Viability of Saccharomyces cerevisiae incorporated within silica and polysaccharide hosts monitored via time-resolved fluorescence.

The viability of Saccharomyces cerevisiae in biocompatible polymers under different growth conditions and studied using time-resolved fluorescence techniques is presented. Two fluorophores, the viscosity sensitive probe 4-(4-(dimethylamino)styryl)-N-methyl-pyridiniumiodine (DASPMI) and the yeast viability stain 2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene)-1-phenylquinolinium iodide (FUN-1) are used to elucidate information on the incorporated yeast cell viability. Variations in cell viscosity, which are indicative of the cell state, were obtained using DASPMI. Prior to observing FUN-1 in yeast cells using fluorescence lifetime imaging, its photophysics in solution and heterogeneous media were investigated. Time-resolved emission spectra were measured and analysed to associate lifetimes to the spectral emission. Preliminary results show that monitoring the fluorescence lifetime of FUN-1 may give a useful insight into cellular metabolism. The results indicate that both fluorophores may be used to monitor the entrapped yeast cell viability, which is important for in vitro studies and applications, such as that in the biofuel industry, where Saccharomyces cerevisiae are required to remain active in high ethanol environments.

The influence of silver nanostructures formed in situ in silica sol-gel derived films on the rate of Förster resonance energy transfer.

The efficiency of Förster resonance energy transfer (FRET) can be enhanced in the presence of a metal. Herein, we demonstrate the increased efficiency for a novel model sensor system where FRET is shown to occur between Rhodamine 6G in the bulk sol-gel matrix and Texas Red, which is held a fixed distance away by covalent attachment onto a silane spacer. Silver colloids are formed using light to initiate the reduction of a silver salt, which can be achieved at controlled locations within the film. Both the fluorescence intensity and lifetime maps and analysis indicate that an enhanced FRET efficiency has been achieved in the presence of silver nanoparticles. An increase in efficiency of 1.2-1.5 times is demonstrated depending on the spacer used. The novelty of our approach lies in the method of silver-nanoparticle formation, which allows for the accurate positioning of the silver nanoparticles and hence selective fluorescence enhancement within a biocompatible host material. Our work gives a practical demonstration of metal-enhanced FRET and demonstrates the ability of such systems to be developed for molecular-recognition applications that could find use in lab-on-a-chip technologies.

In situ formation of silver nanostructures produced via laser irradiation within sol-gel derived films and their interaction with a fluorescence tagged protein.

The presence of a conducting metal surface is known to affect the emission of a fluorophore in its proximity. This can lead to an enhancement in its fluorescence intensity along with a decrease in the fluorescence lifetime. This phenomenon, sometimes known as metal enhanced fluorescence, has implications in the area of sensing and "lab on a chip" applications. Here controlled, localised use of metallic structures can be advantageous in enhancing the detection of a fluorescent signal. The sol-gel technique has been demonstrated as a useful method by which to produce a biocompatible material. The versatility of the reaction allows for the inclusion of metal ions, which can form metallic nanostructures permitting the potential enhancement of fluorescence to be exhibited. In this work we incorporate silver nitrate within silica sol-gel derived films produced using a simple procedure at relative low temperatures (close to ambient). A compact time-resolved fluorescence microscope equipped with a semiconductor laser was used to photoactivate the silver ions to form localised metallic structures within the films. Patterning was achieved by computer control of the microscope stage and using the laser in CW mode. The films were characterised using AFM and UV-vis spectroscopy to ascertain the presence of the photoactivated silver nanostructures. The effect of the presence of these structures was elucidated by studying the time-resolved fluorescence of FITC labelled bovine serum albumin adsorbed to the films, where a decrease in the lifetime of the FITC label was observed in the location of the nanostructures.

Monitoring sol-to-gel transitions via fluorescence lifetime determination using viscosity sensitive fluorescent probes.

The sol-to-gel transition was monitored via the use of time-resolved recording of the fluorescence emission of viscosity-sensitive probes. Two dyes were chosen for the study, water-soluble DASPMI and a hydrophobic BODIPY, and steady-state, time-resolved and time-tagged fluorescence measurements were performed. These techniques, coupled with the probes different solubility, allowed complementary fluorescence lifetime and intensity data to be obtained from the dyes introduced into the matrix-forming mixture to produce sol-gel derived monoliths. Two different precursors were used as examples. A hydrogel was formed from a commercially available gellan gum (Gelrite), and a glass-like monolith was formed using tetraethyl orthosilicate. Changes in fluorescence lifetime could be related to those in the local viscosity sensed by the probe. The combination of this type of probe with time-resolved measurements is extremely useful in monitoring the microscopic changes that occur during the sol-to-gel transition within this important class of materials.

Effect of pH on aqueous phenylalanine studied using a 265-nm pulsed light-emitting diode.

Recently, we described the characteristics and application of a 265-nm AlGaN light-emitting diode (LED) operated at 1-MHz repetition rate, 1.2-ns pulse duration, 1.32-microW average power, 2.3-mW peak power, and approximately 12-nm bandwidth. The LED enables the fluorescence decay of weakly emitting phenylalanine to be measured routinely in the condensed phase, even in dilute solution. For a pH range of 1-11, we find evidence for a biexponential rather than a monoexponential decay, whereas at pH 13, only a monoexponential decay is present. These results provide direct evidence for the dominance of two phenylalanine rotamers in solution with a photophysics closer to the other two fluorescent amino acids, tyrosine and tryptophan, than has previously been reported. Although phenylalanine fluorescence is difficult to detect in most proteins because of its low quantum yield and resonance energy transfer from phenylalanine to tyrosine and tryptophan, the convenience of the 265-nm LED may well take protein photophysics in new directions, for example, by making use of this resonance energy transfer or by observing phenylalanine fluorescence directly in specific proteins where resonance energy transfer is inefficient.

Pre-denaturing transitions in human serum albumin probed using time-resolved phosphorescence.

The investigation of protein dynamics has long been of interest, since protein interactions and functions can be determined by their structure and changes in conformation. Although fluorescence, occurring on the nanosecond timescale, from intrinsic fluorescent amino acids has been extensively used, in order to fully access conformational changes longer timescales are required. Phosphorescence enables processes on the microsecond to second timescale to be accessed. However, at room temperature this emission can be weak and non trivial to measure. It requires the removal of oxygen - a common triplet state quencher and appropriate instrumentation. In this work we make use of a chemical deoxygenator to study room temperature phosphorescence from tryptophan in human serum albumin excited using a pulsed UV light emitting diode. This is extended to monitor the phosphorescence emission upon increasing temperature, allowing pre-denaturing transitions to be observed. Time-resolved data are analysed, both as the sum of exponential decays and using a distribution analysis based on non extensive decay kinetics. These results are compared to a fluorescence study and both the average lifetime and contribution of the different emitting components were found to give more dramatic changes on the phosphorescence timescale.