest throughout the cell, hindering our ability to focus on specific trafficking pools of proteins. We developed a family of excitation ratiometric, activatable pH responsive tandem dyes, consisting of a pH sensitive Cy3 donor linked to a fluorogenic malachite green acceptor. These cell-excluded dyes are targeted and activated upon binding to a genetically expressed fluorogen-activating protein and are suitable for selective labeling of surface proteins for analysis of endocytosis and recycling in live cells using both confocal and superresolution microscopy. Quantitative profiling of the endocytosis and recycling of tagged β2-adrenergic receptor (B2AR) at a single-vesicle level revealed differences among B2AR agonists, consistent with more detailed pharmacological profiling.

Monitoring C5aR2 Expression Using a Floxed tdTomato-C5aR2 Knock-In Mouse.

The biological significance of C5a receptor [(C5aR)2/C5L2], a seven-transmembrane receptor binding C5a and C5adesArg, remains ill-defined. Specific ligation of C5aR2 inhibits C5a-induced ERK1/2 activation, strengthening the view that C5aR2 regulates C5aR1-mediated effector functions. Although C5aR2 and C5aR1 are often coexpressed, a detailed picture of C5aR2 expression in murine cells and tissues is still lacking. To close this gap, we generated a floxed tandem dye (td)Tomato-C5aR2 knock-in mouse that we used to track C5aR2 expression in tissue-residing and circulating immune cells. We found the strongest C5aR2 expression in the brain, bone marrow, and airways. All myeloid-derived cells expressed C5aR2, although with different intensities. C5aR2 expression in blood and tissue neutrophils was strong and homogeneous. Specific ligation of C5aR2 in neutrophils from tdTomato-C5aR2 mice blocked C5a-driven ERK1/2 phosphorylation, demonstrating functionality of C5aR2 in the reporter mice. In contrast to neutrophils, we found tissue-specific differences in C5aR2 expression in eosinophils, macrophages, and dendritic cell subsets. Naive and activated T cells stained negative for C5aR2, whereas B cells from different tissues homogeneously expressed C5aR2. Also, NK cell subsets in blood and spleen strongly expressed C5aR2. Activation of C5aR2 in NK cells suppressed IL-12/IL-18-induced IFN-γ production. Intratracheal IL-33 challenge resulted in decreased C5aR2 expression in pulmonary eosinophils and monocyte-derived dendritic cells. In summary, we provide a detailed map of murine C5aR2 immune cell expression in different tissues under steady-state conditions and upon pulmonary inflammation. The C5aR2 knock-in mouse will help to reliably track and conditionally delete C5aR2 expression in experimental models of inflammation.

Multiexcitation Fluorogenic Labeling of Surface, Intracellular, and Total Protein Pools in Living Cells.

Malachite green (MG) is a fluorogenic dye that shows fluorescence enhancement upon binding to its engineered cognate protein, a fluorogen activating protein (FAP). Energy transfer donors such as cyanine and rhodamine dyes have been conjugated with MG to modify the spectral properties of the fluorescent complexes, where the donor dyes transfer energy through Förster resonance energy transfer to the MG complex resulting in binding-conditional fluorescence emission in the far-red region. In this article, we use a violet-excitable dye as a donor to sensitize the far-red emission of the MG-FAP complex. Two blue emitting fluorescent coumarin dyes were coupled to MG and evaluated for energy transfer to the MG-FAP complex via its secondary excitation band. 6,8-Difluoro-7-hydroxycoumarin-3-carboxylic acid (Pacific blue, PB) showed the most efficient energy transfer and maximum brightness in the far-red region upon violet (405 nm) excitation. These blue-red (BluR) tandem dyes are spectrally varied from other tandem dyes and are able to produce fluorescence images of the MG-FAP complex with a large Stokes shift (>250 nm). These dyes are cell-permeable and are used to label intracellular proteins. Used together with a cell-impermeable hexa-Cy3-MG (HCM) dye that labels extracellular proteins, we are able to visualize extracellular, intracellular, and total pools of cellular protein using one fluorogenic tag that combines with distinct dyes to effect different spectral characteristics.

Tandem Dye-Sensitized Solar Cells Based on TCO-less Back Contact Bottom Electrodes

Mechanically stacked and series connected tandem dye sensitized solar cells (T-DSSCs) are fabricated in novel architectures. The architecture consist of TCO tandem DSSCs stacked with TCO-less back contact DSSCs as bottom electrodes (TCO-less tandem DSSCs). Resulting TCO-less tandem DSSCs architecture finds its usefulness in efficient photon harvesting due to improved light transmission and enhanced photons reaching to the bottom electrodes. The fabricated tandem performance was verified with visible light harvesting model dyes D131 and N719 as a proof of concept and provided the photoconversion efficiency (PCE) of 6.06% under simulated condition. Introduction of panchromatic photon harvesting by utilizing near infrared light absorbing Si-phthalocyanine dye in combination with the modified tandem DSSC architecture led to enhancement in the PCE up to 7.19%.

Issue highlights--November 2015 (88:B6).

Issue highlights--November 2015 (88:B6).

Combining novel device architecture and NIR dye towards the fabrication of transparent conductive oxide-less tandem dye sensitized solar cells

Tandem solar cells with different device architectures utilizing a back contact transparent conductive oxide-less bottom electrode (TCO-less tandem DSSC) that has better control of the optical transmission losses incurred by intermediate TCO layers with flexible Pt/tin-doped indium oxide (ITO)-polyethylene terephthalate film as an intermediate layer are reported. The proposed device architecture suppresses the optical loss to a greater extent (around 30%). Sensitizers were ruthenium-based N719 dye in the top electrode, while the newly synthesized phthalocyanine dye (PC25) corresponding to photon harvesting in the near infrared (NIR) region (up to 900 nm) was used in the TCO-less bottom cell. Open circuit voltage (Voc) ≥ 1.18 V (sum of top cell and bottom cell Voc) justifies the TCO-less tandem DSSC formation.

Impact of Fe doping on performances of CuGaO2 p-type dye-sensitized solar cells

Delafossite materials like CuGaO2 have been receiving much attention due to their potential role in the elaboration of future tandem dye sensitized solar cells. P-type CuGa1−xFexO2 (0≤x≤0.05) with delafossite structure has been obtained by hydrothermal method. Fe-doped and undoped CuGaO2 thin films have been investigated as cathode elements in N719 dye-sensitized solar cells. Integration of iron dopants in the delafossite structure improves the photovoltaic performance of CuGaO2 thin films both due to excellent optical transparency of CuGaO2 in the visible range and its high electrical conductivity. Also Fe leads to a pronounced hydrophilic character and to a higher dye uptake than the undoped sample.

One‐pot preparation of multicolor polymeric nanoparticles with high brightness by single wavelength excitation

ABSTRACTFluorescent nanoparticles with multiplex distinct emission signatures and high brightness by a single wavelength excitation are substantially needed in multiplex bioassays and imaging. In this study, we synthesized fluorescent polymeric nanoparticles incorporated with three polymerizable organic dyes via a one‐pot miniemulsion. By altering the doping ratio of three tandem dyes, the nanoparticles display abundant multiple fluorescence such as blue, cyan, green, orange, pink, red etc., together with distinguishable emission signatures under a single wavelength excitation, which were arising from the effective fluorescence resonance energy transfer (FRET) between the three energy‐matched dyes. Meanwhile, a large Stokes shift (up to 250 nm) can be generated by taking place multiple FRET cascade mechanism between donor and acceptor fluorophores in nanoparticles, which also suggests broad applications in biological labeling and imaging. Moreover, these nanoparticles are uniform in size, highly bright, excellently photostable, and shown prominent longterm stability. Overall, the novel multicolor fluorescent polymeric nanoparticles augur well for their potential applications in multiplexed bioanalysis and emitting displays. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015, 132, 41492.

Tandem dyes: Stability in cocktails and compensation considerations

The stability and performance of tandem-conjugated antibodies can be impaired when stored in antisera cocktails (Biancotto et al., J Immunol Methods 2011;363:245-261; Rawstron et al., Leukemia 2013;27:142-149). This, and the need for frequent re-compensation due to the possible spectral spillover variation between tandem lots, reduces the robustness of clinical flow cytometry panels that include tandems. Since tandems are required for standard 8-10 color screens, further studies of the stability of tandems in cocktails and their spillover variability are warranted. The performance of PE- and APC-tandems stored in cocktails was tested on fresh bone marrow, preserved blood and lyophilized cell samples over 1-, 6-, or 8-week periods, respectively, and their spillover matrices were compared. The observed correction factor differences were used as the basis for analyzing how the application of an incorrect compensation matrix could influence data interpretation. Signal intensities and background fluorescence remained constant for all fluorochromes in the cocktails tested. Spillover correction factors for different PE-Cy7 mAbs did not exceed or were only marginally higher than those for non-tandem organic dye-conjugated mAb. By applying the correction factor differences observed between tandem mAb lots to clinical data, it was found that the over and under compensation would not alter the clinical interpretation. Tandems can be safely stored and used in cocktails. However, each cocktail should be tested on relevant material prior to use. Exact compensation settings are a requirement for accurate data. Provided that careful evaluation of tandem compensation requirements is carried out, certain tandems may use a generic compensation matrix.