Structured illumination microscopy(SIM)is suitable for biological samples because of its relatively low-peak illumination intensity requirement and high imaging speed.The system resolution is affected by two typical d...Structured illumination microscopy(SIM)is suitable for biological samples because of its relatively low-peak illumination intensity requirement and high imaging speed.The system resolution is affected by two typical detection modes:Point detection and area detection.However,a systematic analysis of the imaging performance of the different detection modes of the system has rarely been conducted.In this study,we compared laser point scanning point detection(PS-PD)and point scanning area detection(PS-AD)imaging in nonconfocal microscopy through theoretical analysis and simulated imaging.The results revealed that the imaging resolutions of PSPD and PS-AD depend on excitation and emission point spread functions(PSFs),respectively.Especially,we combined the second harmonic generation(SHG)of point detection(P-SHG)and area detection(A-SHG)with SIM to realize a nonlinear SIM-imaging technique that improves the imaging resolution.Moreover,we analytically and experimentally compared the nonlinear SIM performance of P-SHG with that of A-SHG.展开更多
We describe a multiphoton(mP)-structured illumination microscopy(SIM)technique,which demonstrates substantial improvement in image resolution compared with linear SIM due to the nonlinear response of fluorescence.This...We describe a multiphoton(mP)-structured illumination microscopy(SIM)technique,which demonstrates substantial improvement in image resolution compared with linear SIM due to the nonlinear response of fluorescence.This nonlinear response is caused by the effect of nonsinusoidal structured illumination created by scanning a sinusoidally modulated illumination to excite an mP fluorescence signal.The harmonics of the structured fluorescence illumination are utilised to improve resolution.We present an mP-SIM theory for reconstructing the super-resolution image of the system.Theoretically,the resolution of our m P-SIM is unlimited if all the high-order harmonics of the nonlinear response of fluorescence are considered.Experimentally,we demonstrate an 86 nm lateral resolution for two-photon(2P)-SIM and a 72 nm lateral resolution for second-harmonic-generation(SHG)-SIM.We further demonstrate their application by imaging cells stained with F-actin and collagen fibres in mouse-tail tendon.Our method can be directly used in commercial mP microscopes and requires no specific fluorophores or high-intensity laser.展开更多
Structured illumination microscopy(SIM)is an essential super-resolution microscopy technique that enhances resolution.Several images are required to reconstruct a super-resolution image.However,linear SIM resolution e...Structured illumination microscopy(SIM)is an essential super-resolution microscopy technique that enhances resolution.Several images are required to reconstruct a super-resolution image.However,linear SIM resolution enhancement can only increase the spatial resolution of micros-copy by a factor of two at most because the frequency of the structured illumination pattern is limited by the cutoff frequency of the excitation point spread function.The frequency of the pattern generated by the nonlinear response in samples is not limited;therefore,nonlinear SIM(NL-SIM),in theory,has no inherent limit to the resolution.In the present study,we describe a two-photon nonlinear SIM(2P-SIM)technique using a multiple harmonics scanning pattern that employs a composite structured illumination pattern,which can produce a higher order harmonic pattern based on the fluorescence nonlinear response in a 2P process.The theoretical models of super-resolution imaging were established through our simulation,which describes the working mechanism of the multi-frequency structure of the nonsinusoidal function to improve the reso-lution.The simulation results predict that a 5-fold improvement in resolution in the 2P-SIM is possible.展开更多
Multifocal multiphoton microscopy(MMM)has recently become an important tool in biomedicine for performing three-dimensional fastfluorescence imaging.Using various beamsplitting techniques,MMM splits the near-infrared ...Multifocal multiphoton microscopy(MMM)has recently become an important tool in biomedicine for performing three-dimensional fastfluorescence imaging.Using various beamsplitting techniques,MMM splits the near-infrared laser beam into multiple beamlets and produces a multifocal array on the sample for parallel multiphoton excitation and then recordsfluorescence signal from all foci simultaneously with an area array detector,which significantly improves the imaging speed of multiphoton microscopy and allows for high efficiency in use of the excitation light.In this paper,we discuss the features of several MMM setups using different beamsplitting devices,including a Nipkow spinning disk,a microlens array,a set of beamsplitting mirrors,or a diffractive optical element(DOE).In particular,we present our recent work on the development of an MMM using a spatial light modulator(SLM).展开更多
Using the combination of a refective blazed grating and a reflective phase-only difractive spatiallight modulator(SLM),scanless multitarget-matching multiphoton excitation fuorescence mi.croscopy(SMTM-MP M)was achieve...Using the combination of a refective blazed grating and a reflective phase-only difractive spatiallight modulator(SLM),scanless multitarget-matching multiphoton excitation fuorescence mi.croscopy(SMTM-MP M)was achieved.The SLM shaped an incoming mode-locked,near-infraredTi:sapphire laser beam into an excitation pattern with addressable shapes and sizes that matchedthe samples of interest in the field of view.Temporal and spatial focusing were simultaneouslyrealized by combining an objective lens and a blazed grating.The fluorescence signal fromilluminated areas was recorded by a two-dimensional sCMOS camera.Compared with a conventional temporal focusing multiphoton microscope,our microscope achieved effective use of thelaser power and decreased photodamage with higher axial resolution.展开更多
Mesenchymal stem cells(MSCs)have been cited as contributors to heart repair through cardiogenic differentiation and multiple cellular interactions,including the paracrine effect,cell fusion,and mechanical and electric...Mesenchymal stem cells(MSCs)have been cited as contributors to heart repair through cardiogenic differentiation and multiple cellular interactions,including the paracrine effect,cell fusion,and mechanical and electrical couplings.Due to heart–muscle complexity,progress in the development of knowledge concerning the role of MSCs in cardiac repair is heavily based on MSC–cardiomyocyte coculture.In conventional coculture systems,however,the in vivo cardiac muscle structure,in which rod-shaped cells are connected end-to-end,is not sustained;instead,irregularly shaped cells spread randomly,resulting in randomly distributed cell junctions.Consequently,contact-mediated cell–cell interactions(e.g.,the electrical triggering signal and the mechanical contraction wave that propagate through MSC–cardiomyocyte junctions)occur randomly.Thus,the data generated on the beneficial effects of MSCs may be irrelevant to in vivo biological processes.In this study,we explored whether cardiomyocyte alignment,the most important phenotype,is relevant to stem cell cardiogenic differentiation.Here,we report(i)the construction of a laser-patterned,biochip-based,stem cell–cardiomyocyte coculture model with controlled cell alignment;and(ii)single-cell-level data on stem cell cardiogenic differentiation under in vivo-like cardiomyocyte alignment conditions.展开更多
Optothermal nanotweezers have emerged as an innovative optical manipulation technique in the past decade,which revolutionized classical optical manipulation by efficiently capturing a broader range of nanoparticles.Ho...Optothermal nanotweezers have emerged as an innovative optical manipulation technique in the past decade,which revolutionized classical optical manipulation by efficiently capturing a broader range of nanoparticles.However,the optothermal temperature field was merely employed for in-situ manipulation of nanoparticles,its potential for identifying bio-nanoparticles remains largely untapped.Hence,based on the synergistic effect of optothermal manipulation and CRIPSR-based bio-detection,we developed CRISPR-powered optothermal nanotweezers(CRONT).Specifically,by harnessing diffusiophoresis and thermo-osmotic flows near the substrate upon optothermal excitation,we successfully trapped and enriched DNA functionalized gold nanoparticles,CRISPR-associated proteins,as well as DNA strands.Remarkably,we built an optothermal scheme for enhancing CRISPR-based single-nucleotide polymorphism(SNP)detection at single molecule level,while also introducing a novel CRISPR methodology for observing nucleotide cleavage.Therefore,this innovative approach has endowed optical tweezers with DNA identification ability in aqueous solution which was unattainable before.With its high specificity and feasibility for in-situ bio-nanoparticle manipulation and identification,CRONT will become a universal tool in point-of-care diagnosis,biophotonics,and bio-nanotechnology.展开更多
Optical tweezers system has emerged as an efficient tool to manipulate tiny particles in a non-invasive way.Trapping stiffness,as an essential parameter of an optical potential well,represents the trapping stability.A...Optical tweezers system has emerged as an efficient tool to manipulate tiny particles in a non-invasive way.Trapping stiffness,as an essential parameter of an optical potential well,represents the trapping stability.Additionally,trapping inorganic nanoparticles such as metallic nanoparticles or other functionalized inorganic nanoparticles is important due to their properties of good stability,high conductivity,tolerable toxicity,etc.,which makes it an ideal detection strategy for bio-sensing,force calculation,and determination of particle and environmental properties.However,the trapping stiffness measurement(TSM)methods of inorganic nanoparticles have rarely been analyzed and summarized.Here,in this review,the principle and methods of TSM are analyzed.We also systematically summarize the progress in acquiring inorganic particles trapping stiffness and its promising applications.In addition,we provide prospects of the energy and environment applications of optical tweezering technique and TSM.Finally,the challenges and future directions of achieving the nanoparticles trapping stiffness are discussed.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
Optical tweezers that rely on laser irradiation to capture and manipulate nanoparticles have provided powerful tools for biological and biochemistry studies.However,the existence of optical diffraction-limit and the t...Optical tweezers that rely on laser irradiation to capture and manipulate nanoparticles have provided powerful tools for biological and biochemistry studies.However,the existence of optical diffraction-limit and the thermal damage caused by high laser power hinder the wider application of optical tweezers in the biological field.For the past decade,the emergence of optothermal tweezers has solved the above problems to a certain extent,while the auxiliary agents used in optothermal tweezers still limit their biocompatibility.Here,we report a kind of nanotweezers based on the sign transformation of the thermophoresis coefficient of colloidal particles in low-temperature environment.Using a self-made microfluidic refrigerator to reduce the ambient temperature to around 0℃in the microfluidic cell,we can control a single nanoparticle at lower laser power without adding additional agent solute in the solution.This novel optical tweezering scheme has provided a new path for the manipulation of inorganic nanoparticles as well as biological particles.展开更多
基金supported by the National Natural Science Foundation of China (62275168,62275164,61905145)Guangdong Natural Science Foundation and Province Project (2021A1515011916)+1 种基金Shenzhen Science and Technology R&D and Innovation Foundation (JCYJ20200109105608771)the Science and Technology Planning Project of Shenzhen Municipality (ZDSYS20210623092006020).
文摘Structured illumination microscopy(SIM)is suitable for biological samples because of its relatively low-peak illumination intensity requirement and high imaging speed.The system resolution is affected by two typical detection modes:Point detection and area detection.However,a systematic analysis of the imaging performance of the different detection modes of the system has rarely been conducted.In this study,we compared laser point scanning point detection(PS-PD)and point scanning area detection(PS-AD)imaging in nonconfocal microscopy through theoretical analysis and simulated imaging.The results revealed that the imaging resolutions of PSPD and PS-AD depend on excitation and emission point spread functions(PSFs),respectively.Especially,we combined the second harmonic generation(SHG)of point detection(P-SHG)and area detection(A-SHG)with SIM to realize a nonlinear SIM-imaging technique that improves the imaging resolution.Moreover,we analytically and experimentally compared the nonlinear SIM performance of P-SHG with that of A-SHG.
基金supported by the Project from the National Key Research and Development Program of China(2017YFB0403804)the National Natural Science Foundation of China(61775148 and61527827)the Shenzhen Science and Technology R&D and Innovation Foundation(JCYJ20180305124754860 and JCYJ20200109105608771)。
文摘We describe a multiphoton(mP)-structured illumination microscopy(SIM)technique,which demonstrates substantial improvement in image resolution compared with linear SIM due to the nonlinear response of fluorescence.This nonlinear response is caused by the effect of nonsinusoidal structured illumination created by scanning a sinusoidally modulated illumination to excite an mP fluorescence signal.The harmonics of the structured fluorescence illumination are utilised to improve resolution.We present an mP-SIM theory for reconstructing the super-resolution image of the system.Theoretically,the resolution of our m P-SIM is unlimited if all the high-order harmonics of the nonlinear response of fluorescence are considered.Experimentally,we demonstrate an 86 nm lateral resolution for two-photon(2P)-SIM and a 72 nm lateral resolution for second-harmonic-generation(SHG)-SIM.We further demonstrate their application by imaging cells stained with F-actin and collagen fibres in mouse-tail tendon.Our method can be directly used in commercial mP microscopes and requires no specific fluorophores or high-intensity laser.
基金This work Was supported by National Natural Science Foundation of China(grant nos.61775148,61527827,and 61905145)Guangdong Natural Science Foundation and Province Project(2021A1515011916)Shenzhen Science and Technology R&D and Innovation Foundation(grant nos.JCYJ20200109105608771.J CYJ20180305124754860 and JCYJ20180228162956597).
文摘Structured illumination microscopy(SIM)is an essential super-resolution microscopy technique that enhances resolution.Several images are required to reconstruct a super-resolution image.However,linear SIM resolution enhancement can only increase the spatial resolution of micros-copy by a factor of two at most because the frequency of the structured illumination pattern is limited by the cutoff frequency of the excitation point spread function.The frequency of the pattern generated by the nonlinear response in samples is not limited;therefore,nonlinear SIM(NL-SIM),in theory,has no inherent limit to the resolution.In the present study,we describe a two-photon nonlinear SIM(2P-SIM)technique using a multiple harmonics scanning pattern that employs a composite structured illumination pattern,which can produce a higher order harmonic pattern based on the fluorescence nonlinear response in a 2P process.The theoretical models of super-resolution imaging were established through our simulation,which describes the working mechanism of the multi-frequency structure of the nonsinusoidal function to improve the reso-lution.The simulation results predict that a 5-fold improvement in resolution in the 2P-SIM is possible.
基金This work has been partially supported by NIH(SC COBRE P20RR021949 and Career Award 1k25hl088262-01)NSF(MRI CBET-0923311 and SC EPSCoR RII EPS-0903795 through SC GEAR program)+3 种基金The National Natural Science Foundation of China(31171372,61078067)Guangdong Province Science and Technology Project(2010B060300002)Shenzhen University Application Technology Development Project(201136,CXB201104220021A,JC201005250032A,200854)the Fundamental Research Funds for the Central Universities(K50510050006).
文摘Multifocal multiphoton microscopy(MMM)has recently become an important tool in biomedicine for performing three-dimensional fastfluorescence imaging.Using various beamsplitting techniques,MMM splits the near-infrared laser beam into multiple beamlets and produces a multifocal array on the sample for parallel multiphoton excitation and then recordsfluorescence signal from all foci simultaneously with an area array detector,which significantly improves the imaging speed of multiphoton microscopy and allows for high efficiency in use of the excitation light.In this paper,we discuss the features of several MMM setups using different beamsplitting devices,including a Nipkow spinning disk,a microlens array,a set of beamsplitting mirrors,or a diffractive optical element(DOE).In particular,we present our recent work on the development of an MMM using a spatial light modulator(SLM).
基金supported by Specially Funded Program on National Key Scienti¯c Instruments and Equipment Development(61527827),Program 973(2015CB352005)the National Natural Science Foundation of China(31171372/61525503/61378091/61620106016),Guangdong Natural Science Foundation(2014A030312008/2015A020214023/2015KGJHZ002)Shenzhen Science and Technology R&D Foundation(JCYJ20160422151611496).
文摘Using the combination of a refective blazed grating and a reflective phase-only difractive spatiallight modulator(SLM),scanless multitarget-matching multiphoton excitation fuorescence mi.croscopy(SMTM-MP M)was achieved.The SLM shaped an incoming mode-locked,near-infraredTi:sapphire laser beam into an excitation pattern with addressable shapes and sizes that matchedthe samples of interest in the field of view.Temporal and spatial focusing were simultaneouslyrealized by combining an objective lens and a blazed grating.The fluorescence signal fromilluminated areas was recorded by a two-dimensional sCMOS camera.Compared with a conventional temporal focusing multiphoton microscope,our microscope achieved effective use of thelaser power and decreased photodamage with higher axial resolution.
基金This work was partially supported by NIH(SC COBRE P20RR021949,Career Award 5k25hl088262-04 and 5R01 HL085847)NSF(MRI,CBET-0923311 and SC EPSCoR RII EPS-0903795 through SC GEAR program)+2 种基金Guangdong Provincial Department of Science and Technology,China(2011B050400011)BZG also acknowledges the support from the grant established by the State Key Laboratory of Precision Measuring Technology and Instruments(Tianjin University)ZM acknowledges his Siebel Institute Postdoctoral Fellowship(41523-31595-44-OYZHMA-IQKEH).
文摘Mesenchymal stem cells(MSCs)have been cited as contributors to heart repair through cardiogenic differentiation and multiple cellular interactions,including the paracrine effect,cell fusion,and mechanical and electrical couplings.Due to heart–muscle complexity,progress in the development of knowledge concerning the role of MSCs in cardiac repair is heavily based on MSC–cardiomyocyte coculture.In conventional coculture systems,however,the in vivo cardiac muscle structure,in which rod-shaped cells are connected end-to-end,is not sustained;instead,irregularly shaped cells spread randomly,resulting in randomly distributed cell junctions.Consequently,contact-mediated cell–cell interactions(e.g.,the electrical triggering signal and the mechanical contraction wave that propagate through MSC–cardiomyocyte junctions)occur randomly.Thus,the data generated on the beneficial effects of MSCs may be irrelevant to in vivo biological processes.In this study,we explored whether cardiomyocyte alignment,the most important phenotype,is relevant to stem cell cardiogenic differentiation.Here,we report(i)the construction of a laser-patterned,biochip-based,stem cell–cardiomyocyte coculture model with controlled cell alignment;and(ii)single-cell-level data on stem cell cardiogenic differentiation under in vivo-like cardiomyocyte alignment conditions.
基金supported by the National Natural Science Foundation of China(62275164,61905145,62275168,61775148)National Key Research and Development Program of China(No.2022YFA1206300)+8 种基金Guangdong Natural Science Foundation and Province Project(2021A1515011916,2023A1515012250)Foundation from Department of Science and Technology of Guangdong Province(2021QN02Y124)Shenzhen Science and Technology R&D and Innovation Foundation(JCYJ20200109105608771)Shenzhen Key Laboratory of Photonics and Biophotonics(ZDSYS20210623092006020)The Research Grants Council(RGC)of Hong Kong China(RGC14207920)Innovation Team Project of Department of Education of Guangdong Province(2018KCXTD026)Deanship of Scientific Research(DSR)at King Abdulaziz University,Jeddah(KEP-MSc-70-130-42)King Khalid University through Research Center for Advanced Materials Science(RCAMS)(RCAMS/KKU/006/21)Medical-Engineering Interdisciplinary Research Foundation of Shenzhen University。
文摘Optothermal nanotweezers have emerged as an innovative optical manipulation technique in the past decade,which revolutionized classical optical manipulation by efficiently capturing a broader range of nanoparticles.However,the optothermal temperature field was merely employed for in-situ manipulation of nanoparticles,its potential for identifying bio-nanoparticles remains largely untapped.Hence,based on the synergistic effect of optothermal manipulation and CRIPSR-based bio-detection,we developed CRISPR-powered optothermal nanotweezers(CRONT).Specifically,by harnessing diffusiophoresis and thermo-osmotic flows near the substrate upon optothermal excitation,we successfully trapped and enriched DNA functionalized gold nanoparticles,CRISPR-associated proteins,as well as DNA strands.Remarkably,we built an optothermal scheme for enhancing CRISPR-based single-nucleotide polymorphism(SNP)detection at single molecule level,while also introducing a novel CRISPR methodology for observing nucleotide cleavage.Therefore,this innovative approach has endowed optical tweezers with DNA identification ability in aqueous solution which was unattainable before.With its high specificity and feasibility for in-situ bio-nanoparticle manipulation and identification,CRONT will become a universal tool in point-of-care diagnosis,biophotonics,and bio-nanotechnology.
基金supported by the National Natural Science Foundation of China(62275164,61905145,62275168)National Key Research and Development Program of China(No.2022YFA1200116)+1 种基金Guangdong Natural Science Foundation and Province Project(2021A1515011916)Shenzhen Science and Technology Planning Project(ZDSYS20210623092006020).
文摘Optical tweezers system has emerged as an efficient tool to manipulate tiny particles in a non-invasive way.Trapping stiffness,as an essential parameter of an optical potential well,represents the trapping stability.Additionally,trapping inorganic nanoparticles such as metallic nanoparticles or other functionalized inorganic nanoparticles is important due to their properties of good stability,high conductivity,tolerable toxicity,etc.,which makes it an ideal detection strategy for bio-sensing,force calculation,and determination of particle and environmental properties.However,the trapping stiffness measurement(TSM)methods of inorganic nanoparticles have rarely been analyzed and summarized.Here,in this review,the principle and methods of TSM are analyzed.We also systematically summarize the progress in acquiring inorganic particles trapping stiffness and its promising applications.In addition,we provide prospects of the energy and environment applications of optical tweezering technique and TSM.Finally,the challenges and future directions of achieving the nanoparticles trapping stiffness are discussed.
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the National Natural Science Foundation of China(Nos.62275164,61905145,and 62275168)National Key Research and Development Program of China(No.2022YFA1200116)+1 种基金Guangdong Natural Science Foundation and Province Project(No.2021A1515011916)Shenzhen Science and Technology Planning Project(No.ZDSYS20210623092006020).
文摘Optical tweezers that rely on laser irradiation to capture and manipulate nanoparticles have provided powerful tools for biological and biochemistry studies.However,the existence of optical diffraction-limit and the thermal damage caused by high laser power hinder the wider application of optical tweezers in the biological field.For the past decade,the emergence of optothermal tweezers has solved the above problems to a certain extent,while the auxiliary agents used in optothermal tweezers still limit their biocompatibility.Here,we report a kind of nanotweezers based on the sign transformation of the thermophoresis coefficient of colloidal particles in low-temperature environment.Using a self-made microfluidic refrigerator to reduce the ambient temperature to around 0℃in the microfluidic cell,we can control a single nanoparticle at lower laser power without adding additional agent solute in the solution.This novel optical tweezering scheme has provided a new path for the manipulation of inorganic nanoparticles as well as biological particles.
