Cyanine Fluorophores Research Articles

Mitigating Unwanted Photophysical Processes for Improved Single-Molecule Fluorescence Imaging

Organic fluorophores common to fluorescence-based investigations suffer from unwanted photophysical properties, including blinking and photobleaching, which limit their overall experimental performance. Methods to control such processes are particularly important for single-molecule fluorescence and fluorescence resonance energy transfer imaging where uninterrupted, stable fluorescence is paramount. Fluorescence and FRET-based assays have been carried out on dye-labeled DNA and RNA-based systems to quantify the effect of including small-molecule solution additives on the fluorescence and FRET behaviors of both cyanine and Alexa fluorophores. A detailed dwell time analysis of the fluorescence and FRET trajectories of more than 200,000 individual molecules showed that two compounds identified previously as triplet state quenchers, cyclooctatetraene, and Trolox, as well as 4-nitrobenzyl alcohol, act to favorably attenuate blinking, photobleaching, and influence the rate of photoresurrection in a concentration-dependent and context-dependent manner. In both biochemical systems examined, a unique cocktail of compounds was shown to be optimal for imaging performance. By simultaneously providing the most rapid and direct access to multiple photophysical kinetic parameters, smFRET imaging provides a powerful avenue for future investigations aimed at discovering new compounds, and effective combinations thereof. These efforts may ultimately facilitate tuning organic dye molecule performance according to each specific experimental demand.

An analysis of synaptic transmission mediated with AMPA receptor channel by laser photolysis (II)

Light provides a uniquely powerful stimulus to help visualize and/or perturb biological systems. The use of tissue penetrant near-IR wavelengths enables in vivo applications, however the design of molecules that function in this range remains a substantial challenge. Heptamethine cyanine fluorophores are already important tools for near-IR optical imaging. These molecules are susceptible to photobleaching through a photooxidative cleavage reaction. This review details efforts to define the mechanism of this reaction and two emerging fields closely tied to this process. In the first, efforts that slow photooxidation enable the creation of photobleaching resistant fluorophores. In the second, cyanine photooxidation has recently been employed as the cornerstone of a near-IR uncaging strategy. This review seeks to highlight the utility of mechanistic organic chemistry insights to help tailor cyanine scaffolds for new, and previously intractable, biological applications.

Orientation dependence in fluorescent energy transfer between Cy3 and Cy5 terminally attached to double-stranded nucleic acids

We have found that the efficiency of fluorescence resonance energy transfer between Cy3 and Cy5 terminally attached to the 5' ends of a DNA duplex is significantly affected by the relative orientation of the two fluorophores. The cyanine fluorophores are predominantly stacked on the ends of the helix in the manner of an additional base pair, and thus their relative orientation depends on the length of the helix. Observed fluorescence resonance energy transfer (FRET) efficiency depends on the length of the helix, as well as its helical periodicity. By changing the helical geometry from B form double-stranded DNA to A form hybrid RNA/DNA, a marked phase shift occurs in the modulation of FRET efficiency with helix length. Both curves are well explained by the standard geometry of B and A form helices. The observed modulation for both polymers is less than that calculated for a fully rigid attachment of the fluorophores. However, a model involving lateral mobility of the fluorophores on the ends of the helix explains the observed experimental data. This has been further modified to take account of a minor fraction of unstacked fluorophore observed by fluorescent lifetime measurements. Our data unequivocally establish that Förster transfer obeys the orientation dependence as expected for a dipole-dipole interaction.

Silica nanoparticles encapsulating near-infrared emissive cyanine dyes

We show that efficient near-infrared (NIR) cyanine fluorophores ( 1 and 2) can be encapsulated into silica nanoparticles providing a highly versatile and unique platform for in vivo diagnostics. Utilizing this platform, multiple fluorophores can be loaded within a single particle allowing the light absorption and emission properties of the nanoparticle to be controlled independent of particle size. Furthermore, such dyed nanoparticles may have extinction coefficients as high as about 100 × 10 6 L mol −1 cm −1 in the NIR (on a per mole of particles basis), with quantum yields from about 8–10%. A simple synthetic method for varying particle size and dye-loading level is presented, and a modified Stober synthesis reduces deleterious exposure of the dye to the highly alkaline conditions used. The cyanine dyes are encapsulated in silica in a non-aggregated state and the fluorescence brightness is largely maintained to nominal dye concentrations approaching 50 μM. The ability to control light absorption and emission properties independent of particle size, and convenient access to particle sizes in the range of 20–100 nm (a size regime difficult to access with other nanoparticle approaches such as quantum dots), are important features for anatomical targeting in in vivo diagnostics and targeted therapeutic applications.

Long time scale blinking kinetics of cyanine fluorophores conjugated to DNA and its effect on Förster resonance energy transfer

The blinking kinetics of individual Cy5 fluorophores conjugated to DNA are directly measured using single-molecule spectroscopy. Under deoxygenated aqueous conditions, Cy5 fluorescence exhibits spontaneous and reversible on/off fluctuations with a period lasting seconds. This blinking is observed when directly exciting Cy5 with 640 nm light and by Forster resonance energy transfer (FRET). We find that Cy5 blinking is influenced by the proximity of the donor, the structure of the donor, the presence of 514 nm excitation, and FRET. In the context of single-molecule FRET, blinking of the acceptor produces anticorrelated donor-acceptor intensity fluctuations, which can be difficult to discern from variations in the interdye distance. Slow blinking is, in particular, problematic because it overlaps with biologically relevant time scales. By employing an alternating 514640 nm laser excitation scheme, we show that the dark states can be readily resolved and discriminated from FRET distance fluctuations.

Optical characteristics of responsive biopolymers; co-polycondensation of tri-functional amino acids and Cy-3 bis-amine with diacylchlorides

The synthesis and fluorescence behaviour of a range of amphiphilic poly( l-lysine co-bis-amine-Cy-3 iso-phthalamide) polymers that incorporate low levels of Cy-3 dye co-monomer (0.5–2.1% (w/w)) in their backbone have been investigated in aqueous solution over a range of pH values. The desired functionality of these biocompatible, hydrophobically modified polyelectrolytes was achieved by incorporating pendant hydrophilic carboxyl groups along the polymer backbone, via the l-lysine moiety, balanced by a degree of hydrophobicity introduced via the iso-phthaloyl moiety. Thus, the polymer changes from an extended conformation at high degrees of ionisation to a compact conformation stabilised by hydrophobic association at low degrees of ionisation and eventually precipitates from solution. At intermediate degrees of ionisation, such polymers exhibit amphiphilic properties. A bis-functional cyanine fluorophore derivative, co-polymerised within the polymer backbone, is demonstrated to act as a fluorescent reporter on the conformational state of the polymer. The materials have higher intrinsic fluorescence intensity per fluorophore monomer over a broad range of concentrations than bis-amine Cy-3 and their maximum fluorescence intensity is up to eight fold higher than the maximum intensity of bis-amine Cy-3, which is limited by quenching. It is also shown that the free fluorophore can be used to probe the conformation of unlabelled poly( l-lysine iso-phthalamide) through electrostatic interaction with the polymer. The technique allows rapid spectrophotometric determination of polymer conformation and offers the potential of an environmentally sensitive molecular pH probe for in vivo use.

Flame hydrolysis deposition of glass on silicon for the integration of optical and microfluidic devices

Flame hydrolysis deposition (FHD) of glasses has previously found applications in the telecommunications industry. This paper shows how the technology can be used to deposit silica with different refractive indices and thereby produce low-loss planar waveguides for use in analytical applications. We also show that the glasses can be patterned using a new reactive ion etch and sealed using a modification of anodic bonding, such that the resulting microstructures can be readily incorporated within a lithographically defined "chip", integrating both optical and fluidic circuitry on the same device. In the example described in this paper, waveguides, analytical microtiter chambers and fluidic capillary channels, with the necessary high aspect ratio features (and with depths up to 40 microm) were all produced in glass, using the appropriate deposition and etching technologies. The performance of the chip was assessed in the framework of a low-volume fluorescence assay, using waveguides to address miniaturized microtiter chambers with volumes of 230 and 570 pL. Devices featuring different optical detection configurations, including both in-line and orthogonal waveguide geometries, were fabricated. In the optimal configuration, the experimental detection limit was determined as ca. 20 pM (equivalent to 10 zmol) of a cyanine fluorophore, Cy5. The applicability of the device as a biochip platform was further illustrated by analytical measurements on fluorescently labeled oligodeoxynucleotides.

Near-Infrared Heptamethine Cyanine Dyes: A New Tracer for Solid-Phase Immunoassays

Near-infrared (near-IR) fluorescence has been used to develop a solid-phase immunoassay that detects trace amounts of human immunoglobulin (HuIgG). Various concentrations of HuIgG bound to a nitrocellulose surface were determined from the fluorescence generated by near-IR labeled goat anti-human antibody (GAHG) bound to the HuIgG. The GAHG was labeled with a heptamethine cyanine fluorophore that has spectral properties in the near-IR region (above 780 nm). These fluorophores are versatile because they can be modified for several bioanalytical applications. Fluorescence was detected with a near-IR fluorescence instrument previously developed in the laboratory. Two cyanine fluorophore labels were evaluated for the ability to selectively bind to GAHG on a nitrocellulose matrix with a minimal amount of background interference. After the most appropriate near-IR fluorophore was selected, the labeling of GAHG was optimized under aqueous conditions. The most effective GAHG–dye conjugates were used to develop an immunoassay to detect various concentrations of HuIgG. The results are presented, here. Solutions of HuIgG with concentrations as low as 10−10 molar have been detected with a minimum of interference.

A cyanine fluorophore with a hydrazide functionality as labelling reagent for aldehydes in liquid chromatography

A new hydrazide derivative of a cyanine dye is synthesized for the chemical derivatization of aldehydes. With this red absorbing fluorophore it is possible to apply visible diode laser induced fluorescence (DIO-LIF) detection in conventional-size liquid chromatography (LC). The dye is based on CY5.3a-IA, a sulfonated dicarbocyanine with a iodoacetamido reactive group. The detection limit of n-octylaldehyde is 7 × 10 −8 M, limited by the reactivity of the label. Mixtures comprised of strongly resembling aldehydes were derivatized with the label and analyzed by LC-DIO-LIF. The potential applicability in biological problems is shown by the derivatization of 4-hydroxy-2-nonenal (HNE).

Semiconductor laser-induced fluorescence detection in capillary electrophoresis using a cyanine dye

Cy5, an activated carboxyl cyanine fluorophore, was characterized by capillary electrophoresis (CE) using a semiconductor laser at 652 nm to induce fluorescence. Hydrolysis of the activated Cy5 in the presence of ammonia results in the formation of a mono- and diamide and a dicarboxylic acid. A Cy5-labeled oligonucleotide M 13 primer for DNA sequencing (M13mp18 template) was synthesized with a purity of better than 95%. The labeled primer was analyzed by liquid chromatography, using UV-visible detection, and by CE, monitored by laser-induced fluorescence (LIF) detection. Analysis of the Cy5-labeled oligonucleotide primer by CE-LIF in a 9% polyacrylamide gel-filled capillary indicated the purity of the major Cy5-oligonucleotide primer was greater than 90%. The detection sensitivity for Cy5-based CE-LIF detection system with a 2.5-mW red semiconductor laser is about 10 −10 M.