The use of fluorescent fatty acid analogs as labels in trematode experimental infections

We examined the utility of fluorescent fatty acid analog dyes for labeling larval trematodes to use in experimental infections. Our goals were to identify two dyes that label larval trematodes belonging to the species Maritrema novaezealandensis and Coitocaecum parvum, determine if the dyes influence survival and infectivity of larval trematodes and/or host mortality, and if larval trematodes labeled with alternative dyes could be distinguished post-infection. The two dyes tested, BODIPY FL C₁₂ and BODIPY 558/568 C₁₂, successfully labeled all treated larval trematodes, did not influence cercariae survival or infectivity, and did not influence host mortality in either host-parasite system. All larval parasites were fluorescent and distinguishable after 5 days in amphipod intermediate hosts. In addition, larval Acanthoparyphium sp. were strongly fluorescent with both dyes after 5 weeks within cockle hosts. This method should be extremely useful for experimental studies using trematode-host systems as models for addressing a range of ecological and evolutionary questions.     

Custom fluorescent-nucleotide synthesis as an alternative method for nucleic acid labeling

The variety of potentially useful dyes or haptenes available for fluorescent nucleic acid hybridization assays is far greater than what can be obtained from commercial sources. Since this diversity could be useful in many laboratory applications, we have developed a simple and inexpensive procedure for preparing nonpurified labeled nucleotides, for use in common nucleic acid labeling reactions, such as PCR and nick translation. The modified nucleotides were synthesized by coupling allylamine-dUTP to the succinimidyl-ester derivatives of the fluorescent dyes or haptenes such as biotin or digoxigenin, which require fluorescently labeled proteins for detection. This method allows custom preparation of most common fluorescent nucleotides and rapid testing of new ones, while reducing the cost of procedures such as multiplex fluorescent in situ hybridization (M-FISH) by 100-200 fold.     

Carbocyanine dyes stain the sarcoplasmic reticulum of beating heart cells

In search of a fluorescent dye suitable for monitoring membrane potentials of beating heart cells, we noticed that the carbocyanine dyes, CC₅ and CC₆, show a unique pattern of intracellular distribution in vital and glutaraldehyde-fixed cardiomyoblasts. This distribution is clearly different from that observed in fibroblasts. In heart cells, it parallels the localization of actin-myosin containing myofilaments as visualized by fluorescent antibody staining but it does not correspond to the localization of actin filaments or the microtubules. In fibroblasts these dyes stain only fine filaments and granules in the perinuclear space which correspond to the endoplasmic reticulum. This observation is evidence in support of the hypothesis that carbocyanine dyes accumulate selectively in the sarcoplasmic reticulum. It indicates that certain carbocyanine dyes may be useful tools to differentiate between muscle cells and connective tissue cells in cell cultures.     

Triazole-containing BODIPY dyes as novel fluorescent probes for soluble oligomers of amyloid Aβ1-42 peptide

A straightforward functionalization of BODIPY dyes via incorporation of a triazole moiety produced fluorescent dyes that were capable of distinguishing between secondary structure conformations of soluble oligomeric species of Aβ1-42 peptide. Small concentrations of the dyes, relative to Aβ1-42, provided up to an 8-fold and 35-fold fluorescence increase in the presence of the unordered and ordered, β-sheet-rich conformations of soluble Aβ1-42 oligomers, respectively. These triazole-containing dyes could prove to be useful probes for monitoring amyloid conformational transitions in vitro.     

Engineered T4 viral nanoparticles for cellular imaging and flow cytometry

Viruses are of particular interest as scaffolds for biotechnology applications given their wide range of shapes and sizes and the possibility to modify them with a variety of functional moieties to produce useful virus-based nanoparticles (VNPs). In order to develop functional VNPs for cell imaging and flow cytometry applications, we used the head of the T4 bacteriophage as a scaffold for bioconjugation of fluorescent dyes. Bacteriophage T4 is a double-stranded DNA virus with an elongated icosahedron head and a contractile tail. The head is ～100 nm in length and ～90 nm in width. The large surface area of the T4 head is an important advantage for the development of functional materials since it can accommodate significantly larger numbers of functional groups, such as fluorescent dyes, in comparison with other VNPs. In this study, Cy3 and Alexa Fluor 546 were chemically incorporated into tail-less T4 heads (T4 nanoparticles) for the first time, and the fluorescent properties of the dye-conjugated nanoparticles were characterized. The T4 nanoparticles were labeled with up to 19?000 dyes, and in particular, the use of Cy3 led to fluorescent enhancements of up to 90% compared to free Cy3. We also demonstrate that the dye-conjugated T4 nanoparticles are structurally stable and that they can be used as molecular probes for cell imaging and flow cytometry applications.     

Fluorescent protein biosensors applied to microphysiological systems

This mini-review discusses the evolution of fluorescence as a tool to study living cells and tissues in vitro and the present role of fluorescent protein biosensors (FPBs) in microphysiological systems (MPSs). FPBs allow the measurement of temporal and spatial dynamics of targeted cellular events involved in normal and perturbed cellular assay systems and MPSs in real time. FPBs evolved from fluorescent analog cytochemistry (FAC) that permitted the measurement of the dynamics of purified proteins covalently labeled with environmentally insensitive fluorescent dyes and then incorporated into living cells, as well as a large list of diffusible fluorescent probes engineered to measure environmental changes in living cells. In parallel, a wide range of fluorescence microscopy methods were developed to measure the chemical and molecular activities of the labeled cells, including ratio imaging, fluorescence lifetime, total internal reflection, 3D imaging, including super-resolution, as well as high-content screening. FPBs evolved from FAC by combining environmentally sensitive fluorescent dyes with proteins in order to monitor specific physiological events such as post-translational modifications, production of metabolites, changes in various ion concentrations, and the dynamic interaction of proteins with defined macromolecules in time and space within cells. Original FPBs involved the engineering of fluorescent dyes to sense specific activities when covalently attached to particular domains of the targeted protein. The subsequent development of fluorescent proteins (FPs), such as the green fluorescent protein, dramatically accelerated the adoption of studying living cells, since the genetic “labeling” of proteins became a relatively simple method that permitted the analysis of temporal–spatial dynamics of a wide range of proteins. Investigators subsequently engineered the fluorescence properties of the FPs for environmental sensitivity that, when combined with targeted proteins/peptides, created a new generation of FPBs. Examples of FPBs that are useful in MPS are presented, including the design, testing, and application in a liver MPS.     

Diolistics: incorporating fluorescent dyes into biological samples using a gene gun

The hand-held gene gun provides a rapid and efficient method of incorporating fluorescent dyes into cells, a technique that is becoming known as diolistics. Transporting fluorescent dyes into cells has, in the past, used predominantly injection or chemical methods. The use of the gene gun, combined with the new generation of fluorescent dyes, circumvents some of the problems of using these methods and also enables the study of cells that have proved difficult traditionally to transfect (e.g. those deep in tissues and/or terminally differentiated); in addition, the use of ion- or metabolite-sensitive dyes provides a route to study cellular mechanisms. Diolistics is also ideal for loading cells with optical nanosensors--nanometre-sized sensors linked to fluorescent probes. Here, we discuss the theoretical considerations of using diolistics, the advantages compared with other methods of inserting dyes into cells and the current uses of the technique, with particular consideration of nanosensors.     

Cyanine dye conjugates as probes for live cell imaging

A series of fluorescent compounds suitable for live cell imaging is described. Functionalized forms of four different asymmetric cyanine dyes are reported that are amenable to peptide conjugation. The photophysical properties of the modified dyes and conjugates and the use of the compounds as cellular imaging agents are described. The results obtained indicate that these spectrally versatile compounds, which have absorption and emission profiles spanning the visible spectrum, are useful probes for cellular imaging.     

Dendrimer-based fluorescent indicators: in vitro and in vivo applications

BackgroundThe development of fluorescent proteins and synthetic molecules whose fluorescence properties are controlled by the environment makes it possible to monitor physiological and pathological events in living systems with minimal perturbation. A large number of small organic dyes are available and routinely used to measure biologically relevant parameters. Unfortunately their application is hindered by a number of limitations stemming from the use of these small molecules in the biological environment.ConclusionWe believe that the proposed architecture can represent a useful and novel tool in fluorescence imaging that can be widely applied in conjunction with a broad range of sensing dyes and experimental setups.     

Cascade blue derivatives: water soluble, reactive, blue emission dyes evaluated as fluorescent labels and tracers.

Fluorescent dyes based on the pyrenyloxytrisulfonic acid (Cascade Blue) structure were prepared and evaluated. The dyes contain functional groups that react with amines, thiols, acids, aldehydes, and ketones, forming covalently bonded, fluorescent derivatives of molecules with broad biological interest. Reactive groups in the Cascade Blue dyes include carboxylic acids and activated esters, amines, hydrazides, alcohols, photoaffinity reagents, acrylamides, and haloacetamides. The dyes exhibited absorption maxima at 374-378 nm and 399-403 nm, with extinction coefficients in the range of 1.9 x 10(4)-2.4 x 10(4) M-1cm-1 and 2.3 x 10(4)-3.0 x 10(4) M-1cm-1, respectively. Emission maxima ranged from 422-430 nm. The spectral properties of the fluorescent dyes are sufficiently different from fluorescein to permit simultaneous use of both dyes with minimum spectral interference. The Cascade Blue derivatives have narrower spectral bandwidths and smaller Stokes' shifts than other reactive dyes with similar spectral properties, do not show appreciable sensitivity to pH, have higher solubilities in aqueous solution, and have good to excellent quantum yields. Cascade Blue conjugates of a number of histochemically and biologically useful molecules were prepared, including dextrans, albumins, Fc receptor binding proteins, antibodies, lectins, membrane receptor binding proteins, and biotin binding proteins, as well as biological particles and bacteria. Cascade Blue conjugates of secondary and tertiary labels yielded specific fluorescence localization in the indirect immunofluorescent staining of human epithelial cell (HEp-2) nuclei.     

The use of fluorescent dyes to measure membrane potentials: a response

The use of fluorescent cyanine dyes to estimate membrane potential in cell suspensions has been considered. Several problems related tot he application of the dyes have been reviewed. These problems include: 1) alteration of the membrane potential (Em) and factors involved in establishing Em by the dyes themselves, 2) the effects of altered energy metabolism on the fluorescent response of the dyes and on Em, and 3) calibration of dye fluorescence. Recent reports that advocate the use of the fluorescent dyes are misleading.     

Masked red-emitting carbopyronine dyes with photosensitive 2-diazo-1-indanone caging group

Caged near-IR emitting fluorescent dyes are in high demand in optical microscopy but up to now were unavailable. We discovered that the combination of a carbopyronine dye core and a photosensitive 2-diazo-1-indanone residue leads to masked near-IR emitting fluorescent dyes. Illumination of these caged dyes with either UV or visible light (λ < 420 nm) efficiently generates fluorescent compounds with absorption and emission at 635 nm and 660 nm, respectively. A high-yielding synthetic route with attractive possibilities for further dye design is described in detail. Good photostability, high contrast, and a large fluorescence quantum yield after uncaging are the most important features of the new compounds for non-invasive imaging in high-resolution optical microscopy. For use in immunolabelling the caged dyes were decorated with a (hydrophilic) linker and an (activated) carboxyl group.     

Fluorescent dyes for lymphocyte migration and proliferation studies

Fluorescent dyes are increasingly being exploited to track lymphocyte migration and proliferation. The present paper reviews the properties and performance of some 14 different fluorescent dyes that have been used during the last 20 years to monitor lymphocyte migration. Of the 14 dyes discussed, two stand out as being the most versatile in terms of long-term tracking of lymphocytes and their ability to quantify lymphocyte proliferation. They are the intracellular covalent coupling dye carboxyfluorescein diacetate succinimidyl ester (CFSE) and the membrane inserting dye PKH26. Both dyes have the advantage that they can be used to track cell division, both in vitro and in vivo, due to the progressive halving of the fluorescence intensity of the dyes in cells after each division. However, CFSE appears to have the edge over PKH26 based on homogeneity of lymphocyte staining and cost. Two other fluorescent dyes, although not suitable for lymphocyte proliferation studies, are valuable tracking dyes for short-term (up to 3 day) lymphocyte migration experiments, namely the DNA-binding dye Hoechst 33342 and the cytoplasmic dye calcein. In the future it is highly likely that additional fluorescent dyes, with different spectral properties to CFSE, will become available, as well as membrane inserting fluorescent dyes that more homogeneously label lymphocytes than PKH26.     

Long Fluorescence Lifetime Molecular Probes Based on Near Infrared Pyrrolopyrrole Cyanine Fluorophores for In Vivo Imaging

Fluorescence lifetime (FLT) properties of organic molecules provide a new reporting strategy for molecular imaging in the near infrared (NIR) spectral region. Unfortunately, most of the NIR fluorescent dyes have short FLT typically clustered below 1.5 ns. In this study, we demonstrate that a new class of NIR fluorescent dyes, pyrrolopyrrole cyanine dyes, have exceptionally long FLTs ranging from 3 to 4 ns, both in vitro (dimethyl sulfoxide and albumin/water solutions) and in vivo (mice). These results provide a new window for imaging molecular processes, rejecting backscattered light and autofluorescence, and multiplexing imaging information with conventional NIR fluorescent dyes that absorb and emit light at similar wavelengths.     

New repertoire of ‘donor-two-acceptor’ NIR fluorogenic dyes

Dye molecules with various fluorescent wavelengths are widely used for diagnostic and optical imaging applications. Accordingly, there is a constant demand for fluorogenic dyes with new properties. We have recently developed a novel strategy for the design of long-wavelength fluorescent dyes with a turn-ON option. The design is based on a donor-two-acceptor π-electron system that can undergo an internal charge transfer to form a new fluorochrome with an extended π-conjugated system. Here, we describe a series of such dyes based on two novel latent donors, naphthol and hydroxycoumarin. One of the dyes has showed excellent near-infrared fluorescent characteristics and specifically was demonstrated as a mitochondrial imaging reagent in live cells. This unique strategy for fluorogenic dye design has opened new doors for further near-infrared fluorescence probe discovery.     

Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures

A prerequisite for many studies of neurons in culture is a means of determining their original identity. We needed such a technique to study the interactions in vitro between a class of spinal cord neurons, sympathetic preganglionic neurons, and their normal target, neurons from the sympathetic chain. Here, we describe how we use two highly fluorescent carbocyanine dyes, which differ in color but are otherwise similar, to identify neurons in culture. The long carbon chain carbocyanine dyes we use are lipid-soluble and so become incorporated into the plasma membrane. Neurons can be labeled either retrogradely or during dissociation. Some of the labeled membrane gradually becomes internalized and retains its fluorescence, allowing identification of cells for several weeks in culture. These dyes do not affect the survival, development, or basic physiological properties of neurons and do not spread detectably from labeled to unlabeled neurons. It seems likely that cells become retrogradely labeled mainly by lateral diffusion of dye in the plane of the membrane. If so, carbocyanine dyes may be most useful for retrograde labeling over relatively short distances. An additional feature of carbocyanine labeling is that neuronal processes are brightly fluorescent for the first few days in culture, presumably because dye rapidly diffuses into newly inserted membrane. We have used carbocyanine dyes to identify sympathetic preganglionic neurons in culture. Our results indicate that preganglionic neurons can survive in the absence of their target cells and that several aspects of their differentiation in the absence of target appear normal.     

Fluorescent complexes of DNA with DAPI 4′,6-diamidine-2-phenyl indole.2HCl or DCI 4′,6-dicarboxyamide-2-phenyl indole

4',6-Dioarboxyamide-2-phenyl indole (DCI), a non-ionic structural analogue of 4',6-diamidine-2-phenyl indole.2HCl (DAPI), was synthesized in order to verify the hypothesis of intercalation of both dyes into the DNA double helix.The influence of pH, viscosity, and different concentrations of SDS (sodium dodecylsulphate) or NaCl on the optical and fluorescent properties and the changes in thermal transition of both dye complexes with DNA confirm the affinity of the dyes to the double helix as well as their stabilizing influence on the secondary DNA structure.The results of binding studies, carried out by fluorescent methods have shown that the dyes are strongly bound to DNA, though the number of binding sites is small. According to the experimental data, the fluorescent properties of DAPI and DCI complexes with DNA are connected with the intercalating binding mechanism of these dyes. On the other hand, the eventual ionic or hydrogen bonds of dyes outside the DNA helix do not change noticeably their fluorescent properties.     

Forskolin-Induced Clearance of the Fluorescent Dye Sulforhodamine from Rat Parotid Intralobular Duct Lumen: Visualization of the Secretory Function under a Confocal Laser Scanning Microscope

Cyclic AMP evokes fluid secretion with bicarbonate in exocrine ducts. Clearance of fluorescent dyes from rat parotid intralobular ducts by forskolin was visualized as a fluorescence change in the duct luminal space by optical sectioning under a confocal laser scanning microscope to clarify the secretory function in the ducts. When the isolated rat parotid intralobular duct segments were superfused with membrane-impermeable fluorescent dyes during the experimental period, fluorescent dyes were passively moved into the duct space. Forskolin and isobutylmethylxanthine decreased the fluorescence of anionic dye, sulforhodamine B, and neutral dye, dextran tetramethyl-rhodamine, in the duct space, suggesting that the forskolin-induced clearance of fluorescent dyes might be the result of fluid secretion in the ducts. Methazolamide inhibited a forskolin-induced sustained decrease in duct fluorescence and intracellular acidification. Low concentrations of external Cl?, DIDS, bumetanide and amiloride did not markedly inhibit a forskolin-induced decrease in duct fluorescence. These findings suggest that a major portion of the steady decrease in duct fluorescence by forskolin was related to intracellular HCO3? production, not the uptake mechanism of external Cl?. Glibenclamide, NPPB, DPC and DMA inhibited the forskolin-induced decrease. Forskolin evokes the clearance of fluorescent dyes from duct space possibly due to fluid secretion in rat parotid ducts, associated with secretion through CFTR and DPC-sensitive anion channels of carbonic anhydrase-dependent bicarbonate linked with the Na+/H+ exchange mechanism.     

pH-dependent fluorescence of uncharged benzothiazole-based dyes binding to DNA.

Uncharged benzothiazole-based dyes were synthesized, and their fluorescence properties were examined. These fluorescent dyes showed an intense fluorescence in aqueous solution in the presence of DNA. In addition, the fluorescence intensities were pH dependent, while those of the positively charged dyes were nearly independent of pH. The pH-dependent photophysical behavior suggests that interaction of protonated dyes with DNA results in high intensities of fluorescent emission.     

Red laser-induced fluorescence energy transfer in an immunosystem

We describe two near-infrared fluorescent squaraine dyes (Sq635 and Sq660), their spectra, their covalent linkage to proteins, and their use as donor and acceptor, respectively, in a fluorescence resonance energy transfer (FRET) immunoassay based on the use of red lasers. The dyes show quantum yields of around 10% in the free form and up to 68% when bound to proteins. If converted into their N-hydroxysuccinimide esters, they can be linked to free amino groups of proteins. To improve water solubility, two sulfo groups were introduced. The emission spectrum of Sq635 overlaps the absorption spectrum of Sq660, a fact that makes them a useful pair of dyes for use in FRET immunoassay which is demonstrated for human serum albumin/anti-human serum albumin.