Light-induced Fluorescence Changes Research Articles

Complex Photophysical Properties of K114 Make for a Versatile Fluorescent Probe for Amyloid Detection.

Protein aggregation is a hallmark of Alzheimer's disease (AD) and many other neurodegenerative disorders. Small organic fluorophores such as Congo Red preferentially bind to cross-β-sheet-rich deposits and have been used to label amyloid plaques and tau tangles in histological samples. However, distinguishing between different conformations of protein aggregates is not trivial. Using silkworm and spider silks (prototypical amyloids) and transgenic AD mouse (5XFAD) and human AD brain samples, we report how spectral confocal microscopy allowed for improved detection and differentiation of protein aggregates based on the unexpected photophysical behavior of the amyloid-specific dye K114. The pH and excitation power had pronounced effects on the emission spectrum and intensity of amyloid-bound K114 fluorescence. When bound to β-sheet-rich assemblies, the emission spectrum of K114 was governed by the local pH of the binding pockets much more than by the pH of the mounting medium, likely due to ionization of titratable phenols. Unexpectedly, exposure to high excitation power caused a permanent increase in fluorescence intensity and a spectral blue-shift. These light-induced fluorescence changes were dependent in a complex manner on laser power, exposure time, pH, and amyloid type examined. The above-mentioned phenomena were observed in silk fibers and Alzheimer brain sections from mouse and human, indicating that this may be a general characteristic of K114 when bound to tightly aggregated macromolecules. Potential mechanisms are discussed, likely involving photoinduced electron transfer. Our findings illustrate how the complex photophysical behavior of amyloid-bound K114 can be exploited for improved detection and differentiation of protein aggregates.

Tuning Light-Driven Motion and Bending in Macroscale-Flexible Molecular Crystals Based on a Cocrystal Approach.

Flexible molecular crystals with stimuli-responsive properties are highly desirable; however, uncovering them is still a challenging goal. Herein, we report a cocrystal approach to obtain elastic molecular crystals that exhibit light-induced fluorescence changes and dynamic mechanical responses at the macroscale level. Cocrystals of naphthylvinylpyridine and tetrafluoroterephthalic acid were fabricated in different stoichiometry ratios (2:1 and 1:1), which present different shapes [two-dimensional (2D) and one-dimensional (1D) morphologies], photoemission, and mechanical properties (rigidity and flexibility). Moreover, obviously different photomechanical energy conversions (light-driven cracking/popping and bending/motion) occur for the 2D and 1D cocrystals, respectively. Nuclear magnetic resonance (NMR) spectra show the occurrence of photoinduced [2 + 2] cycloaddition in both cocrystals, which is the primary mechanism for their photoactuating behaviors. Crystal structure analysis and theoretical calculation further reveal that protonation and the hydrogen-bonding network play important roles in light-stimulus-bendable 1D cocrystal. Thus, the transformation from rigidity to flexibility based on cocrystallization with different stoichiometry may offer an effective means to tune the dynamic light-driven responses for smart crystalline materials.

Teaching about photosynthesis with simple equipment: analysis of light-induced changes in fluorescence and reflectance of plant leaves

Solar energy absorbed by plants results in either reflection or absorption. The latter results in photosynthesis, fluorescence, or heat. Measurements of fluorescence changes have been used for monitoring processes associated with photosynthesis. A simple method to follow changes in leaf fluorescence and leaf reflectance associated with nonphotochemical quenching and light acclimation of leaves is described. The main equipment needed consists of a green-light emitting laser pointer, a digital camera, and a personal computer equipped with the camera acquisition software and the programs ImageJ and Excel. Otherwise, only commonly available cheap materials are required.

Sensitivity of CFP/YFP and GFP/mCherry pairs to donor photobleaching on FRET determination by fluorescence lifetime imaging microscopy in living cells

Fluorescent protein-based FRET is a powerful method for visualizing protein-protein interactions and biochemical reactions in living cells. It can be difficult, however, to avoid photobleaching when observing fluorescent cells under the microscope, especially those expressing CFP. We compared the sensitivity of two protein-based FRET pairs to light-induced fluorescence changes in the donor, on FRET determination by fluorescence lifetime imaging microscopy (FLIM). Thanks to the very low excitation light levels of the time- and space-correlated single photon counting (TSCSPC) method, FLIM acquisitions were achieved without donor photobleaching. Here, we show that photobleaching of CFP by a mercury lamp under the microscope induced a decrease in the mean fluorescence lifetime, which interfered with FRET determination between CFP and YFP. Importantly, the range of light-induced variation of the mean fluorescence lifetime of CFP was not proportional to the decrease in the steady state fluorescence intensity and varied from cell to cell. The choice of the CFP/YFP pair therefore requires that the cells be observed and analyzed at very low light levels during the whole FRET experiment. In contrast, the GFP/mCherry pair provided an accurate FRET measurement by FLIM, even if some GFP photobleaching took place. We thus demonstrate that CFP can be an unreliable donor for FRET determination in living cells, due to its photosensitivity properties. We demonstrate that the GFP/mCherry pair is better suited for FRET measurement by FLIM in living cells than the CFP/YFP pair.

Light-induced fluorescence changes in Phycomyces: evidence for blue light-receptor associated flavo-semiquinones.

Light-induced fluorescence changes (LIFCs) were detected in sporangiophores of the blue-light-sensitive fungus Phycomyces blakesleeanus (Burgeff). The LIFCs can be utilized as a spectrophotometric assay for blue-light photoreceptors and for the in vivo characterization of their photochemical primary reactions. Blue-light irradiation of sporangiophores elicited a transient decrease and subsequent regeneration of flavin-like fluorescence emission at 525 nm. The signals recovered in darkness in about 120 min. In contrast to blue light, near-UV (370 nm) caused an increase in the fluorescence emission at 525 nm. Because the LIFCs were altered in a light-insensitive madC mutant with a defective photoreceptor, the fluorescence changes must be associated with early photochemical events of the transduction chain. Action spectra for the fluorescence changes at 525 nm showed major peaks near 470 and 600 nm. Double-pulse experiments involving two consecutive pulses of either blue and near-UV, blue and red, or near-UV and red showed that the responses depended on the sequence in which the different wavelengths were applied. The results indicate a blue-light receptor with intermediates in the near-UV, blue and red spectral regions. We explain the results in the framework of a general model, in which the three redox states of the flavin photoreceptor, the oxidized flavin (Fl), the flavo-semiquinone (FlH*), and the flavo-hydroquinone (FlH2) are each acting as chromophores with their own characteristic photochemical primary reactions. These consist of the photoreduction of the oxidized flavin generating semiquinone, the photoreduction of the semiquinone generating hydroquinone, and the photooxidation of the flavo-hydroquinone regenerating the pool of oxidized flavins. The proposed mechanism represents a photocycle in which two antagonistic photoreceptor forms, Fl and FlH2, determine the pool size of the biological effector molecule, the flavo-semiquinone. The redox changes that are associated with the photocycle are maintained by redox partners, pterins, that function in the near-UV as secondary chromophores.

Changes in light-induced fluorescence of cervical collagen in guinea pigs during gestation and after sodium nitroprusside treatment.

Light-induced fluorescence (LIF) of collagen was used to investigate in vivo changes in cervical collagen in guinea pigs during gestation and following sodium nitroprusside treatment. Natural fluorescence of collagen is due to collagen cross-linking molecules that connect single collagen fibers and therefore provide rigidity of the cervical stroma. LIF of cervical collagen was measured from the surface of the exocervix in anesthetized nonpregnant and timed pregnant guinea pigs at different times of gestation with an instrument designed in our lab (Collascope). Measurements were also performed in guinea pigs at midgestation before and 8 hours after intracervical treatment with sodium nitroprusside. Collagen fluorescence decreased significantly as pregnancy progressed, reached lowest values at delivery, and increased gradually postpartum. Treatment with sodium nitroprusside, but not with the vehicle, caused a significant decrease in LIF (p = 0.007). We conclude, that LIF changes in the cervix reflect the gradual cervical softening (ripening) during pregnancy and the return to the rigid state of the cervix postpartum. Cervical softening during pregnancy, and after sodium nitroprusside treatment, is associated with a decrease in collagen cross-links. Measurements of LIF can be used to investigate cervical softening in vivo.

Bacterial rhodopsins monitored with fluorescent dyes in vesicles and in vivo.

Three retinal-containing pigments have been detected in Halobacterium halobium membranes: bacteriorhodopsin (bR), halorhodopsin (hR), and slow-cycling rhodopsin (sR). The first two hyperpolarize the cell membrane by electrogenic transport of H+ and Cl-, respectively. The third pigment, sR, may be a photosensory receptor since mutants lacking bR and hR retain their retinal-dependent phototaxis responses. We monitored light-induced changes in fluorescence of several voltage-sensitive dyes in cells and membrane vesicles. Red light-induced potential changes generated by bR and hR were similar to signals described previously. Signals generated by hR could be identified using four criteria: wavelength dependence, Cl- dependence, shunting by valinomycin and K+, and the absence of these signals in hR-deficient mutants. The absence (detection limit approximately 0.5 mV) of hyperpolarization signals in bR-hR-sR+ vesicles and cells shows that sR photochemical reactions are nonelectrogenic. Two signals independent of bR and hR were measured: blue light caused a decrease and red light an increase in dye fluorescence. Both signals appear to derive from sR on the basis of their retinal-dependence and action spectra. In a retinal-deficient mutant strain (Flx3R), both sR signals appeared after addition of all-trans retinal. In this strain retinal also restores phototaxis sensitivity within the same time scale. The retinal concentration dependence for all four parameters monitored--the attractant (red) and repellent (blue) phototaxis, and the red light and blue light-induced fluorescence signals--is the same. This correlation is consistent with the hypothesis that both attractant and repellent responses are mediated by sR, as suggested by Bogomolni and Spudich (Proc. Natl. Acad. Sci. USA. 79:6250-6254 (1982)).

Light-induced proton translocation through thylakoid and cytoplasmic membranes of Plectonema boryanum

Light-induced fluorescence changes of 9-aminoacridine, an indicator of proton gradient in energy-transducing membranes, were studied in Plectonema boryanum and other cyanobacteria. The fluorescence changes observed in cell suspensions resulted from a superposition of fluorescence quenching and enhancement as the analysis of the kinetic data shows. Both components of the fluorescence changes are abolished by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) and m-chlorocarbonylcyanide phenylhydrazone. The inhibitory effect of DCMU is removed by 2,3,5,6- or N,N,N′,N′-tetramethyl-p-phenylenediamine. The fluorescence quenching sensitive to substrates and uncouplers of the photophosphorylation is only observed in membrane vesicles obtained by osmotic shock of P. boryanum spheroplasts. Presumably, light-induced quenching of the dye fluorescence in the cells of cyanobacteria is due to the proton transport from the cytoplasm in the inner space of thylakoids while fluorescence enhancement is due to the proton efflux from the cytoplasm into the incubation medium.

The permeability of Rhodospirillum rubrum chromatophores to thiocyanate and perchlorate as detected by light-induced fluorochrome fluorescence changes and by photophosphorylation

The addition of NaSCN and NaClO 4 to Rhodospirillum rubrum chromatophores increased the quenching of atebrin fluorescence and decreased the enhancement of 8-anilinonaphthalene-1-sulfonic acid fluorescence without affecting ATP formation. In the presence of NH 4SCN and NH 4ClO 4 the photophosphorylation as well as the light-induced fluorescence changes of both fluorochromes were inhibited. The comparison of these results with the effects of Cl - and NO - 3 leads to the conclusion that the chromatophore membrane is much more permeable to SCN - and ClO - 4 than to Cl - or NO - 3.

A STUDY OF CHLOROPHYLL FLUORESCENCE IN VIVO BY A FLOW METHOD

Abstract— A flow method has been devised for the study of fluorescence transients during the induction period of photosynthetic systems. The principle of transformation of time‐transients into space‐transients is quantitatively worked out and the characteristics and mode of operation of the apparatus is described and illustrated with experimental results.With this method spectroscopic and kinetic investigation of in vivo chlorophyll fluorescence have been undertaken. The fluorescence emission can be split into two componmts—a variable part and a constant part—with different spectral distributions. The excitation spectrum of the light induced fluorescence changes shows that the variable part is sensitized by system 11, whereas the constant part mainly sensitized by system II has a small component originating from system I. The light‐induced fluorescence change decays in darkness with half‐time of about 10‐2s; this decay is highly sensitive to the action of inhibitors of oxygen evolution.The flow method can be extended to measurements of oxygen, luminescence, absorption differences. It permits using high‐intensity analytic beams with no actinic action; it is well suited for the integration of low level signals.

LIGHT-INDUCED CHANGES IN ABSORBANCY AND FLUORESCENCE OF CHLOROPHYLLOUS PIGMENTS ASSOCIATED WITH THE PIGMENT SYSTEMS 1 AND 2 IN BLUE-GREEN ALGAE.

1. 1. The kinetics and the difference spectra of light-induced changes in absorbancy and of changes in the near infrared fluorescence were measured in the blue-green algae Schizothrix calcicola and Anacystis nidulans . 2. 2. The action spectrum of the photooxidation of P 700 in Schizothrix was measured in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea at 2°. 3. 3. The following quantum requirements q ( λ ), for actinic wavelength λ, were estimated for P 700 and cytochrome oxidation and for P 700 reduction. Schizothrix: P 700 oxidation, q (680 m μ ) varies between 2.0 and 2.6, and q (430 m μ ) between 5.6 and 6.4; cytochrome oxidation, q (680 m μ ) = 3.0−3.5; P 700 reduction, q (680 m μ ) = 3.0. Anacystis: cytochrome oxidation, q (680 m μ ) = 2.8. The requirements per quantum absorbed by the photochemical system responsible for the reaction will be lower. The results are not inconsistent with the assumption that the true quantum requirement for the photochemical transport of 1 electron is 1 for each of the two pigment systems. 4. 4. A new “differential” flow attachment for a fluorescence apparatus was constructed by means of which small changes in fluorescence could be measured with increased precision, because the noise due to actinic light was eliminated. 5. 5. In addition to the earlier observed light-induced changes in the fluorescence of chlorophyll a 2 , a relatively small light-induced increase in red fluorescence, excited by blue light, was observed in Schizothrix calcicola, Porphyridium cruentum and Porphyra sp. It was activated by Pigment System 1, was not reversed by light of System 2, and was not associated with redox changes of P 700. The difference spectrum of this fluorescence was different from that of chlorophyll a 2 , in having a higher hump at 720 mμ.