Optical and spectroscopic methods for biofilm examination and monitoring

Optical and spectroscopic methods for biofilmexamination and monitoring are reviewed.Biofilm examination techniques includemicroscopic methods, coupled with imageanalysis and with oligonucleotide ribosomal RNAprobing methods (fluorescence in situhybridization). Microscopic examinationtechniques are especially advantageous inextracting biofilm structural and architecturalparameters, as well as structure-functionrelationships of the biofilm microbialpopulation. Spectroscopic techniques are ableto elicit biofilm chemical and metabolicpatterns, as well as biofilm activity. They areof outstanding importance for on-line,non-invasive biofilm monitoring, especiallywhen coupled with chemometric algorithms forspectra calibration and pattern recognition.The paper emphasises the importance of thecombination of novel and established analyticaltechniques, as well as their integration withpowerful computational methods for theautomation of biofilm monitoring.     

Fluorescence decay studies of modified dinucleoside monophosphates containing 1-N6-ethenoadenosine

Five dinucleoside monophosphates containing I-N⁶-ethenoadenosine (?A) have been studied using fluorescence measurements. The fluorescence spectra of these dinucleoside monophosphates are almost the same as the fluorescence spectrum of ?AMP. Fluorescence quantum yields of these dimers are greatly reduced compared to that of ?AMP. Intramolecular base-base interactions may be responsible for fluorescence quenching. It is found that the fluorescence decay kinetics does not obey a simple decay law but that the decay data can be well described as a sum of three exponentials. This implies that these dimers cannot be characterized as a two-state system, but can be described as systems consisting of three or more conformational states. Sequence effects upon the fluorescence behavior are observed. The fluorescence quenching and decay parameters of Gp?A and Up?A indicate a higher degree of base-base interaction than in their ?ApG and ?ApU counterparts.     

Detection of three rotational correlation times for a rigid asymmetric molecule using frequency-domain fluorometry

We measured the frequency response of the polarized emission of Y_t-base in propylene glycol at 10 ° C. Data were obtained for excitation wavelengths of 290, 312 and 346 nm, for which the fundamental anisotropies are 0.05, 0.19 and 0.32, respectively. Additionally, data were obtained using CCl₄, to decrease the mean decay time from 9.1 to 4.2 ns. These nine sets of data were analyzed globally to recover the anisotropy decay law. Three correlation times were needed to fit the data, 0.8, 3.0 and 5.6 ns, a range of only 7-fold. We believe this is the first reported detection of three correlation times for a rigid molecule.     

Fluorescence decay studies of poly(riboadenylic acid) containing 1-N6-ethenoadenosine

The fluorescence properties of the 1-N6-etheno derivatives of poly(riboadenylic acid) (poly(rA, epsilon rA)) have been examined. The fluorescence quantum yield of poly(rA, epsilon rA) decreases with an increase in the degree of the epsilon A substitution and is much smaller than that for epsilon AMP even for low degrees of epsilon A substitution. The nearest-neighbor interactions such as epsilon-adenine-adenine and epsilon-adenine-epsilon-adenine may be responsible for this behavior. It is found that the fluorescence decay kinetics obeys a three-exponential decay law for poly(rA, epsilon rA), suggesting that there exist at least three different stacked conformational states.     

Ca2+ interaction with phospholipid bilayers studied by multifrequency phase fluorometry

Calcium interaction with phospholipid membranes containing phosphatidic acid is studied by multifrequency phase fluorometry, using DPH as fluorescent molecule. DPH decay is analysed by a continuous distribution of lifetimes. The results suggest an increase of membrane heterogeneity at low calcium concentrations, without changes in the polarity of the environment surrounding the probe.     

9-amino-2-methoxy-6-chloroacridine monocation fluorescence analysis by phase-modulation fluorometry

The fluorescence of the 9-amino-2-methoxy-6-chloroacridinic monocation (ACMA) in various alcohol-water solutions was studied by phasemodulation fluorometry. Apparent phase and modulation lifetimes were determined at different observation wavelengths for three modulation frequencies. The results are explained by an orientational relaxation of the solvent cage subsequent to ACMA photoexcitation.     

Diurnal movements of chloroplasts in Halophila stipulacea and their effect on PAM fluorometric measurements of photosynthetic rates

Since diurnal chloroplast movements in Halophila stipulacea were described by Drew in 1979, this phenomenon has not been studied further for seagrasses. In addition to an apparent photoprotective role, such movements may affect the measurements of photosynthetic rates based on pulse amplitude modulated (PAM) fluorometry. This is because calculations of electron transport rates (ETR) are directly affected by the light absorption of the leaves (or the so-called absorption factor, AF), the latter of which changes with the movements of the chloroplasts. In this work, we therefore determined chloroplast clumping and dispersal, and measured AFs, chlorophyll contents and PAM fluorescence diurnally for H. stipulacea grown under two irradiance regimes. Diurnal chloroplast clumping occurred in high-light grown (HL) plants (∼450 μmol photons m⁻² s⁻¹ during midday), which was accompanied by a decrease in AF values (from 0.56 in the early morning to 0.34 at midday) but not in the chlorophyll content. Also, non-photochemical quenching (measured as NPQ) increased during the day in these plants. No such chloroplast movements and, thus, no diurnal changes in AF values (0.60 ± 0.04 throughout the day), and no changes in NPQ, were found in low-light grown (LL) plants (∼150 μmol photons m⁻² s⁻¹ during midday). As a consequence of the chloroplast clumping in HL plants, and its effect on AF values, maximal ETRs did not differ significantly between HL and LL plants. This finding thus shows the importance of taking into account changing AF values along the day when calculating ETRs of H. stipulacea, and other seagrasses potentially featuring diurnally changing AFs, under high-irradiance conditions.