Spectral Properties of Fluorescein in Solvent-Water Mixtures: Applications as a Probe of Hydrogen Bonding Environments in Biological Systems

Although fluorescein is a widely used fluorescent probe in the biosciences, the effect of solvent environment on its spectral properties is poorly understood. In this paper we explore the use of fluorescein as a probe of the state of hydrogen bonding in its local environment. This application is based on the observation, originally made by Martin ( Chem. Phys. Lett . 35, 105–111, 1975), that the absorption maximum of fluorescein undergoes substantial shifts in organic solvents related to the hydrogen bonding power of the solvents. We have extended this work by studying the spectral properties of the dianion form of the probe in solvent–water mixtures. We show that the magnitude of the shift correlates with the α and β parameters of Kamlet and Taft ( J. Am. Chem. Soc . 98, 377–383; 2886–2894, 1976), which provide a scale of the hydrogen bond donor acidities and acceptor basicities, respectively, of the solvents. In solvent–water mixtures, these shifts reflect general effects of the solvents on the hydrogen bonding environment of the fluorescein through water–solvent hydrogen bonding and specific effects due to fluorescein–solvent hydrogen bonding. Indeed, both the absorption and fluorescence properties appear to be dominated by these effects indicating that the spectral shifts of the dianion can be used as an indicator of its hydrogen bonding environment. We discuss the application of fluorescein as a probe of hydrogen bonding in the microenvironment immediately surrounding the fluorophore, and we illustrate the effect with reference to the fluorescein–antifluorescein antibody complex where it appears that antibodies selected during the immune response possess binding sites that are increasingly dehydrated and hydrophobic.     

Markovian trees: properties and algorithms

In this paper we introduce a structure called the Markovian tree (MT). We define the MT and explore its alternative representation as a continuous-time Markovian Multitype Branching Process. We then develop two algorithms, the Depth and Order algorithms to determine the probability of eventual extinction of the MT process. We show that both of these algorithms have very natural physically intuitive interpretations and are analogues of the Neuts and U algorithms in Matrix-analytic Methods. Furthermore, we show that a special case of the Depth algorithm sheds new light on the interpretation of the sample paths of the Neuts algorithm.     

Effect of solvent-water mixtures on the prototropic equilibria of fluorescein and on the spectral properties of the monoanion

A spectral resolution procedure was used to resolve the absorption, excitation and emission spectra of the fluorescein monoanion in a number of solvent-water mixtures. This permitted an analysis of the effect of the solvent environment on the spectral properties of the monoanion and on the lactone/monoanion/dianion transitions of fluorescein. The monoanion excitation and emission spectra show relatively small changes with changing environment, a behavior that is related to the hydrogen-bonding environment of the solvent-water mixtures. There is also a general increase in the quantum yield of the monoanion from 0.36 in water to values up to 0.49 in the solvent-water mixtures. The presence of solvent also results in a general increase in the lactone content and in the monoanion:dianion and lactone:monoanion ratios. General polarity effects alone cannot account for the observed effects on the prototropic transitions indicating that specific solute-solvent effects involving hydrogen bonding perturb the prototropic equilibria of fluorescein.     

Characterization of a Series of Far Red Absorbing Perylene Diones: A New Class of Fluorescent Probes for Biological Applications

The far red region of the spectrum is increasingly being utilised in many applications in the biosciences. However, apart from the cyanine group of dyes, there are relatively few far red fluorescent probes available which are of practical use. We have synthesised and characterised a new class of far red fluorescent probes based on the perylene dione chromophore. The 2,10 di-substituted perylene diones possess broad absorption spectra (>90 nm bandwidths), large Stokes shifts (>60 nm) and quantum yields of up to 0.5 with a maximum absorption at 610–640 nm in organic solvents or in solutions of non-ionic detergents. A number of derivatives have been synthesised that can be used as membrane probes, as chromogenic substrates for alkaline phosphatase, and for the labelling of macromolecules such as proteins and DNA. These novel far red fluorophores have potential applications in diagnostic and research applications.     

The thiourea group modulates the fluorescence emission decay of fluorescein-labeled molecules

We have investigated the spectral properties and emission characteristics of fluorescein-5-thiocarbamoyl-N,N'-caproate (FITC-ACA) to examine the origin of the complex emission decay often observed in fluorescein-labeled molecules. The covalent attachment of fluorescein to epsilon-amino-n-caproic acid does not perturb the prototropic transitions of the chromophore or the general fluorescence characteristics of the various prototropic forms. However, both the monoanion and dianion forms of FITC-ACA are quenched relative to free fluorescein and exhibit a complex emission decay that is described by two discrete lifetimes. The thiourea group that links the chromophore to the caproic acid is shown to modulate the emission properties of the FITC-ACA. We show that the emission decay can also be analyzed using the asymmetric distribution model of Alcala et al. In this analysis, the tauL and tauu parameters that represent the lower and upper lifetime limits of the distribution reflect the quenched (0 ns) and unquenched lifetimes, respectively. The beta parameter that describes the distribution of lifetimes between the two limiting states can be related to the quenching efficiency of the thiourea group and to the structure and dynamics of the FITC-ACA molecule.     

Space reduction and an extension for a hidden line elimination algorithm

A simple intersection sensitive algorithm for the hidden line elimination problem, was presented by Nurmi in 1985. This algorithm has O((n + I) logn) time and space complexities, where n is the number of edges in the input scene and I is the number of their intersections on the projection plane. We describe a method that reduces the space requirements of the algorithm to O(n) while retaining the time complexity of O((n+I) logn). Furthermore we show that the algorithm can be easily extended to handle the more general problem of hidden surface removal.     

Using agents for solving a multi-commodity-flow problem

We investigate a commodity trading problem in a flow network with arbitrary topology where sinks combine commodities into bundles in order to generate profits. Our focus is the profit maximization problem for the trading network under both central and distributed control. We compute solutions for the central control problem using an integer linear program while we compute solutions for the distributed case by implementing the nodes in the network as software-agents that exchange messages in order to establish profitable trades. We report on computational results using both methods and demonstrate that there is a connection between agent profits and a centrality measure developed for the problem. We also demonstrate that with our current agent strategy, there is a trade-off between the agents acting too quickly before enough information is available and waiting too long and thus giving each agent too much information and thus too much power over the outcome.     

Spectral properties of the prototropic forms of fluorescein in aqueous solution

The commonly used fluorescent probe, fluorescein, can exist in seven prototropic forms. We have used global analysis procedures to reanalyze the absorption data of Diehl and Horchak-Morris (Talanta 34, 739–741, 1987) in terms of five alternative ionization models. We identify the forms of fluorescein present in aqueous solution and the pK _a of each ionisation transition. The pK_a values of the neutral xanthene, carboxylic acid, and cationic xanthene groups are 6.3, 3.1–3.4, and 3.1–3.4, respectively, and the pK_a value of lactonization is 2.4. As a consequence, the neutral form of fluorescein is a mixture of the lactone (70%), zwitterionic (15%), and quinoid (15%) forms. A knowledge of the forms present in solution permits the characterization of their spectral properties. It is shown that the quinoid and monoanion forms have similar absorption spectra, while the zwitterion spectrum is similar to that of the cation but blue-shifted by 3 nm. The emission spectra of the monoanion and quinoid forms are also identified and shown to be similar but not identical. A model for the excited-state reactions of fluorescein is presented.