A comparison of neural networks and partial least squares for deconvoluting fluorescence spectra

This article compares backpropagation neural networks (BNN) with partial least squares (PLS) techniques in terms of their ability to deconvolute fluorescence spectra. Both actual experimental and simulated spectral data are studied for 2 binary systems. These systems consist of mixtures of tryptophan and tyrosine, and NADH and tryptophan over a total concentration range of 10(-7) to 10(-4) M. It is shown that BNN is superior to PLS for both systems.     

A naphthyl analog of the aminostyryl pyridinium class of potentiometric membrane dyes shows consistent sensitivity in a variety of tissue,cell, and model membrane preparations

The fast potentiometric indicator di-4-ANEPPS is examined in four different preparations: lipid vesicles, red blood cells, squid giant axon, and guinea pig heart. The dye gives consistent potentiometric responses in each of these systems, although some of the detailed behavior varies. In lipid vesicles, the dye displays an increase in fluorescence combined with a red shift of the excitation spectrum upon hyperpolarization. Similar behavior is found in red cells where a dual wavelength radiometric measurement is also demonstrated. The signal-to-noise ratio of the potentiometric fluorescence response is among the best ever recorded on the voltage-clamped squid axon. The dye is shown to be a faithful and persistent monitor of cardiac action potentials with no appreciable loss of signal or deterioration of cardiac activity for periods as long as 2 hr with intermittent illumination every 10 min. These results, together with previously published applications of the dye to a spherical lipid bilayer model and to cells in culture, demonstrate the versatility of di-4-ANEPPS as a fast indicator of membrane potential.     

Measurements of pH in the apoplast of sunflower leaves by means of fluorescence

A method was elaborated by which the pH in leaf apoplast can be measured. The technique is based on the pH dependent fluorescence of 5-carboxyfluorescein (5-CF) or fluorescein isothiocyanate (FITC). The fluorescein isothiocyanate is coupled with a macromolecular dextran molecule (FITC-dextran). For eliminating the effect of the absolute dye concentration the dual excitation technique was applied. It was shown that the ratio of fluorescence excited by light of 491 nm and 463 nm was virtually independent of the concentration of 5-CF and that this fluorescence ratio was related to the pH. The plasmalemma is practically impermeable to FITC-dextran and in the test we carried out over a period of 6 h not the slightest indication was found that it may penetrate the plasma membrane. For 5-CF this cannot be ruled out completely. It is possible that at pH values below 4.5 it may penetrate biological membranes at low rates.
Experiments with leaves of sunflower ( Helianthus animus cv. Erika) perfused with 5-carboxyfluorescein and supplied with different nitrogen forms showed that NH⁺₄ application resulted in a decrease and NO⁺₃ application in an increase of the leaf apoplast pH. Leaf spraying with fasicoccin was followed by a pH decrease, while leaf spraying with the protonophores p -trifluoromethoxy carbonytcyanide phenylhydra-zon (FCCP) or nigericin resulted in neutral apoplastic pH. These results provide evidence that the method is well suited for measuring the response of the leaf apoplast pH to changing physiological conditions.     

Recovery of photosynthesis and phtosystem II fluorescence in Chlamydomonas reinhardtii after exposure to three levels of high light

Recovery from 60 min of photoinhibitory treatment at photosynthetic photon flux densities of 500, 1400 and 2200 μMmol m^?2 s^? was followed in cells of the green alga Chlamydomonas reinhardtii grown at 125 μMmol m^?2 s^?1. These light treatments represent photoregulation, moderate photoinhibition and strong photoinhibition, respectively. Treatment in photoregulatory light resulted in an increased maximal rate of oxygen evolution (P_max) and an increased quantum yield (Φ), but a 15% decrease in F_v/F_M. Treatment at moderately photoinhibitory light resulted in a 30% decrease in F_v/F_M and an approximately equal decrease in Φ. Recovery in dim light restored F_v/F_M within 15 and 45 min after high light treatment at 500 and 1400 μMmol m^?2 s^?1, respectively. Convexity (Θ), a measure of the extent of co-limitation between PS II turnover and whole-chain electron transport, and Φ approached, but did not reach the control level during recovery after exposure to 1400 μMmol m^?2 s^?1, whereas P_max increased above the control. Treatment at 2200 μMmol m^?2 s^?1 resulted in a strong reduction of the modeled parameters Φ, Θ and P_max. Subsequent recovery was initially rapid but the rate decreased, and a complete recovery was not reached within 120 min. Based on the results, it is hypothesized that exposure to high light results in two phenomena. The first, expressed at all three light intensities, involves redistribution within the different aspects of PS II heterogeneity rather than a photoinhibitory destruction of PS II reaction centers. The second, most strongly expressed at 2200 μmol m^?2 s^?1, is a physical damage to PS II shown as an almost total loss of PS II_α and PS II Q_B-reducing centers. Thus recovery displayed two phase, the first was rapid and the only visible phase in algae exposed to 500 and 1400 μmol m^?2 s^?1. The second phase was slow and visible only in the later part of recovery in cells exposed to 2200 μmol m^?2 s^?1.     

Analysis of dark-relaxation kinetics of variable fluorescence in intact leaves

The dark-relaxation kinetics of variable fluorescence, F_v, in intact green leaves of Pisum stativum L. and Dolichos lablab L. were analyzed using modulated fluorometers. Fast (t_1/2 = 1 s) and slow (t_1/2 = 7–8 s) phases in f_v dark-decay kinetics were observed; the rate and the relative contribution of each phase in total relaxation depended upon the fluence rate of the actinic light and the point in the induction curve at which the actinic light was switched off. The rate of the slow phase was accelerated markedly by illumination with far-red light; the slow phase was abolished by methyl viologen. The halftime of the fast phase of F_v dark decay decreased from 250 ms in dark-adapted leaves to 12–15 ms upon adaptation to red light which is absorbed by PSII. The analysis of the effect of far-red light, which is absorbed mainly by PSI, on F_v dark decay indicates that the slow phase develops when a fraction of Q_A ^– (the primary stable electron acceptor of PSII) cannot transfer electrons to PSI because of limitation on the availability of P700⁺ (the primary electron donor of PSI). After prolonged illumination of dark-adapted leaves in red (PSII-absorbed) light, a transient. F_v rise appears which is prevented by far-red (PSI-absorbed) light. This transient f_v rise reflects the accumulation of Q_A ^– in the dark. The observation of this transient F_v rise even in the presence of the uncoupler carbonylcyanide m-chlorophenyl hydrazone (CCCP) indicates that a mechanism other than ATP-driven back-transfer of electrons to QA may be responsible for the phenomenon. It is suggested that the fast phase in F_v dark-decay kinetics represents the reoxidation of Q_A ^– by the electron-transport chain to PSI, whereas the slow phase is likely to be related to the interaction of Q_A ^– with the donor side of PSII.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - F_O initial fluorescence level - F_v variable fluorescence - P700 primary electron donor of PSI - PSI, II photosystem I, II - Q_A (Q_A ^–) Q_B (Q_B ^–) primary and secondary stable electron acceptor of PSII in oxidized (reduced) state Supported by grant B6.1/88 DST, Govt. of India.     

Relationship of actin organization to growth in the two forms of the dimorphic yeastCandida tropicalis

Summary Candida tropicalis is a dimorphic yeast capable of growing both as a budding yeast and as filamentous hyphae depending upon the source of the carbon used in the culture medium. The organization of F-actin during growth of the yeast form (Y-form) and the hyphal form (H-form) was visualized by rhodamine-conjugated phalloidin by using a conventional fluorescence microscope as well as a laser scanning confocal fluorescence microscope. In single cells without a bud or non-growing hyphae, actin dots were evenly distributed throughout the cytoplasm. Before the growth of the bud or hypha, the actin dots were concentrated at one site. During bud growth, actin dots were located solely in the bud. They filled the small bud and then filled the apical two-thirds of the cytoplasm of the middlesized bud. During growth of the large bud, actin dots which had filled the apical half of the cytoplasm gradually moved to the tip of the bud. In the formation of the septum, actin dots were arranged in two lines at the conjunction of the bud and the mother cell. During hyphal growth, the majority of actin dots were concentrated at the hyphal apex. A line of clustered spots or a band of actin was observed only at the site where the formation of a new septum was imminent. This spatial and temporal organization of actin in both categories of cells was demonstrated to be closely related to the growth and local deposition of new cell wall material by monitoring the mode of growth with Calcofluor staining. Treatment of both forms of cells with cytochalasin A (CA) confirmed the close relationship between actin and new cell wall deposition. CA treatment revealed lightly stained unlocalized actin which was associated with abnormal cell wall deposition as well as changes in morphology. These results suggest that actin is required for proper growth and proper deposition of cell wall material and also for maintaining the morphology of both forms of cells.Abbrevations FM fluorescence microscopy - EM electron microscopy - rh rhodamine - CA cytochalasin A - CD cytochalasin D - PBS phosphate-buffered saline - DMSO dimethylsulfoxide - GA glutaraldehyde     

Long-lived primary radical pair state detected by time-resolved fluorescence and absorption spectroscopy in an isolated Photosystem two core

A Photosystem two (PS II) core preparation containing the chlorophyll a binding proteins CP 47, CP 43, D1 and D2, and the non-chlorophyll binding cytochrome-b559 and 33 kDA polypeptides, has been isolated from PS II-enriched membranes of peas using the non-ionic detergent heptylthioglucopyranoside and elevated ionic strengths. The primary radical pair state, P680⁺Pheo^-, was studied by time-resolved absorption and fluorescence spectroscopy, under conditions where quinone reduction and water-splitting activities were inhibited. Charge recombination of the primary radical pair in PS II cores was found to have lifetimes of 17.5 ns measured by fluorescence and 21 ns measured by transient decay kinetics under anaerobic conditions. Transient absorption spectroscopy demonstrated that the activity of the particles, based on primary radical pair formation, was in excess of 70% (depending on the choice of kinetic model), while time-resolved fluorescence spectroscopy indicated that the particles were 91% active. These estimates of activity were further supported by steady-state measurements which quantified the amount of photoreducible pheophytin. It is concluded that the PS II core preparation we have isolated is ideal for studying primary radical pair formation and recombination as demonstrated by the correlation of our absorption and fluorescence transient data, which is the first of its kind to be reported in the literature for isolated PS II core complexes from higher plants.Abbreviations CP 43 and CP 47 chlorophyll binding proteins of PS II having apparent molecular weights on SDS-PAGE of 43 kDa and 47 kDa, respectively - D1 and D2 polypeptides PS II reaction centre polypeptides encoded by the psbA and psbD genes, respectively - HPLC high performance liquid chromatography - PS II Photosystem two - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - P680 primary electron donor of PS II - Pheo phenophytin a - SPC single photon counting - PBQ phenyl-p-benzoquinone - DPC 1,5-diphenylcarbazide AFRC Photosynthesis Research Group, Department of Biochemistry     

Chloroplast movements in leaves: Influence on chlorophyll fluorescence and measurements of light-induced absorbance changes related to ΔpH and zeaxanthin formation

Light-induced chloroplast movements were found to cause changes in chlorophyll fluorescence emission, closely matching those in leaf absorptance, both in terms of the kinetics and the maximum extent of the changes observed in different species. The results demonstrate that chloroplast movements can have a significant effect on the efficiency of light utilization in photosynthesis. They further show that chloroplast movements need to be taken into account in measurements of fluorescence quenching and especially in measurements of light-induced optical changes used to monitor zeaxanthin formation and pH associated light scattering in leaves. Means of minimizing and of adjusting for the influences of chloroplast movements in such measurements are discussed.Abbreviations F fluorescence emission - PFD photon flux density - R reflectance - T transmittance - absorptance C.I.W.-D.P.B. Publication No. 1116.     

Analysis of fluorescence induction in thylakoids with the method of moments reveals two different active Photosystem II centres

There is presently a debate concerning the number of phases in fluorescence induction and on the identification of the several possible heterogeneities in PS II centres. However, the usual methods of analysis present numerical problems, including a lack of robustness (robustness being defined as the ability to give the correct answer in the presence of distortions or artefacts). We present here the adaptation of the method of moments, which was developed for robustness, to the analysis of fluorescence induction. We were thus able to identify three phases in the fluorescence induction in the presence of DCMU. The slowest phase was attributed to the centres inactive in plastoquinone reduction by using duroquinone as electron acceptor. In order to compare fluorescence with and without DCMU, we introduced the rate of photochemistry, defined as the product of the area times the rate constant of an exponential. This quantity is invariant for a given centre no matter what the size of the electron acceptor pool is. The two fastest phases in the presence of DCMU were attributed to active centres because their rate of photochemistry was the same as that of the plastoquinone-reducing phases in the absence of DCMU. Because their reduction of plastoquinone showed different kinetics, these two types of active centres were either separated by more than 250 nm or were associated with discrete plastoquinone pools having restricted diffusion domains.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMBQ 2,5-dimethyl-p-benzoquinone - MOPS 3-[N-Morpholino]propanesulphonic acid - PpBQ Phenyl-p-benzoquinone     

Sulfite inhibition of photochemical activity of intact pea leaves

Sulfite treatment of pea leaf disks in light caused a significant decrease in the relative quantum yield of photosynthetic oxygen evolution and energy storage (ES) as measured by photoacoustic (PA) spectroscopy. The inhibition was concentration dependent and was less in darkness than in light, indicating light-dependent inhibitory site(s) on the photosynthetic electron transport chain. Further, in darksulfite-treated leaves, the energy storage was more affected than the relative quantum yield of oxygen evolution, suggesting that photophosphorylation and/or cyclic electron transport around PS I are sites of sulfite action in darkness. The R_fd values, the ratio of fluorescence decrease (fd) to the steady-state fluorescence (fs), decreased significantly in leaves treated with sulfite in light but were not affected in dark-treated ones, confirming the photoacoustic observations. Similarly, the ratio of variable fluorescence (F_v) to maximum fluorescence (F_m), a measure of PS II photochemical efficiency, was affected by sulfite treatment in light and not changed by treatment in darkness. An attempt was made to explain the mechanism of sulfite action on photosynthetic electron transport in light and in darkness.Abbreviations A_PT amplitude of photothermal signal - A_ox amplitude of oxygen signal - ES energy storage - fd fluorescence decrease - fs steady-state fluorescence - F_m maximum fluorescence - F_v variable fluorescence - PA photoacoustic(s)