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 widefield photon counting imaging with a fast CMOS camera applied to luminescence microscopy is demonstrated.

The demand for practical and convenient enzyme assays for histone lysine methyltransferases (HKMTs) emerges along with the rapid development of this young class of enzymes. A supramolecular reporter pair composed of p-sulfonatocalix[4]arene (CX4) and the fluorescent dye lucigenin (LCG) has been used to monitor enzymatic trimethylation of lysine residues in peptide substrates. The assay affords a switch-ON fluorescence response and operates in a continuous, real-time, and label-free fashion. The underlying working principle relies on the higher affinity of the macrocycle towards the trimethylated product of the enzymatic reaction as compared to the substrate, which allows the assay to be carried out in the product-selective mode. The final product incorporates a trimethylammonium moiety, a known high-affinity binding motif for CX4. Two substrates corresponding to the H3 N-terminal tail, namely, S2 (RTKQTARKSTGGKAP) and S6 (QTARKSTGGS), were selected as model compounds for methylation with the Neurospora crassa Dim-5 enzyme and investigated by the newly developed supramolecular tandem HKMTs assay. Only the longer substrate S2 underwent methylation in solution. The potential of the assay for inhibitor screening was demonstrated by means of inhibition studies with 1,10-phenanthroline to afford an inhibition constant of (70±20) μM.

Stillbazolium salts present remarkable potential for application in several scientific areas. Their versatile behavior is explained by invoking the "twisted intramolecular charge-transfer" (TICT) mechanism, a model that describes the multiple fluorescence of DASPMI (4-(4-(dimethylamino)styryl)- N-methylpyridiniumiodine). One feature of their behavior is the sensitivity of the fluorescence lifetime to viscosity, thus identifying them as suitable probes for microheterogeneous systems, such as cells and sol-gel derived media. Because of their optical transparency, sol-gel matrices are light addressable and therefore appropriate for performing spectroscopic studies. The sol-gel process has been successfully used to produce hosts to biomolecules like proteins, for biosensor applications; however, these systems have to be optimized. Therefore, in this study modification of the matrices was performed through the incorporation of either silanes or polymers. (Aminopropyl)triethoxysilane, trimethoxypropylsilane, or (glycidyloxypropyl)triethoxysilane were added. The modification was also extended to the incorporation of the polymers poly(ethylene glycol) (molecular weight 300 and 20000) and Gelrite. The effect of these modifiers upon the gelation and aging processes was examined via the study of the photophysics of p-DASPMI by using both steady-state and time-resolved fluorescence. It was possible to discriminate the dominant dye-host interactions in each of the main steps of the preparation of modified sol-gel matrices.

A suitable matrix to host enzymes for biosensor applications should encage and retain the bioactive species, while allowing it to be accessed to exploit its catalytic properties. Sol-gel derived monoliths are promising in this aspect. Molecular diffusion was monitored using fluorescence labelled proteins and unbound fluorescence dye molecules (representative of enzyme substrates) and their interaction with and mobility within the host assessed using time-resolved fluorescence anisotropy and fluorescence recovery after photobleaching observed via confocal microscopy.