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
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
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.