Mechanism of Cyanine5 to Cyanine3 Photoconversion and Its Application for High-Density Single-Particle Tracking in a Living Cell.

Cyanine (Cy) dyes are among the most useful organic fluorophores that have found a wide range of applications in single-molecule and super-resolution imaging as well as in other biophysical studies. However, recent observations that blueshifted derivatives of Cy dyes are formed via photoconversion have raised concerns as to the potential artifacts in multicolor imaging. Here, we report the mechanism for the photoconversion of Cy5 to Cy3 that occurs upon photoexcitation during fluorescent imaging. Our studies show that the formal C2H2 excision from Cy5 occurs mainly through an intermolecular pathway involving a combination of bond cleavage and reconstitution while unambiguously confirming the identity of the fluorescent photoproduct of Cy5 to be Cy3 using various spectroscopic tools. The carbonyl products generated from singlet oxygen-mediated photooxidation of Cy5 undergo a sequence of carbon-carbon bond-breaking and -forming events to bring about the novel dye-to-dye transformation. We also show that the deletion of a two-methine unit from the polymethine chain, which results in the formation of blueshifted products, commonly occurs in other cyanine dyes, such as Alexa Fluor 647 (AF647) and Cyanine5.5. The formation of a blueshifted congener dye can obscure the multicolor fluorescence imaging, leading to misinterpretation of the data. We demonstrate that the potentially deleterious photoconversion, however, can be exploited to develop a new photoactivation method for high-density single-particle tracking in a living cell without using UV illumination and cell-toxic additives.

Cyanine Nanocage Activated by Near-IR Light for the Targeted Delivery of Cyclosporine A to Traumatic Brain Injury Sites.

More than 2.8 million annually in the United States are afflicted with some form of traumatic brain injury (TBI), where 75% of victims have a mild form of TBI (MTBI). TBI risk is higher for individuals engaging in physical activities or involved in accidents. Although MTBI may not be initially life-threatening, a large number of these victims can develop cognitive and physical dysfunctions. These late clinical sequelae have been attributed to the development of secondary injuries that can occur minutes to days after the initial impact. To minimize brain damage from TBI, it is critical to diagnose and treat patients within the first or "golden" hour after TBI. Although it would be very helpful to quickly determine the TBI locations in the brain and direct the treatment selectively to the affected sites, this remains a challenge. Herein, we disclose our novel strategy to target cyclosporine A (CsA) into TBI sites, without the need to locate the exact location of the TBI lesion. Our approach is based on TBI treatment with a cyanine dye nanocage attached to CsA, a known therapeutic agent for TBI that is associated with unacceptable toxicities. In its caged form, CsA remains inactive, while after near-IR light photoactivation, the resulting fragmentation of the cyanine nanocage leads to the selective release of CsA at the TBI sites.

Identification of Nonradiative Decay Pathways in Cy3.

Photoexcited fluorescent markers are extensively used in spectroscopy, imaging, and analysis of biological systems. The performance of fluorescent markers depends on high levels of emission, which are limited by competing nonradiative decay pathways. Small-molecule fluorescent dyes have been increasingly used as markers due to their high and stable emission. Despite their prevalence, the nonradiative decay pathways of these dyes have not been determined. Here, we investigate these pathways for a widely used indocarbocyanine dye, Cy3, using transient grating spectroscopy. We identify a nonradiative decay pathway via a previously unknown dark state formed within ∼1 ps of photoexcitation. Our experiments, in combination with electronic structure calculations, suggest that the generation of the dark state is mediated by picosecond vibrational mode coupling, likely via a conical intersection. We further identify the vibrational modes, and thus structural elements, responsible for the formation and dynamics of the dark state, providing insight into suppressing nonradiative decay pathways in fluorescent markers such as Cy3.

Dye-Loaded Quatsomes Exhibiting FRET as Nanoprobes for Bioimaging.

Fluorescent organic nanoparticles (FONs) are emerging as an attractive alternative to the well-established fluorescent inorganic nanoparticles or small organic dyes. Their proper design allows one to obtain biocompatible probes with superior brightness and high photostability, although usually affected by low colloidal stability. Herein, we present a type of FONs with outstanding photophysical and physicochemical properties in-line with the stringent requirements for biomedical applications. These FONs are based on quatsome (QS) nanovesicles containing a pair of fluorescent carbocyanine molecules that give rise to Förster resonance energy transfer (FRET). Structural homogeneity, high brightness, photostability, and high FRET efficiency make these FONs a promising class of optical bioprobes. Loaded QSs have been used for in vitro bioimaging, demonstrating the nanovesicle membrane integrity after cell internalization, and the possibility to monitor the intracellular vesicle fate. Taken together, the proposed QSs loaded with a FRET pair constitute a promising platform for bioimaging and theranostics.

A NIR Light Gated DNA Nanodevice for Spatiotemporally Controlled Imaging of MicroRNA in Cells and Animals.

Nanodevices have potential as intelligent sensing systems for detection of microRNAs (miRNAs) in living cells. However, the resolution offered by "always active" nanodevices is often insufficient to manipulate miRNA sensing with high spatiotemporal control. In this work, using DNA nanotechnology we constructed an activatable DNA nanodevice programmed to detect miRNAs in vitro and in vivo with the high spatial and temporal precision of NIR light. Our nanodevice is functionalized on the surface of upconversion nanoparticles (UCNPs) with a rationally designed DNA beacon that displays UV light-activatable miRNA sensing activity. The UCNPs absorb deep-tissue-penetrable NIR light and emit high-energy UV light locally, which serve as transducers to operate the nanodevice in the NIR window. The nanodevice can naturally enter cells and enable remote regulation of its fluorescent imaging activity for miRNAs in living cells by NIR light illumination in a chosen place and time. Furthermore, we demonstrate that the nanodevice can be expanded to activatable imaging of intratumoral miRNAs in living mice. This work illustrates the potential of DNA nanodevices for miRNA detection with high spatiotemporal resolution, which could expand the toolbox of technologies for precise biological and medical analysis.

Coherent Exciton Delocalization in a Two-State DNA-Templated Dye Aggregate System.

Coherent exciton delocalization in dye aggregate systems gives rise to a variety of intriguing optical phenomena, including J- and H-aggregate behavior and Davydov splitting. Systems that exhibit coherent exciton delocalization at room temperature are of interest for the development of artificial light-harvesting devices, colorimetric detection schemes, and quantum computers. Here, we report on a simple dye system templated by DNA that exhibits tunable optical properties. At low salt and DNA concentrations, a DNA duplex with two internally functionalized Cy5 dyes (i.e., dimer) persists and displays predominantly J-aggregate behavior. Increasing the salt and/or DNA concentrations was found to promote coupling between two of the DNA duplexes via branch migration, thus forming a four-armed junction (i.e., tetramer) with H-aggregate behavior. This H-tetramer aggregate exhibits a surprisingly large Davydov splitting in its absorbance spectrum that produces a visible color change of the solution from cyan to violet and gives clear evidence of coherent exciton delocalization.

New Fluorescent Nanoparticles for Ultrasensitive Detection of Nucleic Acids by Optical Methods.

For decades the detection of nucleic acids and their interactions at low abundances has been a challenging task that has thus far been solved by enzymatic target amplification. In this work we aimed at developing efficient tools for amplification-free nucleic acid detection, which resulted in the synthesis of new fluorescent nanoparticles. Here, the fluorescent nanoparticles were made by simple and inexpensive radical emulsion polymerization of butyl acrylate in the presence of fluorescent dyes and additional functionalization reagents. This provided ultra-bright macrofluorophores of 9-84 nm mean diameter, modified with additional alkyne and amino groups for bioconjugation. By using click and NHS chemistries, the new nanoparticles were attached to target-specific DNA probes that were used in fluorimetry and fluorescence microscopy. Overall, these fluorescent nanoparticles and their oligonucleotide derivatives have higher photostability, brighter fluorescence and hence dramatically lower limits of target detection than the individual organic dyes. These properties make them useful in approaches directed towards ultrasensitive detection of nucleic acids, in particular for imaging and in vitro diagnostics of DNA.

Photoswitching mechanism of cyanine dyes.

Cyanine dyes have been shown to undergo reversible photoswitching, where the fluorophore can be switched between a fluorescent state and a dark state upon illumination at different wavelengths. The photochemical mechanism by which switching occurs has yet to be elucidated. In this study, we have determined the mechanism of photoswitching by characterizing the kinetics of dark state formation and the spectral and structural properties of the dark state. The rate of switching to the dark state depends on the concentration of the primary thiol in the solution and the solution pH in a manner quantitatively consistent with the formation of an encounter complex between the cyanine dye and ionized thiol prior to their conjugation. Mass spectrometry suggests that the photoconversion product is a thiol-cyanine adduct in which covalent attachment of the thiol to the polymethine bridge disrupts the original conjugated pi-electron system of the dye.

Development of a novel ozone- and photo-stable HyPer5 red fluorescent dye for array CGH and microarray gene expression analysis with consistent performance irrespective of environmental conditions.

BACKGROUND: Array-based comparative genomic hybridization (CGH) and gene expression profiling have become vital techniques for identifying molecular defects underlying genetic diseases. Regardless of the microarray platform, cyanine dyes (Cy3 and Cy5) are one of the most widely used fluorescent dye pairs for microarray analysis owing to their brightness and ease of incorporation, enabling high level of assay sensitivity. However, combining both dyes on arrays can become problematic during summer months when ozone levels rise to near 25 parts per billion (ppb). Under such conditions, Cy5 is known to rapidly degrade leading to loss of signal from either "homebrew" or commercial arrays. Cy5 can also suffer disproportionately from dye photobleaching resulting in distortion of (Cy5/Cy3) ratios used in copy number analysis. Our laboratory has been active in fluorescent dye research to find a suitable alternative to Cy5 that is stable to ozone and resistant to photo-bleaching. Here, we report on the development of such a dye, called HyPer5, and describe its' exceptional ozone and photostable properties on microarrays. RESULTS: Our results show HyPer5 signal to be stable to high ozone levels. Repeated exposure of mouse arrays hybridized with HyPer5-labeled cDNA to 300 ppb ozone at 5, 10 and 15 minute intervals resulted in no signal loss from the dye. In comparison, Cy5 arrays showed a dramatic 80% decrease in total signal during the same interval. Photobleaching experiments show HyPer5 to be resistant to light induced damage with 3- fold improvement in dye stability over Cy5. In high resolution array CGH experiments, HyPer5 is demonstrated to detect chromosomal aberrations at loci 2p21-16.3 and 15q26.3-26.2 from three patient sample using bacterial artificial chromosome (BAC) arrays. The photostability of HyPer5 is further documented by repeat array scanning without loss of detection. Additionally, HyPer5 arrays are shown to preserve sensitivity and data quality from gene expression experiments. CONCLUSION: HyPer5 is a red fluorescent dye that behaves functionally similar to Cy5 except in stability to ozone and light. HyPer5 is demonstrated to be resistant to ozone at up to 300 ppb, levels significantly higher than commonly observed during summer months. Consequently, HyPer5 dye can be used in parallel with Cy3 under any environmental conditions in array experiments.

Characterization of hemicyanine Langmuir-Blodgett films by picosecond time-resolved fluorescence.

Hemicyanine Langmuir-Blodgett films have been elaborated and characterized using stationary and time-resolved spectroscopic techniques. Depending on the experimental conditions, especially the pH of the water subphase, the absorption spectra of the films indicate the presence of non-fluorescent H-aggregates in the monolayer. Time-resolved fluorescence measurements revealed three mono-exponential decay times: a very short one (20-23 ps) attributed to an excited intramolecular charge transfer state and two longer ones (100-120 ps and 400-590 ps) attributed to the photoisomerization of the chromophores.

Photoisomerization of cyanine derivatives in 1-butyl-3-methylimidazolium hexafluorophosphate and aqueous glycerol: influence of specific interactions.

Photoisomerization of two cyanine derivatives, 3,3(')-diethyloxadicarbocyanine iodide (DODCI) and merocyanine 540 (MC 540), has been investigated in an ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate and aqueous glycerol (93 wt % glycerol +7 wt % water) by measuring fluorescence lifetimes and quantum yields. The aim of this work is to understand how the rates of photoisomerization of DODCI and MC 540 are influenced by specific solute-solvent interactions besides the viscosity of the medium. For DODCI, it has been observed that the nonradiative rate constants, which represent the rates of photoisomerization, are almost identical in the ionic liquid and aqueous glycerol at given temperature, indicating that viscosity is the sole parameter that governs the rate of photoisomerization. In contrast, the photoisomerization rate constants of MC 540 have been found to be a factor of 2 higher in aqueous glycerol compared to the ionic liquid. The observed behavior is due to the zwitterionic character of MC 540, a consequence of which, the twisted state gets stabilized by the solute-solvent hydrogen bonding interactions in aqueous glycerol, thus lowering the barrier for isomerization.

Increasing the sensitivity of DNA microarrays by metal-enhanced fluorescence using surface-bound silver nanoparticles.

The effects of metal-enhanced fluorescence (MEF) have been measured for two dyes commonly used in DNA microarrays, Cy3 and Cy5. Silver island films (SIFs) grown on glass microscope slides were used as substrates for MEF DNA arrays. We examined MEF by spotting biotinylated, singly-labeled 23 bp DNAs onto avidin-coated SIF substrates. The fluorescence enhancement was found to be dependent on the DNA spotting concentration: below approximately 12.5 muM, MEF increased linearly, and at higher concentrations MEF remained at a constant maximum of 28-fold for Cy5 and 4-fold for Cy3, compared to avidin-coated glass substrates. Hybridization of singly-labeled oligonucleotides to arrayed single-stranded probes showed lower maximal MEF factors of 10-fold for Cy5 and 2.5-fold for Cy3, because of the smaller amount of immobilized fluorophores as a result of reduced surface hybridization efficiencies. We discuss how MEF can be used to increase the sensitivity of DNA arrays, especially for far red emitting fluorophores like Cy5, without significantly altering current microarray protocols.

Uptake and phototoxicity of tricarbocyanine indolenine dye covalently bound with glucose (TICS) under acidification of tumor cells environment.

AIM: To evaluate the influence of pH on accumulation and photocytotoxicity of the novel tricarbocyanine indolenine dye covalently bound with glucose (TICS). METHODS: For in vitro experiments, cultured HeLa cells were incubated with TICS at pH 7.1, 6.5 or 6.2 and then scored for dye uptake, viability and sensitivity to laser (740 nm) irradiation. In vivo TICS was injected to rats with implanted subcutaneously SM-1 tumor and then dye accumulation and tumor necrosis depth after laser irradiation were defined. RESULTS: Reduction of medium pH in vitro was shown to increase in cellular TICS contents and to enhance as its "dark" cytotoxicity, and photocytotoxicity. The ratio of "dark" cytotoxicity to photocytotoxicity parameters increased 2-fold with decreasing pH value. In vivo infusion of glucose (10 g/kg) to rats resulted in improved selectivity of TICS accumulation and increase in tumor necrosis depth after laser irradiation. CONCLUSION: pH reduction of tumor cells environment improves efficacy of photodynamic treatment with TICS.