carbocyanines and 4-4-difluoro-4-bora-3a-4a-diaza-s-indacene

Topics: Boron Compounds; Carbocyanines; Fluorescent Dyes; Ions; Metals; Monophenol Monooxygenase; Oxidoreductases; Reactive Oxygen Species; Solubility; Water; Xanthenes

The long wavelength (far-red to NIR) analyte-responsive fluorescent probes are advantageous for in vivo bioimaging because of minimum photo-damage to biological samples, deep tissue penetration, and minimum interference from background auto-fluorescence by biomolecules in the living systems. Thus, great interest in the development of new long wavelength analyte-responsive fluorescent probes has emerged in recent years. This review highlights the advances in the development of far-red to NIR fluorescent probes since 2000, and the probes are classified according to their organic dye platforms into various categories, including cyanines, rhodamine analogues, BODIPYs, squaraines, and other types (240 references).

Topics: Animals; Boron Compounds; Carbocyanines; Cyclobutanes; Fluorescent Dyes; Humans; Optical Imaging; Phenols; Rhodamines

A full understanding concerning the photophysical properties of a fluorescent label is crucial for a reliable and predictable performance in biolabelling applications. This holds true not only for the choice of a fluorophore in general, but also for the correct interpretation of data, considering the complexity of biological environments. In the frame of a case study involving inflammation imaging, we report the photophysical characterization of four fluorescent S100A9-targeting compounds in terms of UV-vis absorption and photoluminescence spectroscopy, fluorescence quantum yields (Φ

Topics: Animals; Boron Compounds; Calgranulin B; Carbocyanines; Fluorescent Dyes; Mice

Fluorescent nucleoside triphosphates are powerful probes of DNA synthesis, but their potential use in living animals has been previously underexplored. Here, we report the synthesis and characterization of 7-deaza-(1,2,3-triazole)-2'-deoxyadenosine-5'-triphosphate (dATP) derivatives of tetramethyl rhodamine ("TAMRA-dATP"), cyanine ("Cy3-dATP"), and boron-dipyrromethene ("BODIPY-dATP"). Upon microinjection into live zebrafish embryos, all three compounds were incorporated into the DNA of dividing cells; however, their impact on embryonic toxicity was highly variable, depending on the exact structure of the dye. TAMRA-EdATP exhibited superior characteristics in terms of its high brightness, low toxicity, and rapid incorporation and depletion kinetics in both a vertebrate (zebrafish) and a nematode (

Topics: Animals; Boron Compounds; Caenorhabditis elegans; Carbocyanines; Deoxyadenine Nucleotides; DNA; DNA Replication; Fluorescent Dyes; Optical Imaging; Rhodamines; Zebrafish

The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is increasingly used for detection of various macromolecules and metabolites in biological samples. Here, we present a detailed analysis of the CuAAC reaction conditions in cells and tissue sections. Using the optimized CuAAC conditions, we have devised a highly sensitive immunostaining technique, based on the tyramide signal amplification/catalyzed reporter deposition (TSA/CARD) method with a novel alkyne tyramide substrate. The described method offers improved detection threshold compared to conventional immunofluorescent staining and produces significantly lower non-specific background than TSA/CARD with fluorescent tyramides.

Topics: Animals; Azides; Boron Compounds; Brain Chemistry; Bromodeoxyuridine; Carbocyanines; Cells, Cultured; Click Chemistry; Copper; Deoxyuridine; DNA; Female; Fluorescent Antibody Technique; Fluorescent Dyes; Horseradish Peroxidase; Humans; Male; Mice; Pluripotent Stem Cells; Sensitivity and Specificity; Tyramine

Nanogels that are amenable to facile multi-functionalization with imaging, therapeutic, and targeting agents are attractive theranostic platforms for addressing challenges in conventional diagnostics and therapy. In this work, reactive copolymers containing poly(ethylene glycol), maleimide, and pendant hydroxyl groups as side chains are used to construct nanogels by employing their thermoresponsive self-assembly in aqueous media. Subsequent cross-linking of these nanosized aggregates with dithiols using thiol-maleimide chemistry yields nanogels containing maleimide, thiol, and hydroxyl groups. The hydroxyl groups are readily activated to N-hydroxysuccinimide based carbonates that undergo conjugation with amine-containing molecules through carbamate linkage under mild conditions. As a demonstration of multi-functionalization, the maleimide, thiol, and activated carbonate groups were functionalized with a thiol-containing cancer cell targeting peptide, a maleimide-containing fluorescent indocyanine Cy5 dye, and an anticancer drug doxorubicin, respectively. It was observed that enhanced drug release from nanogels occurs under acidic conditions. While the parent nanogel vehicles did not possess any toxicity, drug conjugated constructs with and without targeting group were cytotoxic against MDA-MB-231 breast cancer cells. The cyclic peptide containing targeted nanogel exhibited slightly higher cytotoxicity than its counterpart devoid of any targeting group. Furthermore, higher level of drug internalization into MDA-MB-231 cells was observed for the targeting group containing construct. It can be envisioned that facile fabrication and multi-functionalization of these reactive nanogels simultaneously through nonreversible and reversible linkages offers a modular platform that can be configured as a theranostic agent for addressing challenges in conventional therapy of various diseases.

Topics: Antineoplastic Agents; Boron Compounds; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Doxorubicin; Drug Carriers; Drug Delivery Systems; Female; Fluorescent Dyes; Gels; Humans; Nanostructures; Optical Imaging; Polyethylene Glycols; Polymethacrylic Acids; Theranostic Nanomedicine

Multicolor single-molecule localization microscopy (SMLM) expands our understanding of subcellular details and enables the study of biomolecular interactions through precise visualization of multiple molecules in a single sample with resolution of ~10-20 nm. Probe selection is vital to multicolor SMLM, as the fluorophores must not only exhibit minimal spectral crosstalk, but also be compatible with the same photochemical conditions that promote fluorophore photoswitching. While there are numerous commercially available photoswitchable fluorophores that are optimally excited in the standard Cy3 channel, they are restricted to short Stokes shifts (<30 nm), limiting the number of colors that can be resolved in a single sample. Furthermore, while imaging buffers have been thoroughly examined for commonly used fluorophore scaffolds including cyanine, rhodamine, and oxazine, optimal conditions have not been found for the BODIPY scaffold, precluding its routine use for multicolor SMLM. Herein, we screened common imaging buffer conditions including seven redox reagents with five additives, resulting in 35 overall imaging buffer conditions to identify compatible combinations for BODIPY-based fluorophores. We then demonstrated that novel, photoswitchable BODIPY-based fluorophores with varied length Stokes shifts provide additional color options for SMLM using a combination of BODIPY-based and commercially available photoswitchable fluorophores.

Topics: Boron Compounds; Buffers; Carbocyanines; Color; Fluorescent Dyes; Humans; Microscopy, Fluorescence; Microtubules; Photochemistry; Single Molecule Imaging; Tumor Cells, Cultured

Changes in microscopic viscosity and macromolecular crowding accompany the transition of proteins from their monomeric forms into highly organised fibrillar states. Previously, we have demonstrated that viscosity sensitive fluorophores termed 'molecular rotors', when freely mixed with monomers of interest, are able to report on changes in microrheology accompanying amyloid formation, and measured an increase in rigidity of approximately three orders of magnitude during aggregation of lysozyme and insulin. Here we extend this strategy by covalently attaching molecular rotors to several proteins capable of assembly into fibrils, namely lysozyme, fibrinogen and amyloid-β peptide (Aβ(1-42)). We demonstrate that upon covalent attachment the molecular rotors can successfully probe supramolecular assembly in vitro. Importantly, our new strategy has wider applications in cellulo and in vivo, since covalently attached molecular rotors can be successfully delivered in situ and will colocalise with the aggregating protein, for example inside live cells. This important advantage allowed us to follow the microscopic viscosity changes accompanying blood clotting and during Aβ(1-42) aggregation in live SH-SY5Y cells. Our results demonstrate that covalently attached molecular rotors are a widely applicable tool to study supramolecular protein assembly and can reveal microrheological features of aggregating protein systems both in vitro and in cellulo not observable through classical fluorescent probes operating in light switch mode.

Topics: Amyloid beta-Peptides; Boron Compounds; Carbocyanines; Cell Line; Fibrinogen; Fluorescent Dyes; Humans; Insulin; Microscopy, Electron, Transmission; Molecular Probes; Muramidase; Nanoconjugates; Optical Imaging; Peptide Fragments; Protein Aggregates; Viscosity

A selection of NIR-optically responsive neuron probes was produced comprising of a donor julolidyl group connected to a BODIPY core and several different styryl and vinylpyridinyl derived acceptor moieties. The strength of the donor-acceptor interaction was systematically modulated by altering the electron withdrawing nature of the aryl unit. The fluorescence quantum yield was observed to decrease as the electron withdrawing effect of the aryl subunit increased in line with changes of the Hammett parameter. The effectiveness of these fluorophores as optically responsive dyes for neuronal imaging was assessed by measuring the toxicity and signal-to-noise ratio (SNR) of each dye. A great improvement of SNR was obtained when compared to the first-generation BODIPY-based voltage sensitive dyes with concomitant toxicity decrease. The mechanism for the optical response is disparate from conventional cyanine-based dyes, opening up a new way to produce effective voltage sensitive dyes that respond well into the NIR region.

Topics: Animals; Boron Compounds; Brachyura; Carbocyanines; Crystallography, X-Ray; Fluorescent Dyes; Microscopy, Fluorescence; Molecular Conformation; Neurons; Quantum Theory; Signal-To-Noise Ratio; Spectrophotometry, Infrared; Spectrophotometry, Ultraviolet

Topics: Animals; Binding Sites; Boron Compounds; Carbocyanines; Cell Line, Tumor; Fireflies; Firefly Luciferin; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; HEK293 Cells; Humans; Luciferases, Firefly; Mice; Mice, Nude; Molecular Docking Simulation; Protein Structure, Tertiary; Quantum Dots; Research Design; Software; Spectroscopy, Near-Infrared; Transplantation, Heterologous; Transplantation, Homologous

When the fluorescence signal of a dye is being quantified, the staining protocol is an important factor in ensuring accuracy and reproducibility. Increasingly, lipophilic dyes are being used to quantify cellular lipids in microalgae. However, there is little discussion about the sensitivity of these dyes to staining conditions. To address this, microalgae were stained with either the lipophilic dyes often used for lipid quantification (Nile Red and BODIPY) or a lipophilic dye commonly used to stain neuronal cell membranes (DiO), and fluorescence was measured using flow cytometry. The concentration of the cells being stained was found not to affect the fluorescence. Conversely, the concentration of dye significantly affected the fluorescence intensity from either insufficient saturation of the cellular lipids or formation of dye precipitate. Precipitates of all three dyes were detected as events by flow cytometry and fluoresced at a similar intensity as the chlorophyll in the microalgae. Prevention of precipitate formation is, therefore, critical to ensure accurate fluorescence measurement with these dyes. It was also observed that the presence of organic solvents, such as acetone and dimethyl sulfoxide (DMSO), were not required to increase penetration of the dyes into cells and that the presence of these solvents resulted in increased cellular debris. Thus, staining conditions affected the fluorescence of all three lipophilic dyes, but Nile Red was found to have a stable fluorescence intensity that was unaffected by the broadest range of conditions and could be correlated to cellular lipid content.

Topics: Acetone; Boron Compounds; Carbocyanines; Cells, Cultured; Chemical Precipitation; Flow Cytometry; Fluorescent Dyes; Hydrophobic and Hydrophilic Interactions; Lipids; Microalgae; Oxazines; Solvents; Staining and Labeling

Imaging membranes in live cells with nanometer-scale resolution promises to reveal ultrastructural dynamics of organelles that are essential for cellular functions. In this work, we identified photoswitchable membrane probes and obtained super-resolution fluorescence images of cellular membranes. We demonstrated the photoswitching capabilities of eight commonly used membrane probes, each specific to the plasma membrane, mitochondria, the endoplasmic recticulum (ER) or lysosomes. These small-molecule probes readily label live cells with high probe densities. Using these probes, we achieved dynamic imaging of specific membrane structures in living cells with 30-60 nm spatial resolution at temporal resolutions down to 1-2 s. Moreover, by using spectrally distinguishable probes, we obtained two-color super-resolution images of mitochondria and the ER. We observed previously obscured details of morphological dynamics of mitochondrial fusion/fission and ER remodeling, as well as heterogeneous membrane diffusivity on neuronal processes.

Topics: Boron Compounds; Carbocyanines; Cell Membrane; Dendrites; Endoplasmic Reticulum; Fluorescent Dyes; Hippocampus; Lipid Bilayers; Lysosomes; Microscopy, Fluorescence; Mitochondria; Nanostructures; Neurons; Organelles; Pseudopodia; Stochastic Processes

Even though superresolution microscopy indicates that size of plasma membrane rafts is <20 nm, those structures have never been observed. Förster resonance energy transfer (FRET) is therefore still the most powerful optical method for characterization of such domains. In this letter we investigate relation between nanodomain affinity of a donor-acceptor (D/A) pair and the detectable nanodomain size/area. We show that probes with high affinity to the liquid-ordered (L(o)) phase are required for detecting domain sizes of a few nanometers, and/or domains that occupy a few percent of the bilayer area. A combination of donors and acceptors that prefer different phases is the more favorable approach. For instance, a D/A pair with the distribution constant of donors K(D) = 5 and acceptors K(A) = 0.01 can resolve a broad spectrum of nanodomain sizes. On the other hand, currently available donors and acceptors that prefer the same phase, either the liquid-disordered (L(d)) or L(o) phase, are not so convenient for determining domain sizes <20 nm. Here the detection limits of FRET experiments employing several commonly used D/A pairs have been investigated.

Topics: Boron Compounds; Carbocyanines; Cholera Toxin; Electrons; Fluorescence Resonance Energy Transfer; Lipid Bilayers; Membrane Microdomains; Monte Carlo Method; Nanoparticles; Particle Size; Perylene; Phycoerythrin; Reproducibility of Results; Rhodamines; Time Factors

Enhancement of the environmentally responsive fluorescent properties of polydiacetylene (PDA) by combination with lipophilic fluorophores was demonstrated and properties of the PDA/fluorophore systems were explored. Liposomes containing PDA and fluorophores exhibited enhanced Stokes shift and increase in emission as a result of energy transfer from PDA to fluorophore. The effects of fluorophore variation, degree of PDA polymerization and diyne placement in the diacetylene lipid tails on the emission enhancement were studied. It was determined that signal generation was optimized at a relatively low extent of PDA polymerization with the optimal degree of polymerization dependent on the other parameters. Energy transfer was used as a tool to detect fluorophore exchange between polymerized and unpolymerized liposomes and to study the effects of fluorophore structure on exchange from unpolymerized to PDA liposomes. Fluorophores that locate at the aqueous interface with alkyl anchors were slow to transfer while fluorophores that partition into the alkyl regions of the liposomes transfer quickly.

Topics: Biosensing Techniques; Boron Compounds; Carbocyanines; Energy Transfer; Fluorescent Dyes; Liposomes; Models, Molecular; Polyacetylene Polymer; Polymers; Polyynes; Spectrometry, Fluorescence

The effects of annexin A5 on the lateral diffusion of single-molecule lipids and single-molecule proteins were studied in an artificial lipid bilayer membrane. Annexin A5 is a member of the annexin superfamily, which binds preferentially to anionic phospholipids in a Ca2+-dependent manner. In this report, we were able to directly monitor single BODIPY 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DHPE) and ryanodine receptor type 2 (RyR2) labeled with Cy5 molecules in lipid bilayers containing phosphatidylserine (PS) by using fluorescence microscopy. The diffusion coefficients were calculated at various annexin A5 concentrations. The diffusion coefficients of BODIPY-DHPE and Cy5-RyR2 in the absence of annexin A5 were 4.81 x 10(-8) cm(2)/s and 2.13 x 10(-8) cm(2)/s, respectively. In the presence of 1 microM annexin A5, the diffusion coefficients of BODIPY-DHPE and Cy5-RyR2 were 2.2 x 10(-10) cm(2)/s and 9.5 x 10(-11) cm(2)/s, respectively. Overall, 1 microM of annexin A5 was sufficient to induce a 200-fold decrease in the lateral diffusion coefficient. Additionally, we performed electrophysiological examinations and determined that annexin A5 has little effect on the function of RyR2. This means that annexin A5 can be used to immobilize RyR2 in a lipid bilayer when imaging and analyzing RyR2.

Topics: Animals; Annexin A5; Boron Compounds; Carbocyanines; Dogs; Lipid Bilayers; Microscopy, Fluorescence; Phosphatidylethanolamines; Ryanodine Receptor Calcium Release Channel

To obtain detailed information about the three-dimensional (3D) organization of small biomolecular assemblies with a size of less than 100 nanometers, advanced techniques are required that enable the determination of absolute 3D positions and distances between individual fluorophores well below the resolution limit of conventional light microscopy. We show how spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) can provide significant contributions and allow us to determine distances between conventional individual fluorophores (Bodipy 630/650 and Cy5.5) that are less than 20 nm apart. We take advantage of fluorescent dyes (here Cy5.5 and Bodipy 630/650) that can be efficiently excited by a single pulsed diode laser emitting at 635 nm but differ in their fluorescence lifetime and emission maxima. The potential of the method for ultrahigh colocalization studies is demonstrated by measuring the end-to-end distance between single fluorophores separated by double-stranded DNA of various lengths. Combining SFLIM with polarization-modulated excitation allows us to obtain, simultaneously, information about the relative orientation of fluorophores. Furthermore, we show that the environment-dependent photophysics of conventional fluorophores, that is, photostability, blinking pattern, and the tendency to enter irreversible nonfluorescent states, sets certain limitations to their in vitro and in vivo applications.

Topics: Algorithms; Biophysics; Boron Compounds; Carbocyanines; DNA; Electrons; GTP Phosphohydrolase Activators; Image Processing, Computer-Assisted; Microscopy; Microscopy, Confocal; Microscopy, Fluorescence; Molecular Probe Techniques; Normal Distribution; Software; Time Factors

We follow somite segmentation in living chick embryos and find that the shaping process is not a simple periodic slicing of tissue blocks but a much more carefully choreographed separation in which the somite pulls apart from the segmental plate. Cells move across the presumptive somite boundary and violate gene expression boundaries thought to correlate with the site of the somite boundary. Similarly, cells do not appear to be preassigned to a given somite as they leave the node. The results offer a detailed picture of somite shaping and provide a spatiotemporal framework for linking gene expression with cell movements.

Topics: Animals; Boron Compounds; Carbocyanines; Cell Adhesion; Cell Movement; Cell Size; Central Nervous System; Ceramides; Chick Embryo; Epithelial Cells; Gene Expression; Gene Expression Profiling; Mesoderm; Microscopy, Confocal; Models, Biological; Receptor, EphA4; Somites; Time Factors

Localized Ca(2+)-release events, Ca(2+)sparks, have been suggested to be the 'elementary building blocks' of the calcium signalling system in all types of muscles. In striated muscles these occur at regular intervals along the fibre corresponding to the sarcomeric structures which do not exist in smooth muscle. We showed previously that in visceral and vascular myocytes Ca(2+)sparks occurred much more frequently at certain sites (frequent discharge sites [FDSs]). In this paper, we have related the position of FDSs to the distribution of the sarcoplasmic reticulum in the same living myocyte. The three-dimensional distribution of the SR in freshly isolated rabbit portal vein myocytes was visualized by means of high-resolution confocal imaging after staining with DiOC(6)and/or BODIPY TR-X ryanodine. Both fluorochromes revealed a similar staining pattern indicating a helical arrangement of well-developed superficial SR which occupied about 6% of the cell volume. Computing the frequency of spontaneous Ca(2+)sparks detected by means of fluo-4 fluorescence revealed that in about 70% of myocytes there was only one major FDS located on a prominent portion of superficial SR network usually within 1-2 microm of the nuclear envelope, although a few sparks occurred at other sites scattered generally in superficial locations throughout the cell. Polarized mitochondria were readily identified by accumulation of tetramethylrhodamine ethyl ester (TMRE). These were closely associated with the SR network in extra-nuclear regions. TMRE staining, however, failed to reveal any mitochondria near the FDS-related SR element. When observed, propagating [Ca(2+)](i)waves and associated myocyte contractions were initiated at FDSs. This study provide first insight into the three-dimensional arrangement of the SR in living smooth muscle cells and relates the peculiarity of the structural organization of the myocyte to the features of Ca(2+)signalling at subcellular level.

Topics: Aniline Compounds; Animals; Boron Compounds; Calcium; Carbocyanines; Fluorescent Dyes; Image Processing, Computer-Assisted; Microscopy, Confocal; Microscopy, Fluorescence; Mitochondria; Muscle Fibers, Skeletal; Muscle, Smooth, Vascular; Organometallic Compounds; Portal Vein; Rabbits; Ryanodine; Sarcoplasmic Reticulum; Xanthenes

We have developed a novel method for real-time monitoring of RNA synthesis in in vitro transcription reactions using fluorescence resonance energy transfer (FRET). Two 15mer DNAs, either of which was labeled with Bodipy493/503 as a donor or Cy5 as an acceptor, were prepared. When the two fluorescent DNAs hybridized to adjacent locations on Xenopus: elongation factor 1-alpha (xelf1-alpha) RNA, the distance between the two fluorophores became very close, causing FRET to occur and resulting in changes in fluorescence spectra. A high accessibility 30mer site of xelf1-alpha RNA was found and excess amounts of a pair of donor and acceptor DNA probes that were complementary to the site were added to the in vitro transcription reaction solution. Changes in fluorescence spectra were observed in response to progression of xelf1-alpha RNA synthesis that showed that the fluorescent probes hybridized to the synthesized RNA. Furthermore, when probes hybridizing to the synthesized xelf1-alpha RNA with less efficiency were used to monitor the reaction, spectral changes in response to RNA synthesis were also observed. This result suggests that the probes hybridized to synthesizing RNA molecules before they folded to form secondary structure and that there is no need to select sites on the RNA for the probes, which is required for probes hybridizing to folded RNA molecules.

Topics: Animals; Boron Compounds; Carbocyanines; DNA Probes; Energy Transfer; Gene Expression Profiling; Nucleic Acid Conformation; Nucleic Acid Hybridization; Peptide Elongation Factor 1; RNA, Messenger; Spectrometry, Fluorescence; Templates, Genetic; Transcription, Genetic; Xenopus

To understand the mechanisms for endocytic sorting of lipids, we investigated the trafficking of three lipid-mimetic dialkylindocarbocyanine (DiI) derivatives, DiIC16(3) (1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), DiIC12(3) (1,1'- didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), and FAST DiI (1,1'-dilinoleyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate), in CHO cells by quantitative fluorescence microscopy. All three DiIs have the same head group, but differ in their alkyl tail length or unsaturation; these differences are expected to affect their distribution in membrane domains of varying fluidity or curvature. All three DiIs initially enter sorting endosomes containing endocytosed transferrin. DiIC16(3), with two long 16-carbon saturated tails is then delivered to late endosomes, whereas FAST DiI, with two cis double bonds in each tail, and DiIC12(3), with saturated but shorter (12-carbon) tails, are mainly found in the endocytic recycling compartment. We also find that DiOC16(3) (3,3'- dihexadecyloxacarbocyanine perchlorate) and FAST DiO (3, 3'-dilinoleyloxacarbocyanine perchlorate) behave similarly to their DiI counterparts. Furthermore, whereas a phosphatidylcholine analogue with a BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore attached at the end of a 5-carbon acyl chain is delivered efficiently to the endocytic recycling compartment, a significant fraction of another derivative with BODIPY attached to a 12-carbon acyl chain entered late endosomes. Our results thus suggest that endocytic organelles can sort membrane components efficiently based on their preference for association with domains of varying characteristics.

Topics: Animals; Boron Compounds; Carbocyanines; Cell Membrane; CHO Cells; Cricetinae; Endocytosis; Endosomes; Fluorescent Dyes; Humans; Lipid Metabolism; Lipids; Lysosomes; Models, Biological; Receptors, Transferrin

Investigations of fluorescence lifetimes of the dye 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoic acid (NBD-HA) and of DNA M13 primers labeled with NBD-HA, Cy3, rhodamine green and 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene++ +-3-propionic acid (BODIPY-FL) dyes in polyacrylamide gels of various degrees of crosslinking and different crosslinkers, and in a cellulose sieving buffer with different organic modifiers, are described. The dependence of fluorescence lifetime on gel matrix and on experimental conditions was studied in order to identify which factors may be important for optimization of multiplex fluorescence lifetime detection. Lifetimes were determined in both batch solution and on-the-fly, on-column in CE. Results show that lifetimes of the primer-attached dyes remain constant in gels of different composition. Additionally, multiexponential fluorescence decays are observed for primer-attached dyes in batch solutions of the cellulose sieving buffers but are reduced to monoexponential decays when measured on-the-fly, on-column in CE. Lifetime detectability can be improved by addition of an organic modifier to the gel matrix.

Topics: Acrylic Resins; Boron Compounds; Buffers; Carbocyanines; Cellulose; DNA Primers; Electrophoresis, Capillary; Fluorescence; Fluorescent Dyes; Gels; Time

In the vertebrate olfactory system, sensory neurons with common odorant specificities project to specific glomeruli in the olfactory bulb. How do olfactory sensory neurons find their glomerular targets? To address this question, we have visualized the genesis of the peripheral olfactory system in living zebrafish embryos. Dye labelings reveal that a primordial yet stereotyped map of glomeruli is apparent during embryogenesis. By labeling a small number of cells with an ectopically expressed green fluorescent protein reporter, we can observe the dynamic growth behaviors of individual olfactory neuron growth cones as they project to their glomeruli. We find that olfactory axons extend directly to their partner glomeruli, suggesting that these cells' growth cones rely upon pathfinding cues to reach their targets.

Topics: Afferent Pathways; Animals; Animals, Genetically Modified; Axons; Boron Compounds; Carbocyanines; Fluorescent Dyes; Genes, Reporter; Green Fluorescent Proteins; Luminescent Proteins; Microscopy, Video; Neurites; Neuronal Plasticity; Olfactory Bulb; Olfactory Receptor Neurons; Plasmids; Zebrafish

We have refined prismless total internal reflection fluorescence microscopy with extremely low background to visualize single fluorophores attached to protein molecules interacting with a filamentous biopolymer labelled with different colour fluorophores. By using Stokes and anti-Stokes fluorescence, two different colour fluorescences from two different colour fluorophores excited with a single wavelength laser can be observed simultaneously. This microscopy was applied to visualize motor proteins, actin and myosin molecules. Single myosin molecules labelled with a tetramethylrhodamine-5-iodoacetamide interacting with a BODIPY FL-labelled actin filament, a filamentous polymer of actin molecules, were observed clearly and simultaneously in aqueous solution. Individual hydrolysis reactions of Cy3-labelled ATP by single myosin molecules and sliding of a BODIPY FL-labelled actin filament along the myosin molecules could also be observed simultaneously. Thus, this technique is useful for observing single molecular processes of proteins interacting with a biological macromolecule such as an actin filament and a DNA.

Topics: Actins; Actomyosin; Adenosine Triphosphate; Boron Compounds; Carbocyanines; Fluorescent Dyes; Lasers; Microscopy, Fluorescence; Microscopy, Video; Myosin Subfragments; Rhodamines; Spectrometry, Fluorescence

Fluorescein isothiocyanate (FITC) is largely used in immunofluorescence methods. We propose to analyse the quality of some recent fluorochromes using image analysis. Fluorochromes tested include FITC and dichlorotriazinylaminofluorescein (DTAF), dipyrrometheneboron difluoride (BODIPY), Rhodol Green and cyanine 2. RAMOS cells were immunolabelled against the proliferating cell nuclear antigen (PCNA) revealed by the biotin-streptavidin technique. Slides were mounted in anhydrous glycerol or in buffered glycerol (pH 7.0 or pH 8.5). No antifading medium was added. Cell fluorescence emission intensity and bleaching characteristics were measured. Rhodol Green exhibited the highest fluorescence intensity and the best photobleaching resistance. Although BODIPY also resisted well during the photobleaching assay, its fluorescence intensity was weak. FITC, DTAF and cyanine 2 showed intermediate fluorescence intensity and a fast decay of fluorescence. Among the green emitting fluorochromes tested, Rhodol Green appeared to be the best.

Topics: Bacterial Proteins; Boron Compounds; Carbocyanines; Evaluation Studies as Topic; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Antibody Technique; Fluorescent Dyes; Humans; Microscopy, Fluorescence; Proliferating Cell Nuclear Antigen; Spectrometry, Fluorescence; Streptavidin; Tumor Cells, Cultured