As one of the most important realizations of stimulated emission depletion(STED)microscopy,the continuous-wave(CW)STED system,constructed by using CW lasers as the excitation and STED beams,has been investigated and d...As one of the most important realizations of stimulated emission depletion(STED)microscopy,the continuous-wave(CW)STED system,constructed by using CW lasers as the excitation and STED beams,has been investigated and developed for nearly a decade.However,a theoretical model of the suppression factors in CW STED has not been well established.In this investigation,the factors that affect the spatial resolution of a CW STED system are theoretically and numerically studied.The full-width-at-half-maximum(FWHM)of a CW STED with a doughnut-shaped STED beam is also reanalyzed.It is found that the suppression function is dominated by the ratio of the local STED and excitation beam intensities.In addition,the FWHM is highly sensitive to both the fluorescence rate(inverse of fluoresce lifetime)and the quenching rate,but insensitive to the rate of vibrational relaxation.For comparison,the suppression function in picosecond STED is only determined by the distribution of the STED beam intensity scaled with the saturation intensity.Our model is highly consistent with published experimental data for evaluating the spatial resolution.This investigation is important in guiding the development of new CW STED systems.展开更多
Wide-field linear structured illumination microscopy(LSIM)extends resolution beyond the diffraction limit by moving unresolvable high-frequency information into the passband of the microscopy in the form of moiré...Wide-field linear structured illumination microscopy(LSIM)extends resolution beyond the diffraction limit by moving unresolvable high-frequency information into the passband of the microscopy in the form of moiréfringes.However,due to the diffraction limit,the spatial frequency of the structured illumination pattern cannot be larger than the microscopy cutoff frequency,which results in a twofold resolution improvement over wide-field microscopes.This Letter presents a novel approach in point-scanning LSIM,aimed at achieving higher-resolution improvement by combining stimulated emission depletion(STED)with point-scanning structured illumination microscopy(ps SIM)(STED-ps SIM).The according structured illumination pattern whose frequency exceeds the microscopy cutoff frequency is produced by scanning the focus of the sinusoidally modulated excitation beam of STED microscopy.The experimental results showed a 1.58-fold resolution improvement over conventional STED microscopy with the same depletion laser power.展开更多
The significant role of telomeres in cells has attracted much attention since they were discovered.Fluorescence imaging is an effective method to study subcellular structures like telomeres.However,the diffraction lim...The significant role of telomeres in cells has attracted much attention since they were discovered.Fluorescence imaging is an effective method to study subcellular structures like telomeres.However,the diffraction limit of traditional optical microscope hampers further investigation on them.Recent progress on superresolution fluorescence microscopy has broken this limit.In this work,we used stimulated emission depletion(STED) microscope to observe fluorescence-labeled telomeres in interphase cell nuclei.The results showed that the size of fluorescent puncta representing telomeres under the STED microscope was much smaller than that under the confocal microscope.Two adjacent telomeres were clearly separated via STED imaging,which could hardly be discriminated by confocal microscopy due to the diffraction limit.We conclude that STED microscope is a more powerful tool that enable us to obtain detailed information about telomeres.展开更多
We have built an integrated imaging system by combining stimulated emission depletion(STED)microscope and atomic force microscope(AFM).The STED microscope was constructed based on the supercontinuum fiber laser and a ...We have built an integrated imaging system by combining stimulated emission depletion(STED)microscope and atomic force microscope(AFM).The STED microscope was constructed based on the supercontinuum fiber laser and a super lateral resolution of42 nm was achieved.With this integrated imaging system,morphological features,mechanical parameters and fluorescence super resolution imaging were obtained simultaneously for both nanobeads and fixed cell samples.This new integrated imaging system is expected to obtain comprehensive information at the nanoscale for studies in nanobiology and nanomedicine.展开更多
As an essential part in the toolbox of super-resolution microscopy,stimulated emission depletion(STED)nanoscopy has been widely explored in revealing the substructure and bioactivities in fluorescence imaging.Among th...As an essential part in the toolbox of super-resolution microscopy,stimulated emission depletion(STED)nanoscopy has been widely explored in revealing the substructure and bioactivities in fluorescence imaging.Among the applied STED fluorophores,silicon-substituted rhodamines(SiRs)belong to one of the most extensively employed fluorophores.The carboxy-SiR was favored in STED bioimaging with many advantages,including reliable photostability,cell permeability,tunable fluorogenicity,feasible structural decoration and so on.We reviewed the research of carboxy-SiR in the STED nanoscopy and hopefully this can inspire more efforts in the design and application of STED fluorophores.展开更多
A stimulated emission depletion is capable of breaking the diffraction limit by exciting fluorescent molecules with a solid Gaussian beam and quenching the excited molecules with another donut beam through stimulated ...A stimulated emission depletion is capable of breaking the diffraction limit by exciting fluorescent molecules with a solid Gaussian beam and quenching the excited molecules with another donut beam through stimulated emission.The coincidence degree of these two beams in three dimensions will significantly influence the spatial resolution of the microscope.However,the conventional alignment approach based on raster scanning of gold nanoparticles by the two laser beams separately suffers from a mismatch between fluorescence and scattering modes.To circumvent the above problems,we demonstrate a fast alignment design by scanning the second beam over the fabricated sample,which is made of aggregation-induced emission(AIE)dye resin.The relative positions of solid and donut laser beams can be represented by the fluorescent AIE from the labeled spots in the dye resin.This design achieves ultra-high resolutions of 22 nm in the x/y relative displacement and 27 nm in the z relative displacement for fast spatial matching of the two laser beams.This study has potential applications in scenarios that require the spatial matching of multiple laser beams,and the field of views of different objectives,for example,in a microscope with high precision.展开更多
A stimulated emission depletion （STED） microscopy scheme using axially symmetric polarized vortex beams is pro- posed based on unique focusing properties of such kinds of beams. The concept of axially symmetric pola...A stimulated emission depletion （STED） microscopy scheme using axially symmetric polarized vortex beams is pro- posed based on unique focusing properties of such kinds of beams. The concept of axially symmetric polarized vortex beams is first introduced, and the basic principle about the scheme is described. Simulation results for several typical beams are then shown, including radially polarized vortex beams, azimuthally polarized vortex beams, and high-order axi- ally symmetric polarized vortex beams. The results indicate that sharper doughnut spots and thus higher resolutions can be achieved, showing more flexibility than previous schemes based on flexible modulation of both phase and polarization for incident beams.展开更多
A versatile and inexpensive super-resolution fluorescent microscope that functions as easily as a conventional confocal microscope is described. Components of the microscope were designed on a platform which was place...A versatile and inexpensive super-resolution fluorescent microscope that functions as easily as a conventional confocal microscope is described. Components of the microscope were designed on a platform which was placed atop a surplus microscope frame. All optical components and equipments used are given. The excitation and depletion beams are extracted from a compact low-cost supercontinuum light source. The focal spot of the depletion beam at the focal plane is studied and imaged by a 100 nm aperture near-field fiber tip. The collinear excitation and depletion beam focused by a 0.9 numerical aperture microscope objective produce a 90 nm lateral super-resolution as verified by imaging 100 nm diameter fluorescent beads.展开更多
基金Project supported by the National Natural Science Foundation of China(Grant Nos.11672229 and 61378083)International Cooperation Foundation of the National Science and Technology Major Project of the Ministry of Science and Technology of China(Grant No.2011DFA12220)+4 种基金Major Research Plan of the National Natural Science Foundation of China(Grant No.91123030)Natural Science Foundation of Shaanxi Province of China(Grant Nos.2010JS110and 2013SZS03-Z01)Natural Science Basic Research Program of Shaanxi Province-Major Basic Research Project,China(Grant No.2016ZDJC-15)Young Scientist Fund of the National Natural Science Foundation of China(Grant No.11504294)the Youth Talent Plan of the Natural Science Foundation of Shaanxi Province of China(Grant No.2016JQ103)
文摘As one of the most important realizations of stimulated emission depletion(STED)microscopy,the continuous-wave(CW)STED system,constructed by using CW lasers as the excitation and STED beams,has been investigated and developed for nearly a decade.However,a theoretical model of the suppression factors in CW STED has not been well established.In this investigation,the factors that affect the spatial resolution of a CW STED system are theoretically and numerically studied.The full-width-at-half-maximum(FWHM)of a CW STED with a doughnut-shaped STED beam is also reanalyzed.It is found that the suppression function is dominated by the ratio of the local STED and excitation beam intensities.In addition,the FWHM is highly sensitive to both the fluorescence rate(inverse of fluoresce lifetime)and the quenching rate,but insensitive to the rate of vibrational relaxation.For comparison,the suppression function in picosecond STED is only determined by the distribution of the STED beam intensity scaled with the saturation intensity.Our model is highly consistent with published experimental data for evaluating the spatial resolution.This investigation is important in guiding the development of new CW STED systems.
基金supported by the National Natural Science Foundation of China(Nos.62275168,62275164,61775148,and 61905145)the National Key Research and Development Program of China(No.2022YFA1206300)+5 种基金the Guangdong Natural Science Foundation and Province Project(Nos.2021A1515011916 and 2023A1515012250)the Foundation from Department of Science and Technology of Guangdong Province(No.2021QN02Y124)the Foundation from Department of Education of Guangdong Province(No.2023ZDZX2052)the Shenzhen Science and Technology R&D and Innovation Foundation(No.JCYJ20200109105608771)the Shenzhen Key Laboratory of Photonics and Biophotonics(No.ZDSYS20210623092006020)the Medical-Engineering Interdisciplinary Research Foundation of Shenzhen University。
文摘Wide-field linear structured illumination microscopy(LSIM)extends resolution beyond the diffraction limit by moving unresolvable high-frequency information into the passband of the microscopy in the form of moiréfringes.However,due to the diffraction limit,the spatial frequency of the structured illumination pattern cannot be larger than the microscopy cutoff frequency,which results in a twofold resolution improvement over wide-field microscopes.This Letter presents a novel approach in point-scanning LSIM,aimed at achieving higher-resolution improvement by combining stimulated emission depletion(STED)with point-scanning structured illumination microscopy(ps SIM)(STED-ps SIM).The according structured illumination pattern whose frequency exceeds the microscopy cutoff frequency is produced by scanning the focus of the sinusoidally modulated excitation beam of STED microscopy.The experimental results showed a 1.58-fold resolution improvement over conventional STED microscopy with the same depletion laser power.
基金supported by the National Natural Science Foundation of China(61378062,21227804,21390414,61475181)the National Basic ResearchProgram of China(2012CB825805)the Shanghai Municipal Commission for Science and Technology(14ZR1448000)
文摘The significant role of telomeres in cells has attracted much attention since they were discovered.Fluorescence imaging is an effective method to study subcellular structures like telomeres.However,the diffraction limit of traditional optical microscope hampers further investigation on them.Recent progress on superresolution fluorescence microscopy has broken this limit.In this work,we used stimulated emission depletion(STED) microscope to observe fluorescence-labeled telomeres in interphase cell nuclei.The results showed that the size of fluorescent puncta representing telomeres under the STED microscope was much smaller than that under the confocal microscope.Two adjacent telomeres were clearly separated via STED imaging,which could hardly be discriminated by confocal microscopy due to the diffraction limit.We conclude that STED microscope is a more powerful tool that enable us to obtain detailed information about telomeres.
基金supported by the National Basic Research Program of China(2013CB933701)the National Natural Science Foundation of China(91213305,21127901,21121063)Chinese Academy of Sciences
文摘We have built an integrated imaging system by combining stimulated emission depletion(STED)microscope and atomic force microscope(AFM).The STED microscope was constructed based on the supercontinuum fiber laser and a super lateral resolution of42 nm was achieved.With this integrated imaging system,morphological features,mechanical parameters and fluorescence super resolution imaging were obtained simultaneously for both nanobeads and fixed cell samples.This new integrated imaging system is expected to obtain comprehensive information at the nanoscale for studies in nanobiology and nanomedicine.
基金supported by the Natural Science Foundation of Shanghai(Nos.19ZR1480000,20ZR1470200)National Natural Science Foundation of China(No.81830106)the Program for Professor of Special Appointment(Eastern Scholar,No.TP2017039)。
文摘As an essential part in the toolbox of super-resolution microscopy,stimulated emission depletion(STED)nanoscopy has been widely explored in revealing the substructure and bioactivities in fluorescence imaging.Among the applied STED fluorophores,silicon-substituted rhodamines(SiRs)belong to one of the most extensively employed fluorophores.The carboxy-SiR was favored in STED bioimaging with many advantages,including reliable photostability,cell permeability,tunable fluorogenicity,feasible structural decoration and so on.We reviewed the research of carboxy-SiR in the STED nanoscopy and hopefully this can inspire more efforts in the design and application of STED fluorophores.
基金supported by the National Key Research and Development Program of China(No.2018YFB0704103)National Natural Science Foundation of China(No.62175153)。
文摘A stimulated emission depletion is capable of breaking the diffraction limit by exciting fluorescent molecules with a solid Gaussian beam and quenching the excited molecules with another donut beam through stimulated emission.The coincidence degree of these two beams in three dimensions will significantly influence the spatial resolution of the microscope.However,the conventional alignment approach based on raster scanning of gold nanoparticles by the two laser beams separately suffers from a mismatch between fluorescence and scattering modes.To circumvent the above problems,we demonstrate a fast alignment design by scanning the second beam over the fabricated sample,which is made of aggregation-induced emission(AIE)dye resin.The relative positions of solid and donut laser beams can be represented by the fluorescent AIE from the labeled spots in the dye resin.This design achieves ultra-high resolutions of 22 nm in the x/y relative displacement and 27 nm in the z relative displacement for fast spatial matching of the two laser beams.This study has potential applications in scenarios that require the spatial matching of multiple laser beams,and the field of views of different objectives,for example,in a microscope with high precision.
基金Project supported by the National Natural Science Foundation of China(Grant Nos.61108047 and 61475021)the Natural Science Foundation of Beijing,China(Grant No.4152015)+2 种基金the Program for New Century Excellent Talents in Universities of China(Grant No.NCET-13-0667)the Top Young Talents Support Program of BeijingChina(Grant No.CIT&TCD201404113)
文摘A stimulated emission depletion （STED） microscopy scheme using axially symmetric polarized vortex beams is pro- posed based on unique focusing properties of such kinds of beams. The concept of axially symmetric polarized vortex beams is first introduced, and the basic principle about the scheme is described. Simulation results for several typical beams are then shown, including radially polarized vortex beams, azimuthally polarized vortex beams, and high-order axi- ally symmetric polarized vortex beams. The results indicate that sharper doughnut spots and thus higher resolutions can be achieved, showing more flexibility than previous schemes based on flexible modulation of both phase and polarization for incident beams.
文摘A versatile and inexpensive super-resolution fluorescent microscope that functions as easily as a conventional confocal microscope is described. Components of the microscope were designed on a platform which was placed atop a surplus microscope frame. All optical components and equipments used are given. The excitation and depletion beams are extracted from a compact low-cost supercontinuum light source. The focal spot of the depletion beam at the focal plane is studied and imaged by a 100 nm aperture near-field fiber tip. The collinear excitation and depletion beam focused by a 0.9 numerical aperture microscope objective produce a 90 nm lateral super-resolution as verified by imaging 100 nm diameter fluorescent beads.
