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.

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.

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.

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.

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.

Laser Stimulation of the Chloroplast/Endoplasmic Reticulum Nexus in Tobacco Transiently Produces Protein Aggregates （Boluses） within the Endoplasmic Reticulum and Stimulates Local ER Remodeling

Does the ER subdomain that associates with the chloroplast in vivo, hereafter referred to as the chloroplast/ER nexus, play a role in protein flow within the ER？ In studies of tobacco cells either constitutively or transiently expressing ER-retained luminal, GFP-HDEL, or trans-membrane, YFP-RHD3, fluorescent fusion proteins, brief 405-nm （3-6-mW） laser stimulation of the nexus causes a qualitative difference in the movement and behavior of proteins in the ER. Photostimulating the nexus produces fluorescent protein punctate aggregates （boluses） within the lumen and membrane of the ER. The aggregation propagates through the membrane network throughout the cell, but within minutes can revert to normal, with disaggregation propagating back toward the originally photostimulated nexus. In the meantime, the ER grows and anastomoses around the chloroplast, forming a dense cisternal and tubular network. If this network is again photostimulated, bolus formation does not recur and, if the photostimulation results in photobleaching, fluorescence recovery after photobleaching occurs as it would typically in areas away from the nexus. Bolus propagation is not mediated by the actin cytoskeleton, but can be reversed by pre-conditioning the cells for 30 min with high, 40-45℃, temperature （heat stress）. Because it is not reversed with heat stress, the reorganization of the ER at the nexus following photostimulation is a separate event.

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.

New approaches to diagnostics and treatment of cholangiocellular cancer based on photonics methods

Cholangiocellular cancer(CCC)is an oncological disease of the bile ducts characterized by a high mortality rate.To date,the use of standard methods for the diagnosis and treatment of CCC has not been able to reduce mortality from this disease.This work presents the results of fluorescence diagnostics(FD),which consists in using a modified optical fiber and photodynamic therapy(PDT)using a therapeutic laser instead of a low-intensity laser.This technique was tested on 43 patients in a clinical setting.The results obtained indicate a direct correlation between spectroscopic and video FD methods.Furthermore,a direct correlation was found between the photobleaching of a chlorin e6-based photosensitizer,with the commercial names of PhotoIon Radachlorin and Photoran and stricture regression.Our findings demonstrate the possibility of using a therapeutic laser with a wavelength of 660 nm for both diagnosis and treatment of bile ducts cancer,which results in a significant reduction of the operation time without decreasing its effectiveness.

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.

Glyoxylate Reductase Isoform 1 is Localized in the Cytosol and Not Peroxisomes in Plant Cells

Glyoxylate reductase （GLYR） is a key enzyme in plant metabolism which catalyzes the detoxification of both photorespiratory glyoxylate and succinic semialdehdye, an intermediate of the γ-aminobutyrate （GABA） pathway. Two isoforms of GLYR exist in plants, GLYR1 and GLYR2, and while GLYR2 is known to be localized in plastids, GLYR1 has been reported to be localized in either peroxisomes or the cytosol. Here, we reappraised the intracellular localization of GLYR1 in Arabidopsis thaliana L. Heynh （ecotype Lansberg erecta） using both transiently-transformed suspension cells and stably-transformed plants, in combination with fluorescence microscopy. The results indicate that GLYR1 is localized exclusively to the cytosol regardless of the species, tissue and/or cell type, or exposure of plants to environmental stresses that would increase flux through the GABA pathway. Moreover, the C-terminal tripeptide sequence of GLYR1, -SRE, despite its resemblance to a type 1 peroxisomal targeting signal, is not sufficient for targeting to peroxisomes. Collectively, these results define the cytosol as the intracellular location of GLYR1 and provide not only important insight to the metabolic roles of GLYR1 and the compartmentation of the GABA and photorespiratory pathways in plant cells, but also serve as a useful reference for future studies of proteins proposed to be localized to peroxisomes and/or the cytosol.

Removal of chromophoric dissolved organic matter under combined photochemical and microbial degradation as a response to different irradiation intensities

Throughout the freshwater continuum, Dissolved Organic Carbon(DOC) and the colored fraction, Chromophoric Dissolved Organic Material(CDOM), are continuously being added,removed, and transformed, resulting in changes in the chromophoricity and lability of organic matter over time. We examined, experimentally, the effect of increasing irradiationintensities on the combined photochemical and microbial degradation of CDOM and DOC.This was done by using a simulated mixed water column: aged water from a humic lake was exposed to four irradiation-intensities – representing winter, early and late spring, and summer conditions(0.10, 0.16, 0.36, and 0.58 W/m^(2)) – and compared with dark controls over 37days. We found a linear relationship between CDOM degradation and irradiation-intensities up to 0.36 W/m^(2);the degradation rate saturated at higher intensities, both at specific wavelengths and for broader intervals. After 37 days at high irradiation-intensity, CDOM absorption of irradiation at 340 nm had been reduced by 41%;48% of DOC had been removed and DOC degradation continued to increase. Aromaticity(SUVA254) declined significantly over 37 days at the two lowest but not at the two highest UV-intensities;levels in unexposed control water remained constant. Direct observations of the humic lake showed that CDOM absorption of irradiation(340 nm) declined by 27% from winter to summer. A model based on hydrological CDOM input and CDOM degradation calculated from field measurements of UV-radiation and experimental CDOM degradation with UV-exposure from sunlight accurately predicted the annual course as observed in the lake. With no external CDOM input,92% of the CDOM could be degraded in a year. The results support the notion that combined photochemical and microbial CDOM degradation can be remarkably higher in lakes than previously thought and that humic lakes retain their color due to light absorption by ongoing CDOM input.

Improving FRET efficiency measurement in confocal microscopy imaging

Spectral bleedthrough （SBT） ratio is dependent on the level of fluorescence intensity in confocal imaging. Precision Frster resonance energy transfer （FRET） algorithm corrects SBT ratio according to fluorescence intensity and avoids over-or under-estimation of SBT ratio. In this letter, we propose a new method to accurately measure the FRET efficiency of FRET plasmid in single living cells by combining the calculation of SBT in precision FRET algorithm with E-FRET formulae. We also use this method to measure the FRET efficiency of FRET-Bid, and find that in healthy A549 cells it is about 15%, which is verified by FRET acceptor photobleaching method.

In vivo skin imaging prototypes ＂made in Latvia＂

This paper briefly reviews the operational principles and designs of portable in vivo skin imaging prototypes developed at the Biophotonics Laboratory of the Institute of Atomic Physics and Spectroscopy, University of Latvia. Four types of imaging devices are presented. Multi-spectral imagers ensure distant mapping of specific skin parameters （e.g., distribution of skin chromophores）. Autofluorescence photobleaching rate imagers show potential for skin tumor assessment and margin delineation. Photoplethysmography video-imagers remotely detect cutaneous blood pulsations and provide real-time information on the human cardiovascular state. Multimodal skin imagers perform the above-mentioned functions by acquiring several spectral and video images using the same image sensor.