Cyanines emission

As seen from (12) and Fig. 6, the peaks in the excitation anisotropy spectrum indicate a small angle between the absorption and emission transition dipoles suggesting allowed 1PA transitions while valleys indicate large angles between these two dipoles, suggesting a forbidden 1PA transition. Due to selection rules for symmetrical cyanine-like dyes, the valleys in the anisotropy spectrum could indicate an allowed 2PA transition as demonstrated in Fig. 6. Thus, an excitation anisotropy spectrum can serve as a useful guide to suggest the positions of the final states in the 2PA spectra. 

The tunability on emission wavelength of cyanine derivatives is based on the understanding of structure-photophysical property relationships, which allows the development of near-IR fluorophores [80, 84—87]. Enhancement of the rigidity in... 

Lanthanides also have potential as DEFRET energy donors. Selvin et al. have reported the use of carbostyril-124 complexes (53) with europium and terbium as sensitizers for cyanine dyes (e.g., (54)) in a variety of immunoassays and DNA hybridization assays.138-140 The advantage of this is that the long lifetime of the lanthanide excited state means than it can transfer its excitation energy to the acceptor over a long distance (up to 100 A) sensitized emission from the acceptor, which occurs at a wavelength where there is minimal interference from residual lanthanide emission, is then measured. 

Thomas, L, Netzel, K. N. and Zhao, M. (1995). Base-content dependence of emission enhancements, quantum yields, and lifetimes for cyanine dyes bound to double-strand DNA Photophysical properties of monomeric and bichromophoric DNA stains. J. Phys. Chem. 99,17936-17947. 

The longer the polymethine bridge in a cyanine dye, the higher the absorbance and emission wavelengths become. In general, for each incremental increase of n, the absorbance and... 

Figure 9.51 Time-resolved FRET assay systems involve energy transfer between the lanthanide chelate and an organic dye that are brought together as two labeled molecules bind to an analyte. In this illustration, an antibody labeled with a lanthanide chelate is used along with a Cy5-labeled antibody to detect a protein target in solution. Excitation of the lanthanide label results in energy transfer and excitation of the cyanine dye only if they are held within close enough proximity to allow efficient FRET to occur. Under these conditions, excitation of the lanthanide chelate results in cyanine dye emission, which will not occur if the labeled antibodies have not bound to a target.

The second label also may be a fluorescent compound, but doesn t necessarily have to be. As long as the second label can absorb the emission of the first label and modulate its signal, binding events can be observed. Thus, the two labeled DNA probes interact with each other to produce fluorescence modulation only after both have bound target DNA and are in enough proximity to initiate energy transfer. Common labels utilized in such assay techniques include the chemiluminescent probe, N-(4-aminobutyl)-N-ethylisoluminol, and reactive fluorescent derivatives of fluorescein, rhodamine, and the cyanine dyes (Chapter 9). For a review of these techniques, see Morrison (1992). 

Albert H. Coons was the first to attach a fluorescent dye (fluorescein isocyanate) to an antibody and to use this antibody to localize its respective antigen in a tissue section. Fluorescein, one of the most popular fluorochromes ever designed, has enjoyed extensive application in immunofluorescence labeling. For many years, classical fluorescent probes such as FITC or Texas red (TR) have been successfully utilized in fluorescence microscopy. In recent decades, brighter and more stable fluorochromes have continually been developed (see Table 14.1). Marketed by a number of distributors, cyanine dyes, Cy2, Cy3, Cy5, Cy7, feature enhanced water solubility and photostability as well as a higher fluorescence emission intensity as compared to many of the traditional dyes, such as FITC or TR. 

The optical properties of organic dyes (Fig. ld-f, Table 1) are controlled by the nature of the electronic transition(s) involved [4], The emission occurs either from an electronic state delocalized over the whole chromophore (the corresponding fluorophores are termed here as resonant or mesomeric dyes) or from a charge transfer (CT) state formed via intramolecular charge transfer (ICT) from the initially excited electronic state (the corresponding fluorophores are referred to as CT dyes) [4], Bioanalytically relevant fluorophores like fluoresceins, rhodamines, most 4,4 -difluoro-4-bora-3a,4a-diaza-s-indacenes (BODIPY dyes), and cyanines (symmetric... 

Malicka J, Gryczynski I, Gryczynski Z, Lakowicz JR (2003) Effects of fluorophore-to-silver distance on the emission of cyanine-dye-labeled oligonucleotides. Anal Biochem 315 57-66... 

Presently, the only commercially available dyes that are applied because of then-ability to form fluorescent aggregates are trimethine cyanines JC-1 and JC-9 (Fig. 11) [25], the first one being studied much more extensively than the second one. The dye JC-1 is known to form red-fluorescent (emission maximum at 590 nm) J-aggregates in mitochondria possessing strong intramitochondrial negative potential, while upon depolarization of the mitochondrial membrane, the dye monomer green emission (maximum at 527 nm) is observed [25]. JC-9 demonstrates similar properties [25]. Such properties permit the application of these dyes for, e.g., detection of apoptotic electrical depolarization of mitochondria [25]. 

The complexation with CDs also results in spectral shifts of the absorption and emission maxima of cyanine dyes. The complexation of cyanine dyes 1 (X = S, R = Et, n = 1-3) with p-CD red-shifts the emission bands [25]. 

The absorption and emission maxima of trimethinecyanine 9a (551 nm and 565 nm), squaraine 9b (631 nm and 641 nm), and cyanine 9c (784 nm and 805 nm) hardly change after complexation with a-CD and [S-CD in water, but the fluorescence quantum yields increase noticeably [28]. CD complexes of these water-soluble dyes containing reactive carboxylic functionalities have potential use as fluorescent labels. 

Squaraines 17a-17c were encapsulated in these macrocyles to form the corresponding pseudorotaxanes. Squaraine rotaxanes 14 and 15 with a phenylene tetralactam macrocycle have absorption/emission profiles (Table 3) that closely match those of Cy5, whereas squaraine rotaxanes 16 D 17 with an anthrylene macrocycle have a red-shifted absorption/emission that matches that of the homologous cyanine Cy5.5 (Table 4). These rotaxanes should be useful for fluorescence microscopy imaging applications. 

While the covalent attachment of cyanine dyes such as Cy5 or Alexa 647 to proteins does not result in noticeable changes in their spectral properties, squaraine dyes (oxo-squarines and squaraines with substituted squaraine oxygens) behave quite the opposite the absorption and emission maxima of squaraines are in general red-shifted after binding to proteins and the quantum yields and fluorescence lifetimes are manifold increased [68-70]. In general hydrophobic squaraines exhibit more pronounced increases compared to hydrophilic dyes. These effects are even stronger in noncovalent dye-protein complexes. Importantly, the photostability of squaraines also increases after binding to proteins. 

Embedding of pinacyanol in a three-dimensional polyphenylene dendrimer results in a red-shift of absorption (from 600 to 620 nm) and emission (from 625 to 648 nm) maxima and in an increase of the quantum yield from 9 x KT4 for free dye in water to 1.4 x 10 2 after insertion in the dendrimeric architecture [71]. This dendrimer was used to develop two FRET systems utilizing cyanine dyes as the donor (DTCI) and the acceptor (pinacyanol and 26a) molecules [72], The FRET system allows the time-resolved detection, where energy transfer can be observed at the single-molecule level. 

Bringley JF, Penner TL, Wang R, Harder JF, Harrison WJ, Buonemani L (2008) Silica nanoparticles encapsulating near-infrared emissive cyanine dyes. J Colloid Interface Sci 320 132-139... 

A similar sharp red emission exhibit squarylium dyes like the squarylium cyanine dye 64, which emits 650nm [152]. Unfortunately, the Stokes shift is rather small, which leads to high reabsorption. A number of other red emitting dyes have been exploited as well [153-156]. 

Cyanine dyes are in principle capable of shifting the emission into the near-IR region [157], however, their ionic character makes it difficult to dope them into films by vacuum vapor evaporation. Other materials investigated for IR emission are complexes based on rare earth ions (Nd3+, Er3+), which are also used in inorganic amplifiers and lasers [158]. 

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