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...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.展开更多
Fluorescence resonance energy transfer(FRET)technology had been widely used to study proteinprotein interactions in living cells.In this study,we developed a ROI-PbFRET method to real-time quantitate the FRET efficie...Fluorescence resonance energy transfer(FRET)technology had been widely used to study proteinprotein 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.展开更多
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 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.展开更多
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 l...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.展开更多
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 la...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.展开更多
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 pro...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.展开更多
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 dr...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.展开更多
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 inter...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.展开更多
文摘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.
基金This work is supported by the National Natural Science Foundation of China(NSFC)(Grant 81071491)Key Project of the Department of Education and Finance of Guangdong Province(cxzd115).
文摘Fluorescence resonance energy transfer(FRET)technology had been widely used to study proteinprotein 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.
文摘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.
基金This work was supported by grants from the National Research Foundation(NRF)(NRF2019R1A2C2088973)funded by the Ministry of Educationthe Korea Evaluation Institute of Industrial Technology(KEIT)(20011377)funded by the Ministry of Trade,Industry&Energy,Republic of Korea.
文摘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.
文摘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.
基金We thank X.Peng(Shenzhen University)for great assistance in tunneling nanotubes of live cell.This work was partially supported by the National Key R&D Program of China(No.2018YFC0910600)the National Natural Science Foundation of China(Nos.61975132,61775145,61525503,61620106016,61835009,and 81727804)+4 种基金China Postdoctoral Science Foundation(No.2019M650211)Guangdong Province Key Area R&D Program(No.2019B110233004)Project of Department of Education of Guangdong Province(No.2015KGJHZ002/2016KCXTD007)the Shenzhen Basic Research Project(Nos.JCYJ20170412110212234 and JCYJ20170412105003520)the Natural Science Foundation of Shenzhen University(2019108).
文摘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.
文摘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.
基金This work was supported by the National Natural Science Foundation of China(no.31900023)National Key R&D Program of China(no.2018YFC1406701)+4 种基金Program of Shandong Taishan Scholars(no.tspd20181203)Natural Science Foundation of Shandong(no.ZR2017LD013)AoShan Talents Cultivation Program(no.2017ASTCP-OS14)State Key Laboratory of Microbial Technology Open Projects Fund(no.M2019-07)Young Scholars Program of Shandong University(no.2017WLJH22).
文摘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.
