M. Isabel C. Ferreira

Studies on the interaction of Nile red with horseradish peroxidase in solution

The interaction of an extrinsic probe (Nile red) with an enzyme (horseradish peroxidase) in solution was investigated using fluorescence techniques. Nile red fluorescence is very environmentally sensitive and the presence of domains of differing polarity within the enzyme was ascertained by the decomposition of the Nile red emission spectrum. Further evidence for the position of the probe inside the enzyme was obtained from a molecular modeling study. A decrease in the emission intensity of the dye during incubation with horseradish peroxidase was explained by the occurrence of resonance energy transfer between the Nile red and the heme group in the enzyme. This was supported by a calculation of the critical transfer distance and a comparison of the fluorescence intensity of the dye in both the holo‐ and apo‐enzyme. These data were then applied to the study of the effect of temperature on the structure of the enzyme, where changes in conformation were elucidated.

Effect of polymer strengtheners on the local environment of biocompatible glass as probed by fluorescence.

Mixed silica–calcite matrices were prepared by developing a “low” temperature (sol–gel) method in presence of several biocompatible polymers, thus providing samples with adequate porosity for the flow of biological fluids and also mechanically robust. In order to analyse and characterise the sample’s microenvironments, the highly solvatochromic probe Nile red was used, which enabled the role of polymer addition upon local environmental effects in the host media to be elucidated. The polymers used were polyethylene glycol, polymethylmethacrylate and polyethylene. Each matrix was also characterized with respect to microstructure, morphology and pore size via the use of X-ray diffractometry and scanning electron microscopy.The results show that is was possible to obtain, in a controlled way, mixed silica–calcite matrices with a wide range of porosities (important if the material is to be used for scaffold or drug release applications, for example). The spectroscopic behaviour of Nile red when incorporated has confirmed the existence of distinct and specific local polarities within each type of matrix that may determine to a large extent the mechanism of interaction between these matrices and biological molecules.

Probing Local Environments by Time-Resolved Stimulated Emission Spectroscopy

Time-resolved stimulated emission spectroscopy was employed to probe the local environment of DASPMI (4-(4-(dimethylamino)styryl)-N-methyl-pyridinium iodide) in binary solvents of different viscosity and in a sol-gel matrix. DASPMI is one of the molecules of choice to probe local environments, and the dependence of its fluorescence emission decay on viscosity has been previously used for this purpose in biological samples, solid matrices as well as in solution. The results presented in this paper show that time-resolved stimulated emission of DASPMI is a suitable means to probe the viscosity of local environments. Having the advantage of a higher time resolution, stimulated emission can provide information that is complementary to that obtained from fluorescence decay measurements, making it feasible to probe systems with lower viscosity.

Application of Fluorescence Techniques to Characterise the Preparation of Protein-Containing sol – gel Derived Hosts for use as Catalytic Media

In this work we collate and review the usage that we have made of fluorescence techniques employed to follow the sol to gel transition and aging in different tetraethylorthosilicate based materials. The sol-gel method allows porous glasslike of good optical quality to be produced at relatively low (ambient) temperatures, which facilitates the incorporation of a range of molecules; from laser dyes to biomolecules. Here the use of “common” viscosity (DASPMI) and polarity (Nile red) sensitive fluorescence probes to monitor the host manufacture is made. Nile red was also used to label two catalytically active proteins (cytochrome c and subtilisin Carlsberg). These were incorporated into the different host media and the dye used to ascertain changes in protein conformation, both upon incorporation and at the end of an aging period. Complementary measurements of catalytic activity were performed. The probe emission was monitored via steady state and time-resolved fluorescence techniques and comparison made with the catalytic activity measurements to elucidate the amount of accessible and active protein. Overall it was found that the hosts became stable after an aging period approaching 20 days and that the major influence on the catalytic reaction rates was that of host mediated mass transport.

Nile Red Synchronous Scan Fluorescence Spectroscopy to Follow Matrix Modification in Sol–Gel Derived Media and its Effect on the Peroxidase Activity of cytochrome c

The highly solvatochromic dye Nile red is used in conjunction with synchronous scan fluorescence spectroscopy to elucidate changes in the internal environment of cytochrome c, upon incorporation into differently modified sol–gel derived media. Nile red was first studied in a variety of solvents in order to quantify changes in polarity. Matrix modifications involved the addition of several silanes, intended to interact with any unreacted hydroxyl entities left from the matrix forming reaction, while polymers were used to help reduce shrinkage and modify the internal pore environment. Slight unfolding of the protein was observed on incorporation into the sol–gel derived media. During the aging process further changes were monitored by using difference synchronous scan fluorescence spectra and complementary measurements of catalytic activity, expressed as the initial velocity. Combining Nile red synchronous scan fluorescence with cytochrome c activity data lead to a method to elucidate effects linked to protein conformation and those related to the sol–gel derived host.

Time-resolved fluorescence microscopy

Fluorescence imaging techniques are powerful tools in the biological and biomedical sciences, because they are minimally invasive and can be applied to live cells and tissues. The fluorescence emission can be characterized not only by its intensity and position, by also by its fluorescence lifetime, polarization and wavelength. Fluorescence Lifetime Imaging (FLIM) in particular has emerged as a key technique to image the environment and interaction of specific proteins in living cells. Using a time-correlated single photon counting (TCSPC)-based FLIM set-up, we find that the fluorescence lifetime of GFP-tagged proteins in cells is a function of the refractive index of the medium the cells are suspended in. In addition, combining Fluorescence Recovery After Photobleaching (FRAP) of fluorescently labeled proteins of different sizes in sol gels with time-resolved fluorescence anisotropy measurements, we demonstrate that we can measure their lateral and rotational diffusion. This allows us to infer the size and connectivity of the pores in the sol gel matrix. Moreover, wide-field photon counting imaging, originally developed for astronomical applications, is a powerful imaging method because of its high sensitivity and excellent signal-to-noise ratio. It has a distinct advantage over CCD-based imaging due to the ability to time the arrival of individual photons. The potential of time-resolved wide-field photon counting imaging with a fast CMOS camera applied to luminescence microscopy is demonstrated.