The photophysical properties of tyrosine and its derivatives with free and blocked functional groups in water were studied by steady-state and time-resolved fluorescence spectroscopy and global analysis. Tyrosine fluorescence intensity decays in water at pH ) 5.5 in the short-wavelength region (290-320 nm) are monoexponential, whereas, at longer wavelengths, they are biexponential. The monoexponential fluorescence intensity decay of O-methyl tyrosine across the fluorescence band is observed. The fluorescence lifetimes of Tyr calculated using a global analysis are equal to 3.37 ( 0.04 ns at the short-wavelength region and 0.98 ( 0.12 ns at the longer-wavelength region. This observation, together with the decay-associated spectra, indicate that the short-lifetime component can be attributed to tyrosine with phenol hydroxyl groups hydrogen-bonded with water molecules. The rotamer populations calculated from potentials of mean forces, as well as those obtained from 1 H NMR spectroscopy, do not correspond to the pre-exponential factors obtained from fluorescence spectroscopy. The calculated energy barriers of rotations about the C R -C β bond indicate that the interconversion rate constant for tyrosine and N-acetyl-tyrosinamide are much greater than the fluorescence rate constant. Monoexponential fluorescence intensity decay of tyrosine derivatives in acetonitrile solution is observed for all derivatives studied and, contrary to the aqueous solution, the amide group does not quench the fluorescence. Thus, specific conformation(s) stabilized by the hydrogen-bond network seem to be responsible for the heterogeneous fluorescence intensity decay of tyrosine derivatives in aqueous solution.

Fluorescence technologies have been the preferred method for detection, analytical sensing, medical diagnostics, biotechnology, imaging, and gene expression for many years. Fluorescence becomes essential for studying molecular processes with high specificity and sensitivity through a variety of biological processes. A significant problem for practical fluorescence applications is the apparent non-linearity of the fluorescence intensity resulting from inner-filter effects, sample scattering, and absorption of intrinsic components of biological samples. Sample absorption can lead to the primary inner filter effect (Type I inner filter effect) and is the first factor that should be considered. This is a relatively simple factor to be controlled in any fluorescence experiment. However, many previous approaches have given only approximate experimental methods for correcting the deviation from expected results. In this part we are discussing the origin of the primary inner filter effect and presenting a universal approach for correcting the fluorescence intensity signal in the full absorption range. Importantly, we present direct experimental results of how the correction works. One considers problems emerging from varying absorption across its absorption spectrum for all fluorophores. We use Rhodamine 800 and demonstrate how to properly correct the excitation spectra in a broad wavelength range. Second is the effect of an inert absorber that attenuates the intensity of the excitation beam as it travels through the cuvette, which leads to a significant deviation of observed results. As an example, we are presenting fluorescence quenching of a tryptophan analog, NATA, by acrylamide and we show how properly corrected results compare to the initial erroneous results. The procedure is generic and applies to many other applications like quantum yield determination, tissue/blood absorption, or acceptor absorption in FRET experiments.

Of the many optical bioassays available, sensing by fluorescence anisotropy have great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation as the emission lifetime of the label needs to be comparable to the correlation lifetime (tumbling time) of the biomolecule which is labelled. For proteins of moderate size this is in the order of 20–200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatics dyes. These have the significant drawback of UV/blue absorption and emission as well as an often significant solvent sensitivity. Here, we report the synthesis and characterization of a new fluorescent label for high molecular weight biomolecules assay based on the azadioxatriangulenium motif. The NHS ester of the long fluorescence lifetime, red emitting fluorophore: azadioxatriangulenium (ADOTA-NHS) was conjugated to anti-rabbit Immunoglobulin G (antiIgG). The long fluorescence lifetime was exploited to determine the correlation time of the high molecular weight antibody and its complex with rabbit Immuniglobulin G (IgG) with steady-state fluorescence anisotropy and time-resolved methods: solution phase immuno-assay was performed following either steady-state or time-resolved fluorescence anisotropy. By performing a variable temperature experiment it was determined that the binding of the ligand resulted in an increase in correlation time by more than 75 %, and a change in the steady-state anisotropy increase of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay for detecting binding events involving biomolecules of far larger size than what is possible with the other red emitting organic dyes.

The fluorescence of LDS 798 dye in aqueous solution has a very short lifetime of 24 ps, independent of excitation wavelength. The time response of common photon counting detectors depends on the wavelength of the registered photon. In lifetime measurements, the instrument response function (IRF) is usually approximated by the temporal profile of the scattered excitation light. Because lambda(Exc) is typically much shorter than lambda(Em), a systematic error may be present in these measurements. We demonstrate that the fluorescence decay of LDS 798 is a better approximation of IRF, in particular, for avalanche photodiodes used in the near infrared spectral region.