QUANTITATIVE FRET MEASUREMENT BASED ON CONFOCAL MICROSCOPY IMAGING AND PARTIAL ACCEPTOR PHOTOBLEACHING

Fluorescence resonance energy transfer(FRET)technology had been widely used to study protein
protein interactions in living cells.In this study,we developed a ROI-PbFRET method to real-time quantitate the FRET efficiency of FRET construct in living cells by combining the region of interest(ROI)function of confocal microscope and partial acceptor photobleaching.We validated the ROI-PbFRET method using GFPs-based FRET constructs including 18AA and SCAT3,and used it to quantitatively monitor the dynamics of caspase-3 activation in single live cells stably expressing SCAT3 during staurosporine(STS)-induced apoptosis.Our results for thefirst demonstrate that ROI-PbFRET method is a powerful potential tool for detecting the dynamics of molecular interactions in live cells.

Determination of diffusion coefficient by image-based fluorescence recovery after photobleaching and single particle tracking system implemented in a single platform

Fluorescence recovery after photobleaching(FRAP)and single particle tracking(SPT)techni-ques determine the diffusion coefficient from average diffusive motion of high-concentration molecules and from trajectories of low-concentration single molecules,respectively.Lateral dif-fusion coefficients measured by FRAP and SPT techniques for the same biomolecule on cell membrane have exhibited inconsistent values across laboratories and platforms with larger dif-fusion coefficient determined by FRAP,but the sources of the inconsistency have not been investigated thoroughly.Here,we designed an image-based FRAP-SPT system and made a direct comparison between FRAP and SPT for diffusion coefficient of submicron particles with known theoretical values derived from Stokes-Einstein equation in aqueous solution.The combined iFRAP-SPT technique allowed us to measure the diffusion coefficient of the same fluorescent particle by utilizing both techniques in a single platform and to scrutinize inherent errors and artifacts of FRAP.Our results reveal that diffusion coefficient overestimated by FRAP is caused by inaccurate estimation of the bleaching spot size and can be corrected by simple image analysis.Our iFRAP-SPT technique can be potentially used for not only cellular membrane dynamics but also for quantitative analysis of the spatiotemporal distribution of the solutes in small scale analytical devices.

Photobleaching characteristics of traditional Japanese paper in a museum environment

Photobleaching of aged traditional Japanese paper that has been thermally yellowed during storage for 200 years was examined from the standpoint of accumulated light radiation dosage in a museum environment. The light intensity was evaluated using a blue wool reference of the Japan Industrial Standards (JIS) as a dosimeter. The wavelength sensitivity of the photobleaching was compiled under monochromatic light radiation. Color changes in the specimens were measured in tristimuli values in color. by using a color analyzer. The aged pieces of paper were monitored continuously as they were photobleached under three different lighting conditions in a museum environment for 8000 h. The combination of the yellowness index changes of the aged pieces of paper and the color changes of a blue wool reference was interpreted as follows. Photobleaching was governed by accumulated light intensities and was independant upon daily lighting conditions. The wavelength sensitivity of the photobleaching of aged paper showed that the maximum effect occurred at 420 nm in the visible light range. The blue wool reference was confirmed to perform well as a dosimeter.

Protoporphyrin IX photobleaching of subcellular distributed sites of leukemic HL60 cells based on ALA-PDT <i>in vitro</i>

The leukaemia cells HL60, incubated in 10 mM/ml ALA (5-aminolevulinic) for 4 hours, were carried an experimental research with fluorescent probes in photodynamic therapy (PDT) based on ALA by using PDT reaction room (The average fluence rate of the 412 nm source was 5 mW/cm2). Cells viability were determined using a Cell Counting Kit-8 (CCK-8) assay, and PpIX Photobleaching of subcellular distributed sites of HL60 cells in vitro were investigated by fluorescence spectra acquired during treatment. The results showed that the fluorescence intensity of mitochondria, lysosomes, endoplasmic reticulum had decreased by 81.5%, 52.3% and 21.0%, respectively, compared with their initial values after a 45-minute light treatment. The rate of PpIX photobleaching in mitochondria was significantly higher than others. Addi-tionally, the change of the activity of HL60 cells was basically characterized by the change fluorescence intensity in mitochondria, which suggest that mitochondria is one of main therapeutic targets of photodynamic therapy.

PhotobleaChing Influence in Different Phases for Coumarin 307 and Acriflavine Laser Dyes

Under high-excitation irradiance conditions to induce fluorescence, the dependence of photobleaching of Coumarin 307 （C307） and acriflavine （ACF） laser dyes in liquid and solid phases have been studied. A cw LD laser source of 1 mW and 407 nm wavelength was used as an exciting source. For one hour exposure time, it was found that the solid dye samples suffer photobleaching more than the liquid dye samples. This is because in liquid solutions the dye molecules can circulate during the irradiation, while the photobleaching is a serious problem when the dye is incorporated into solid matrix and cannot circulate.

Biocompatible carbon dots with low-saturation-intensity and highphotobleaching-resistance for STED nanoscopy imaging of the nucleolus and tunneling nanotubes in living cells

Many kinds of nano particles and organic dyes as fluorescent probes have been used in the stimulated emission depletion(STED)nanoscopy.Due to high toxicity,photobleaching and non-water solubility,these fluorescent probes are hard to apply in living cell imaging.Here,we reporta new fluorescence carbon dots(FNCDs)with high photoluminescence quantum yield(56%),low toxicity,anti-photobleaching and goodwater-solubility that suitable for live-cell imaging can be obtained by doping fluorine element.Moreover,the FNCDs can stain the nucleolusand tunneling nanotubes(TNTs)in the living cell.More importantly,for STED nanoscopy imaging,the FNCDs effectively depleted backgroundsignals and improved imaging resolution.Furthermore,the lateral resolution of single FNCDs size under the STED nanoscopy is up to 22.1 nm for FNCDs deposited on a glass slide was obtained.And because of their good water dispersibility,the higher resolution of single FNCDs sizein the nucleolus of a living cell can be up to 19.7 nm.After the image optimizati on steps,the fine fluoresce nee images of TNTs diameter with ca.75 nm resolution is obtained living cell,yielding a threefold enhancement compared with that in confocal imaging.Additionally,the FNCDs show excellent photobleaching resistance after 1,000 scan cycles in the STED model.All results show that FNCDs have significant potentialfor application in STED nanoscopy.

Can DyeCycling break the photobleaching limit in single-molecule FRET?

Biomolecular systems,such as proteins,crucially rely on dynamic processes at the nanoscale.Detecting biomolecular nanodynamics is therefore key to obtaining a mechanistic understanding of the energies and molecular driving forces that controlbiomolecular systems.Single-molecule fluorescence resonance energy transfer(smFRET)is a powerful technique to observe inreal-time how a single biomolecule proceeds through its functional cycle involving a sequence of distinct structural states.Currently,this technique is fundamentally limited by irreversible photobleaching,causing the untimely end of the experiment andthus,a narrow temporal bandwidth of≤3 orders of magnitude.Here,we introduce“DyeCycling”,a measurement scheme withwhich we aim to break the photobleaching limit in smFRET.We introduce the concept of spontaneous dye replacement bysimulations,and as an experimental proof-of-concept,we demonstrate the intermittent observation of a single biomolecule forone hour with a time resolution of milliseconds.Theoretically,DyeCycling can provide>100-fold more information per singlemolecule than conventional smFRET.We discuss the experimental implementation of DyeCycling,its current and fundamentallimitations,and specific biological use cases.Given its general simplicity and versatility,DyeCycling has the potential torevolutionize the field of time-resolved smFRET,where it may serve to unravel a wealth of biomolecular dynamics by bridgingfrom milliseconds to the hour range.

Fluorescence recovery after photobleaching:analyses of cyanobacterial phycobilisomes reveal intrinsic fluorescence recovery

Fluorescence recovery after photobleaching(FRAP)has been used to study the dynamics of the cyanobacterial photosynthesis apparatus since 1997.Fluorescence recovery of cyanobacteria during FRAP was conventionally interpreted as a result of phycobilisome(PBS)diffusion on the surface of the thylakoid membrane.The mechanism of state transition in cyanobacteria has been widely attributed to PBS diffusion.However,in red algae,another PBS-containing group,the intrinsic photoprocess was found to contribute greatly to the fluorescence recovery of PBS,which raises questions concerning the role of FRAP in red algal PBS.Therefore,it is important to re-evaluate the nature of PBS fluorescence recovery in cyanobacteria.In the present study,four cyanobacterial strains with different phenotypes and PBS compositions were used to investigate their FRAP characteristics.Fluorescence recovery of PBS was observed in wholly photobleached cells in all four cyanobacterial strains,in which the contribution of PBS diffusion to the fluorescence recovery was not possible.Moreover,the fluorescence recovered in isolated PBSs and PBS-thylakoid membranes after photobleaching further demonstrated the intrinsic photoprocess nature of fluorescence recovery.These findings suggest that the intrinsic photoprocess contributed to the fluorescence recovery following photobleaching when measured by the FRAP method.

Effects of H pylori infection on gap-junctional intercellular communication and proliferation of gastric epithelial cells in vitro

AIM： To explore the effects of H pylori infection on gap-junctional intercellular communication （GJIC） and proliferation of gastric epithelial cells in vitro. METHODS： A human gastric epithelial cell line （SGC- 7901） cultured on coverslips was exposed overnight to intact H pylori （CagA^＋ or CagA^- strains） and sonicated extracts, respectively. GJIC between the cells was detected by fluorescence redistribution after photobleaching （FRAP） technique. Proliferation of SGC cells was determined by methylthiazolyl tetrazolium （MTT） assay. RESULTS： When compared with control in which cells were cultured with simple medium alone, both CagA^＋ and CagA^- H pylori isolates could inhibit GJIC （CagA^＋： F = 57.98, P 〈 0.01; CagA^-： F = 29.59, P 〈 0.01） and proliferation （CagA^＋： F = 42.65, P 〈 0.01; CagA^-： F = 58.14, P 〈 0.01） of SGC-7901 cells. Compared with CagA^- strains, CagA^＋ H pylori more significantly downregulated GJIC of gastric cells （intact Hpylori： t = 13.86, P 〈 0.01; sonicated extracts： t = 11.87, P 〈 0.01） and inhibited proliferation gastric cells to a lesser extent in vitro （intact H pylori： t = 3.06, P 〈 0.05; sonicated extracts： t = 3.94, P 〈 0.01）. CONCLUSION： Compared with CagA^- H pylori strains, CagA^＋ strains down-regulate GJIC of gastric epithelial cells more significantly and inhibit proliferation of gastric cells to a lesser extent in vitro. H pylori, especially CagA^＋ strains, may play an important role in gastric carcinogenesis.

Autumn Photoproduction of Carbon Monoxide in Jiaozhou Bay, China

Carbon monoxide(CO) plays a significant role in global warming and atmospheric chemistry. Global oceans are net natural sources of atmospheric CO. CO at surface ocean is primarily produced from the photochemical degradation of chromophoric dissolved organic matter(CDOM). In this study, the effects of photobleaching, temperature and the origin(terrestrial or marine) of CDOM on the apparent quantum yields(AQY) of CO were studied for seawater samples collected from Jiaozhou Bay. Our results demonstrat that photobleaching, temperature and the origin of CDOM strongly affected the efficiency of CO photoproduction. The concentration, absorbance and fluorescence of CDOM exponentially decreased with increasing light dose. Terrestrial riverine organic matter could be more prone to photodegradation than the marine algae-derived one. The relationships between CO AQY and the dissolved organic carbon-specific absorption coefficient at 254 nm for the photobleaching study were nonlinear, whereas those of the original samples were strongly linear. This suggests that: 1) terrestrial riverine CDOM was more efficient than marine algae-derived CDOM for CO photoproduction; 2) aromatic and olefinic moieties of the CDOM pool were affected more strongly by degradation processes than by aliphatic ones. Water temperature and the origin of CDOM strongly affected the efficiency of CO photoproduction. The photoproduction rate of CO in autumn was estimated to be 31.98 μmol m-2 d-1 and the total DOC photomineralization was equivalent to 3.25%- 6.35% of primary production in Jiaozhou Bay. Our results indicate that CO photochemistry in coastal areas is important for oceanic carbon cycle.

PHOTOCHROMIC PROPERTIES OF (E)-DICYCLOPROPYLMETHYLENE-(2,5-DIMETHYL-3-FURYLETHYLIDENE)-SUCCINIC ANHYDRIDE DOPED IN POLYSTYRENE THIN FILMS

Fulgide 1-E doped in polystyrene polymer films was heated at various annealing temperatures.Upon irradiation with UV light(366 nm),fulgide 1-E undergoes a conrotatory ring closure to the pink colored closed form 1-C.The later color was switched back to the original color when the films were irradiated with white light.The kinetics of photocoloration and photobleaching processes were followed spectrophotometrically by monitoring the absorbance of the ring closed product 1-C at itsλ_(max) of 525 nm.The first-...

VISIBLE LIGHT PHOTOPOLYMERIZATION OF METHYL METHACRYLATE COINITIATED WITH TITANOCENE AND KETOCOUMARIN DYE

The photosensitive system which can initiate methyl methacrylate with visible light was composed of compound 1 bis( eta-5-cyclopentadienyl)-bis[2,6-difluoro-3-(1-H-pyrrolyl)phenyl]titanium (titanocene) and compound 2 [(3,3'-carbonylbis(7-diethylamino coumarin)] (ketocoumarin dye). The high photosensitive initiating efficiency of this photosensitive system could be very promising for efficient system for laser (Ar+ 488 nm) to plate and photocuring for thick coating and ink. The variation of UV-visible spectrum of compound 2 during irradiation indicates that photolysis of compound 2 is through its triplet state and it can be quenched by O-2 The much quicker photobleaching of the photosensitive system suggests that there exists certain quick electron transfer reaction between compounds 1 and 2.

Changes of gap junctional intercellular communication in detrusor instability

Objective: To demonstrate the functional changes of gap junctional mediation of intercellular communication in detrusor instability (DI) and its mechanisms. Methods: The function of gap junctional intercellular communication in the cultured bladder detrusor cells was detected by fluorescence redistribution after photobleaching. Results: At the fourth minute after bleaching, the mean fluorescences recovery rates of DI group bladder detrusor cells were (35 791±0 836)%, that of control group (8 645±0 673)%. The mean fluorescence recovery rates of DI group were significantly higher than those of control group ( P <0 01). Conclusion: It shows that the increase of intercellular excitatory communication is one of the important reasons of pathogenesis of DI.

Tobacco Rattle Virus-induced Phytoene Desaturase (PDS) Silencing in Centaurea cyanus

Virus-induced gene silencing(VIGS)is a genetic tool used to assess gene function.Tobacco rattle virus(TRV)is a VIGS vector commonly used to induce endogenous gene silencing in plants.However,there is no VIGS system established for Centaurea spp.We evaluated the effectiveness of a TRV-based VIGS system using phytoene desaturase(PDS)as a reporter gene in Centaurea cyanus.Three methods including pressure-,vacuum-and apical meristem-infiltrationwere tested to infect C.cyanus seedlings.Photobleached leaveswere only obtained using apicalmeristem-infiltration after a 14 d treatment.The CcPDS transcripts in photobleached leaves were significantly reduced compared with that in green leaves treated with empty TRV.Four C.cyanus cultivars were tested to detect their VIGS responses,and‘Dwarf Tom Pouce Blue’was the most sensitive.The agro-infiltration condition was optimized by screening for the optimal seedling stage as well as the optimum Agrobacterium density for efficient silencing.Seedlings with four true leaves and infiltration with an Agrobacterium density of OD_(600)0.5 were optimal conditions to obtain more photobleached leaves and more intense photobleached phenotype.The results demonstrated the feasibility of TRV-based VIGS for functional analysis of genes in C.cyanus.

Visualization of bHLH transcription factor interactions in living mammalian cell nuclei and developing chicken neural tube by FRET

Members of the basic helix-loop-helix （bHLH） gene family play important roles in vertebrate neurogenesis. In this study, confocal microscopy-based fluorescence resonance energy transfer （FRET） is used to monitor bHLH protein-protein interactions under various physiological conditions. Tissue-specific bHLH activators, NeuroD 1, Mash 1, Neurogenin 1 （Ngn 1）, Neurogenin2 （Ngn2）, and ubiquitous expressed E47 protein are tagged with enhanced yellow fluorescence protein （EYFP） and enhanced cyan fluorescence protein （ECFP）, respectively. The subcellular localization and mobility ofbHLH fusion proteins are examined in HEK293 cells. By transient transfection and in ovo electroporation, four pairs of tissue-specific bHLH activators and E47 protein are over-expressed in HEK293 cells and developing chick embryo neural tube. With the acceptor photobleaching method, FRET could be detected between these bHLH protein pairs in the nuclei of transfected cells and developing neural tubes. Mashl/E47 and Ngn2/E47 FRET pairs show higher FRET efficiencies in the medial and the lateral half of chick embryo neural tube, respectively. It suggests that these bHLH protein pairs formed functional DNA-protein complexes with regulatory elements of their downstream target genes in the specific regions. This work will help one understand the behaviours of bHLH factors in vivo.

Hydrogen self-supplying initiators excited by visible light for the fabrication of transparent films

Unimolecular Type Ⅱ radical photoinitiators(PIs) have received significant attention in photocuring owing to the fact that they improve the sustainability of the overall process compared with traditional Type Ⅱ radical photoinitiators. However, the photopolymerization efficiency of unimolecular Type Ⅱ radical photoinitiators is hindered by their short excitation wavelengths,poor photon capture abilities, and inefficient photobleaching performance. Herein, we report a coumarin-based self-sufficient initiator(C-NA), which is designed by integrating “hydrogen donor” and “hydrogen acceptor” into the coumarin framework and used for single-component visible light curing. C-NA exhibits a visible light absorbance and high molar extinction coefficient and is completely photobleached under the irradiation of 405 nm light-emitting diodes(LEDs). The formation of free radicals arises from the transfer of hydrogen from the diethylamino group to the coumarin framework, together with a highly efficient photodegradation process of C-NA. Finally, C-NA was successfully applied to prepare a transparent film material. Therefore,C-NA offers new insights into the design of promising unimolecular Type Ⅱ radical photoinitiators for photocuring.

Mineralization of 4-Chlorophenol under Visible Light Irradiation in the Presence of Aluminum and Zinc Phthalocyaninesulfonates

Photosensitized oxidation of 4 chlorophenol (4CP) by the title complexes (AlPcS and ZnPcS) in aerated aqueous solution upon visible light irradiation ( λ ≥450 nm) has been investigated using methanol as a disassociating reagent. It is confirmed that the monomeric species of the sensitizer is more active than the corresponding dimer in singlet oxygen generation for 4CP oxidation. However, the monomer is also the main component found in the sensitizer's photobleaching. In this regard, AlPcS is much more stable than ZnPcS, and the photobleaching is observed to proceed via singlet and triplet oxygen, respectively. The final products of 4CP oxidation in alkaline solution are carbon dioxide and chloride ions, while at pH=7 and pH=3 the p benzoquinone is the product. The temperature is found to have influence on both the photosensitized degradation of methyl orange and ZnPcS photobleaching, with an activation energy of 15 8 and 24 2 kJ/mol, respectively.

Virus-induced Phytoene Desaturase(PDS) Gene Silencing Using Tobacco Rattle Virus in Lilium × formolongi

Gene function analysis is challenging for Lilium due to the lack of an efficient method for stable genetic transformation. Virus-induced gene silencing(VIGS) is an attractive tool for determining gene function in plants. This study reported that Tobacco rattle virus(TRV)-based VIGS of a PHYTOENE DESATURASE(PDS) ortholog(LhPDS) can be achieved in Lilium × formolongi seedlings, with a survival rate of 92%, using the inoculation method of rubbing plus injection. Compared with untreated and mock-treated seedlings, the photobleached leaf phenotype of silenced plants significantly correlates with down-regulation of endogenous LhPDS(P ≤ 0.05). In addition, the silencing phenomenon can be observed in the growing points of plants, indicating that systemic viral infection was achieved using the protocol. The results indicate that TRV-based VIGS can be used to characterize gene function in Lilium × formolongi. This work lays the foundation for gene function analysis and molecular breeding in Lilium spp.

Distribution and spectral characteristics of chromophoric dissolved organic matter in a coastal bay in northern China

The absorption spectra of chromophoric dissolved organic matter（CDOM）,along with general physical,chemical and biological variables,were determined in the Bohai Bay,China,in the springs of 2011 and 2012. The absorption coefficient of CDOM at 350 nm（a350） in surface water ranged from 1.00 to 1.83 m-1（mean： 1.35 m-1） in May 2011 and from 0.78 to 1.92 m-1（mean：1.19 m-1） in April 2012. Little surface-bottom difference was observed due to strong vertical mixing. The a350 was weakly anti-correlated to salinity but positively correlated to chlorophyll a（Chl-a） concentration. A shoulder over 260–290 nm,suggestive of biogenic molecules,superimposed the overall pattern of exponentially decreasing CDOM absorption with wavelength. The wavelength distribution of the absorption spectral slope manifested a pronounced peak at ca. 300 nm characteristic of algal-derived CDOM. All a250/a365 ratios exceeded 6,corresponding to CDOM molecular weights（Mw） of less than 1 kDa. Spectroscopically,CDOM in the Bohai Bay differed substantively from that in the Haihe River,the bay＇s dominant source of land runoff; photobleaching of the riverine CDOM enlarged the difference.Results point to marine biological production being the principal source of CDOM in the Bohai Bay during the sampling seasons. Relatively low runoff,fast dilution,and selective photodegradation are postulated to be among the overarching elements responsible for the lack of terrigenous CDOM signature in the bay water.

HID-1 is a peripheral membrane protein primarily associated with the medial-and transGolgi apparatus

Caenorhabditis elegans hid-1 gene was first identified in a screen for mutants with a high-temperature-induced dauer formation(Hid)phenotype.Despite the fact that the hid-1 gene encodes a novel protein(HID-1)which is highly conserved from Caenorhabditis elegans to mammals,the domain structure,subcellular localization,and exact function of HID-1 remain unknown.Previous studies and various bioinformatic softwares predicted that HID-1 contained many transmembrane domains but no known functional domain.In this study,we revealed that mammalian HID-1 localized to the medial-and transGolgi apparatus as well as the cytosol,and the localization was sensitive to brefeldin A treatment.Next,we demonstrated that HID-1 was a peripheral membrane protein and dynamically shuttled between the Golgi apparatus and the cytosol.Finally,we verified that a conserved N-terminal myristoylation site was required for HID-1 binding to the Golgi apparatus.We propose that HID-1 is probably involved in the intracellular trafficking within the Golgi region.