Ratiometric dyes

A different approach to design a self-calibrating dye was proposed [70], in which a viscosity-sensitive molecular rotor (2-cyano-3-(4-dimethylaminophenyl) prop-2-enoic acid) was covalently linked to a reference dye, 7-methoxycoumarin-3-carboxylic acid, which exhibited no viscosity sensitivity (40, Fig. 13). A ratiometric measurement, that is, rotor emission relative to reference emission, was shown to be widely independent of dye concentration [70]. However, the design of such a ratiometric dye poses some challenges because of resonance energy transfer from... 

Fig. 13 Self-calibrating dyes DMA-2,4 and p-A,A-dimethylaminobenzoic acid. Compound 40 is an engineered ratiometric dye composed of a viscosity-sensitive molecular rotor and a nonviscosity-sensitive reference dye [70]...

With further understanding how molecular rotors interact with their environment and with application-specific chemical modifications, a more widespread use of molecular rotors in biological and chemical studies can be expected. Ratiometric dyes and lifetime imaging will enable accurate viscosity measurements in cells where concentration gradients exist. The examination of polymerization dynamics benefits from the use of molecular rotors because of their real-time response rates. Presently, the reaction may force the reporters into specific areas of the polymer matrix, for example, into water pockets, but targeted molecular rotors that integrate with the matrix could prevent this behavior. With their relationship to free volume, the field of fluid dynamics can benefit from molecular rotors, because the applicability of viscosity models (DSE, Gierer-Wirtz, free volume, and WLF models) can be elucidated. Lastly, an important field of development is the surface-immobilization of molecular rotors, which promises new solid-state sensors for microviscosity [145]. 

Paul Do you worry about using non-ratiometric dyes here when these things are supposed to be contracting and oscillating I know people have shown waves with Fura 2, but it always makes me a little nervous when non-ratiometric dyes are used, because some of this could be artefactual. 

Equation (2) shows why r is in fact a ratiometric parameter this is because the variations of intensity influence proportionally the FVv and FVh values. Therefore, the anisotropy allows obtaining self-referencing information on sensing event from a single reporter dye. This information is independent on reporter concentration. 

Square-650-pH having a pKa in the physiological pH range (pKa = 7.1 for free dye and the pKa 6.1 when labeled to an antibody) was recently introduced by SETA BioMedicals [119]. This dye is commercially available as a free carboxylic acid and a mono-NHS ester. Square-650-pH has spectral properties similar to those of the CypHer dyes but is fluorescent in both the protonated and deproto-nated forms. This dye displays reasonable molar absorptivities (135,000 and 48,000 M-1cm-1) and quantum yields (16% and 9%) for the protonated and deprotonated forms, an extremely large Stokes shift of more than 100 nm for the deprotonated form, and enables excitation and emission ratiometric measurement... 

Sreejith S, Divya KP, Ajayaghosh A (2008) A near-infrared squaraine dye as a latent ratiometric fluorophore for the detection of aminothiol content in blood plasma. Angew Chem 120 8001-8005... 

Ratiometric luminescent probes make a smart use of the excitation wavelength effect on the emission intensity for extended optosensor performance. For example, the fluorescence from 8-hydroxy-l,3,5-pyrenetrisulfonate (HPTS) and other pH-sensitive dyes in water comes only from its (photo)excited basic form, but the absorption spectra of HPTS and PTS (pAa 7.3) differ considerably (Figure 3). 

Center for Healthcare Technologies at Lawrence Livermore National Laboratory in Livermore, potentially capable to measure pH at or near the stroke site29. The probe is the distal end of a 125 pm fibre tapered up to a diameter of 50 pm. A fluorescent pH-indicator, seminaphthorhodamine-1-carboxylate, is embedded inside a silica sol-gel matrix which is fixed to the fibre tip. Excitation of the dye takes place at 533 nm and the emission in correspondence of the acid (580 nm) and basic (640 nm) bands are separately detected. The use of this ratiometric technique obviates worrying about source fluctuations, which have the same effects on the two detected signals. The pH sensor developed was first characterised in the laboratory, where it showed fast response time (of the order of tens of seconds) and an accuracy of 0.05 pH units, well below the limit of detection necessary for this clinical application (0.1 pH units). The pH sensor was also tested in vivo on rats, by placing the pH sensor in the brain of a Spraque-Dawley rat at a depth of approximately 5 mm30. 

Kim S, Pudavar FIE, Prasad PN (2006) Dye-concentrated organically modified silica nanoparticles as a ratiometric fluorescent pH probe by one- and two-photon excitation. Chem Commun (Camb) 19 2071-2073... 

Sun H, Scharff-Poulsen AM, Gu H, Almdal K (2006) Synthesis and characterization of ratiometric pH sensing nanoparticles with covalently attached fluorescent dyes. Chem Mater... 

The ratiometric measurements are preferable because the ratio of the fluorescence intensities at two wavelengths is in fact independent of the total concentration of the dye, photobleaching, fluctuations of the source intensity, sensitivity of the instrument, etc. The characteristics of some fluorescent pH indicators allowing ratiometric measurements are given in Table 10.1. 

Fig. 19 A PAMAM dendrimer ratiometric sensor synthesised by conjugation of PAMAM den-drimers to a chloride-sensitive probe (BAC) and to a chloride-insensitive dye (TMR)...

Fig. 5 Data acquisition cycles for ratiometric PDI using luminescent dyes (e.g., europium complexes) with decay times in the ps range...

---