Energy Transfer Dye Research Articles

Energy Transfer Studies in Binary Dye Solution Mixture of Coumarin 440 + Coumarin 540 and its LifetimeCalculations

The sensitized fluorescence emission from the bimixture [Coumarin 440 (donor) + Coumarin 540 (acceptor)] has been measured as a function of dye concentration using a fluorescence spectrophotometer. The concentration of Coumarin 440 (donor) is kept constant at 5×10-5M while the concentration of Coumarin 540 (acceptor) is varied. The spectroscopic parameters for this bimixture have been calculated from the concentration dependence of peak fluorescence intensity curves. Also the data obtained from the Horiba JobinYvon Time Resolved Fluorescence Spectrophotometer was analyzed for the determination of lifetimes at various emission peaks. A comparative study between theoretically calculated lifetimes and experimentally obtained lifetimes shows a good agreement. The results indicate that the energy transfer process between unlike molecules can be studied by lifetime measurements (which could also be determined from the fluorescence emission spectral studies). Many attempts have been made in studies aimed to improve dye lasers efficiency and to extend their spectral range of operation. Energy transfer dye lasers (ETDL) have generated considerable interest as they are useful in obtaining enhanced laser output, wide tuning range, reduced concentration and pump threshold requirements and ultra-short pulse width in contrast to their single dye counterpart [1-7]. Energy Transfer Dye Lasers using numerous donor-acceptor dye pairs have been reported by various investigators duringthe last five decades. Fluorescence energy transfer is the transfer of the excited state energy from a donor (D) to acceptor (A) [8–19]. This transferoccurs without the appearance of photon and is primarily a resultof dipole–dipole interaction between the donor and the acceptor.The rate of energy transfer depends upon the extent of overlapof the emission spectrum of the donor with the absorptionspectrum of the acceptor, the relative orientation of the donorand acceptor transition dipoles and the distance between thesemolecules. The non-radiative energy transfer occurs as a result ofdipole–dipole coupling between the donor and the acceptor,anddoes not involve the emission and reabsorption of photons. In contrast to these trivial factorsnonradiative energy transfer depends upon the molecular detailsof donor–acceptor pairs.Non-radiative energy transfer is effective overdistance ranging of 50A°.The intervening of solvent or other macromoleculeshas little effect on the efficiency of the energy transfer, whichdepends primarily on the D–A distances[18].Dye lasers have some limitations as the dye solution used as an active medium absorbs energy from the excitation source in a very limited range and so the emission band also has these limitations. If a dye laser has to be used as an ideal source its spectral region needs to be extended. In order to extend the spectral region of operation mixtures of different dye solutions/dye molecules embedded in solid matrices arebeingused.The use of such energy transfer in dye lasers is also helpful in minimizing the photoquenching effects and thereby, increasing the laser efficiency. In the present studies the energy transfer mechanism has been investigated in the ethanol solution mixture, i.e. Coumarin 440 and Coumarin 540 from their absorption and emission spectra. The dependence of lifetimes of the dye molecules on their

Photophysical properties, photostability and laser activity of 2,6-bis [β-(2-benzoxazolyl) vinyl] naphthalene (BBVN)

The photophysical properties such as electronic absorption, molar absorptivity, oscillator strength, transition dipole moment, fluorescence spectra and fluorescence quantum yield of BBVN were determined in different solvents and micelles. Both electronic absorption and emission spectra are slightly sensitive to medium polarity which indicates little change in the dipole moment of BBVN molecule upon excitation. The quantum yield for photochemical transformation (Фc) of BBVN was calculated in different organic solvents at room temperature. The dye solutions in DMSO, DMF, EtOH and CHCl3 give laser emission in the blue–green region upon excitation by a 337.1nm nitrogen pulse, the tuning range, gain coefficient and emission cross-section have been determined. Excitation energy transfer from BBVN to rhodamine B was studied to improve the laser emission from this dye when excited by 337nm. Such an energy transfer dye laser system (ETDL) obeys a long range culumbic energy transfer mechanism with a critical transfer distance, R0, of 42Å and kET equal 1.6×1013M−1s−1.

Dye selection for live cell imaging of intact siRNA

Investigations into the fate of small interfering RNA (siRNA) after transfection may unravel new ways to improve RNA interference (RNAi) efficiency. Because intracellular degradation of RNA may prevent reliable observation of fluorescence-labeled siRNA, new tools for fluorescence microscopy are warranted to cover the considerable duration of the RNAi effect. Here, the characterization and application of new fluorescence resonance energy transfer (FRET) dye pairs for sensing the integrity of duplex siRNA is reported, which allows an assessment of the degradation status of an siRNA cell population by live cell imaging. A panel of high-yield fluorescent dyes has been investigated for their suitability as FRET pairs for the investigation of RNA inside the cell. Nine dyes in 13 FRET pairs were evaluated based on the performance in assays of photostability, cross-excitation, bleed-through, as well as on quantified changes of fluorescence as a consequence of, e.g., RNA strand hybridization and pH variation. The Atto488/Atto590 FRET pair has been applied to live cell imaging, and has revealed first aspects of unusual trafficking of intact siRNA. A time-lapse study showed highly dynamic movement of siRNA in large perinuclear structures. These and the resulting optimized FRET labeled siRNA are expected to have significant impact on future observations of labeled RNAs in living cells.

Photophysical Parameters, Excitation Energy Transfer, and Photoreactivity of 1,4-Bis(5-phenyl-2-oxazolyl)benzene (POPOP) Laser Dye

The effect of solvents on the absorption and emission spectra of 1,4-bis(5-phenyl-2-oxazolyl)benzene (POPOP) laser dye has been studied in various solvents at 298 K. A bathochromic shift was observed in absorption and fluorescence spectra upon increase of solvent polarity, which indicates that this transition is π- 𝜋 ∗ . The ground and excited state dipole moments were calculated as 2.23 and 6.34 Debye, respectively. The dye solution in MeOH, n-heptane, and methyl isobutyl ketone gives laser emission in the blue region upon excitation by a 337.1 nm nitrogen pulse; the gain coefficient and emission cross section as well as normalized photostability have been determined. Excitation energy transfer from POPOP to rhodamine B and fluorescine was studied to improve the laser emission from these dyes. Such an energy transfer dye laser system (ETDL) obeys a long range columbic energy transfer mechanism with a critical transfer distance, R0, of 25 and 33 A and kq equal to 1 0 . 4 × 1 0 1 2 and 2 6 . 2 × 1 0 1 2 M − 1 s − 1 for the POPOP/RB and POPOP/fluorescine pair, respectively. The POPOP dye is highly photostable in polar protic and polar aprotic solvents, while it displays photodecomposition in chloromethane solvent via formation of a contact ion pair. The photochemical quantum yield and rate of photodecomposition depend on the electron affinity of solvent.

DNA crunching by a viral packaging motor: Compression of a procapsid-portal stalled Y-DNA substrate

Many large double-stranded DNA viruses employ high force-generating ATP-driven molecular motors to package to high density their genomes into empty procapsids. Bacteriophage T4 DNA translocation is driven by a two-component motor consisting of the procapsid portal docked with a packaging terminase-ATPase. Fluorescence resonance energy transfer and fluorescence correlation spectroscopic (FRET-FCS) studies of a branched (Y-junction) DNA substrate with a procapsid-anchoring leader segment and a single dye molecule situated at the junction point reveal that the "Y-DNA" stalls in proximity to the procapsid portal fused to GFP. Comparable structure Y-DNA substrates containing energy transfer dye pairs in the Y-stem separated by 10 or 14 base pairs reveal that B-form DNA is locally compressed 22-24% by the linear force of the packaging motor. Torsional compression of duplex DNA is thus implicated in the mechanism of DNA translocation.

Single-Molecule and FRET Fluorescence Correlation Spectroscopy Analyses of Phage DNA Packaging: Colocalization of Packaged Phage T4 DNA Ends within the Capsid

Linear DNAs of any sequence can be packaged into empty viral procapsids by the phage T4 terminase with high efficiency in vitro. Packaging substrates of 5 kbp and 50 kbp, terminated by energy transfer dye pairs, were constructed from plasmid and λ phage DNAs. Nuclease and fluorescence correlation spectroscopy (FCS) assays showed that ∼ 20% of the substrate DNA was packaged and that the DNA dye ends of the packaged DNA were protected from nuclease digestion. Upon packaging, both 5-kbp and 50-kbp DNAs produced comparable fluorescence resonance energy transfer (FRET) between Cy5 and Cy5.5 double-dye terminated DNAs. Single-molecule FRET (sm-FRET) and photobleaching analysis shows that FRET is intramolecular rather than intermolecular upon packaging of most procapsids and demonstrates that single-molecule detection allows mechanistic analysis of packaging in vitro. FRET-FCS and sm-FRET measurements are comparable and show that both the 5-kbp and the 50-kbp packaged DNA ends are held within 8–9 nm of each other, within the dimensions of the long axis of the procapsid portal. The calculated distribution of FRET distances is relatively narrow for both FRET-FCS and sm-FRET, suggesting that the two packaged DNA ends are held at the same fixed distance relative to each other in most capsids. Because one DNA end is known to be positioned for ejection through the portal, it can be inferred that both DNAs ends are held in proximity to the portal entrance and ejection channel. The analysis suggests that a DNA loop, rather than a DNA end, is translocated by the packaging motor to fill the procapsid.

Concentration-dependent energy transfer studies in ternary dye mixture of Stilbene-420, Coumarin-540 and Nile Blue

The energy transfer studies in the case of ternary dye mixture [Stilbene-420 (donor) + Coumarin-540 (intermediator) + Nile Blue (acceptor)] have been done and discussed through optical gain characteristics at various acceptor concentrations under nitrogen laser excitation. The concentration of the other two dyes were kept constant. It is observed that the concentration of the acceptor dye plays a very critical role in energy transfer dye laser (ETDL) as small change in its concentration varies the intensity of the laser output in the red region by large amount. Also, the highest laser output in the red region is obtained when the concentration of the acceptor dye is slightly higher than that of the intermediator dye. The present studies are helpful in deciding the optimum concentration of the acceptor dye to be used in ternary dye mixture for maximum gain and tuning range. The ternary dye mixture under study provides an ETDL tuning range up to 700 nm.

Energy transfer and photodegradation of Perylene Red with DTTC and HITC acceptor dyes in PMMA

Energy transfer from Perylene Red to 1,1′,3,3,3′,3′-hexamethylindoletricarbocyanine iodide and 3,3′-diethylthiatricarbocyanine iodide in poly(methyl methacrylate) was investigated by monitoring the extent of fluorescence following irradiation with light of 532 nm. The measurements yielded information about the enhancement of HITC fluorescence induced by the dye, the energy transfer efficiency from the dye to HITC and the relative stability of the fluorescence of dye/HITC (DTTC)/PMMA films. The results of this study have important implications for near-IR emitting energy transfer dye lasers.

Energy transfer mechanism between laser dyes doped in ORMOSILs

Laser dyes perylene orange (p-orange) or perylene red (p-red) or pyrromethene 567 (p567) were co-doped with coumarin 440 (C440) or coumarin 500 (C500) into MTES- and VTES-derived organically modified silicates (ORMOSILs) by sol–gel process. Energy transfer from both C440 and C500, energy donors, to p-red, p-orange and p567, acceptor, respectively, has been observed and studied by steady-state emission measurement. The effect of donor concentration on energy transfer and its mechanism were studied. The potential application as an energy transfer dye laser (ETDL) was also discussed. At least 1-fold increase in the slope efficiency of p-red and 3-fold increase in the photostability of p567 was observed in such ETDLs.

Influence of energy transfer on fluorescence and lasing properties of various laser dyes co-doped in ORMOSILs

Laser dye perylene red (p-red) was co-doped with coumarin 440 (C440) and/or pyrromethene 567 (p567) into MTES-derived organically modified silicates (ORMOSILs) by sol–gel process. Energy transfer among C440, p567 and p-red has been observed. The fluorescence and lasing properties of such energy transfer dye lasers have been investigated. Improved laser efficiency of p-red and broad tunable range as wide as 60 nm were obtained. The effect of dye concentration on energy transfer, laser efficiency and tunable range were also discussed. With the presence of C440 and/or p567, the laser lifetime of p-red exceeded 45 000 pulses at pump intensity beyond 0.5 J/cm2.

Negatively Charged, Dye Labeled-Dideoxynucleotides for “Direct-Load” DNA Sequencing.

For Abstract see ChemInform Abstract in Full Text.

Synthesis and Energy Transfer Efficiency of FRET Terminators Derived from Different Linkers

AbstractFor Abstract see ChemInform Abstract in Full Text.

Synthesis of Novel Tyrosinyl FRET Cassettes, Terminators, and Their Potential Use in DNA Sequencing

AbstractFor Abstract see ChemInform Abstract in Full Text.

Rigid, Conjugated, Fluoresceinated Thymidine Triphosphates: Syntheses and Polymerase Mediated Incorporation into DNA Analogues.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Synthesis of Novel Tyrosinyl FRET Cassettes, Terminators, and Their Potential Use in DNA Sequencing

Fluorescence resonance energy transfer (FRET) dye labeled cassettes and terminators with one or more donor dyes (fluorescein) and acceptor dye (rhodamine dyes) with benzofuran or tyrosine linker moieties were synthesized. These terminators were evaluated for their energy transfer and DNA sequencing potential using thermostable DNA polymerase.

Synthesis and Energy Transfer Efficiency of FRET Terminators Derived from Different Linkers

A number of different energy transfer dye labeled-cassettes were synthesized using aminoacid based trifunctional linkers and coupled to the propargylamino-substituted dideoxynucleoside-5′-triphosphates (ddNTPs). These terminators were evaluated for their energy transfer efficiency and DNA sequencing potential using thermostable DNA polymerase.

Negatively Charged, Dye Labeled-Dideoxynucleotides for “Direct-Load” DNA Sequencing

A four-color set of negatively charged, single dye as well as energy transfer dye labeled-ddNTPs were synthesized and evaluated in combination with a novel polymerase in a “direct-load” DNA sequencing, obviating the laborious and time consuming post-reaction work-up.

Rigid, conjugated, fluoresceinated thymidine triphosphates: syntheses and polymerase mediated incorporation into DNA analogues.

Syntheses of a unique set of energy transfer dye labeled nucleoside triphosphates, compounds 1-3, are described. Attempts to prepare these compounds were only successful if the triphosphorylation reaction was performed before coupling the dye to the nucleobase, and not the other way around. Compounds were prepared as both the 2'-deoxy (a) and 2',3'-dideoxy- (b) forms. They feature progressively longer rigid conjugated linkers connecting the nucleobase and the hydroxyxanthone moiety. UV spectra of the parent nucleosides 12-14 show that as the length of the linker increases so does the absorption of the donor in the 320-330 nm region, but with relatively little red-shift of the maxima. Fluorescence spectra of the same compounds show that radiation in the 320-330 nm region results in predominant emission from the fluorescein. When the linker is irradiated at 320 nm, the only significant emission observed corresponds to the hydroxyxanthone part of the molecules at 520 nm; this corresponds to an effective Stokes' shift of 200 nm. As the absorption at 320-330 nm by the linker increases with length, so does the intensity of the fluorescein emission. A gel assay was used to gauge relative incorporation efficiencies of compounds 1-3, dTTP, ddTTP, and 6-TAMRA-ddTTP. Throughout, the thermostable polymerase TaqFS was used, as it is the one most widely applied in high throughput DNA sequencing. This assay showed that only compounds 3 were incorporated efficiently; these have the longest linkers. Of these, the 2'-deoxy nucleoside 3 a was incorporated and did not prevent the polymerase from extending the chain further. The 2',3'-dideoxy nucleoside 3 b was incorporated only about 430 times less efficiently than ddTTP under the same conditions, and caused chain termination. The implications of these studies on modified sequencing protocols are discussed.

ChemInform Abstract: Fluorescence Resonance Energy Transfer Dye Nucleotide Terminators: A New Synthetic Approach for High‐Throughout DNA Sequencing

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Energy transfer and optical gain studies of FDS: Rh B dye mixture investigated under cw laser excitation

Radiative and non-radiative (Forster type) energy transfer processes in a dye mixture of FDS and Rh B in methanol under cw Ar ion laser excitation were investigated for fixed donor concentration and varying acceptor concentration. It is found that most of the pump power absorbed by FDS is transferred to Rh B as a useful pump power. Transfer probability (PDA), transfer efficiency both radiative (ηR) and non-radiative (ηNR) and optical gain (G) of the system were studied for various pump powers. The gain characteristics of Rh B are found to alter due to the change in the effective fluorescence lifetime caused by energy transfer reaction. Theoretical calculations were also done to find the total transfer efficiency (ηT) at various acceptor concentrations to identify the appropriate energy transfer mechanism responsible for gain enhancement in Rh B. Both radiative and non-radiative transfer processes are taken into consideration in all the calculations. Various energy transfer parameters viz. radiative rate constant (KR), non-radiative rate constant (KNR), critical concentration (C0), critical radius (R0) and half quenching concentration ([A]1/2) are calculated by using the Stern–Volmer plots and concentration dependence of radiative and non-radiative transfer efficiencies. Concentration and pump power dependence of the peak gain and lasing wavelengths of the energy transfer dye lasers (ETDL) have also been studied. The experimental results show that the dominant mechanism responsible for the efficient excitation transfer in this mixture is of radiative nature, whereas the long range dipole–dipole (d–d) interaction (Forster type) is comparatively smaller.