Maintaining the s-polarization state of laser beams is important to achieve high modulation depth in a laser-interference-based super-resolution structured illumination microscope(SR-SIM).However,the imperfect optical...Maintaining the s-polarization state of laser beams is important to achieve high modulation depth in a laser-interference-based super-resolution structured illumination microscope(SR-SIM).However,the imperfect optical components can depolarize the laser beams hence degenerating the modulation depth.Here,we first presented a direct measurement method designed to estimate the modulation depth more precisely by shifting illumination patterns with equal phase steps.This measurement method greatly reduces the dependence of modulation depths on the samples,and then developed a polarization optimization method to achieve high modulation depth at all orientations by actively and quantitatively compensating for the additional phase difference using a combination of waveplate and a liquid crystal variable retarder(LCVR).Experimental results demonstrate that our method can achieve illumination patterns with modulation depth higher than 0.94 at three orientations with only one LCVR voltage,which enables isotropic resolution improvement.展开更多
Super-resolution structured illumination microscopy(SR-SIM)relies heavily on post-processing reconstruction to obtain high-quality SR images from raw data.Although many SIM reconstruction algorithms have been develope...Super-resolution structured illumination microscopy(SR-SIM)relies heavily on post-processing reconstruction to obtain high-quality SR images from raw data.Although many SIM reconstruction algorithms have been developed to recover fine cellular structures with high fidelity even from the noisy data,whether the pixel intensities of reconstructed SR images are still proportional to the original fluorescence intensity has been less explored.The linearity between the intensity before and after reconstruction is de fined as the intensity fidelity.Here,we proposed a method to evaluate the reconstructed SR image intensity fidelity at different spatial frequencies.With the proposed metric,we systematically investigated the impact of the key factors on the intensity fidelity in the standard Wiener-SIM reconstructions with simulated data,then evaluated the intensity fidelity of the SR images reconstructed by representative open-source packages.Our work provides a reference for SR-SIM image intensity fidelity improvement.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
Optical microscopy allows us to observe the biological structures and processes within living cells.However,the spatial resolution of the optical microscopy is limited to about half of the wavelength by the light di&#...Optical microscopy allows us to observe the biological structures and processes within living cells.However,the spatial resolution of the optical microscopy is limited to about half of the wavelength by the light di®raction.Structured illumination microscopy(SIM),a type of new emerging super-resolution microscopy,doubles the spatial resolution by illuminating the specimen with a patterned light,and the sample and light source requirements of SIM are not as strict as the other super-resolution microscopy.In addition,SIM is easier to combine with the other imaging techniques to improve their imaging resolution,leading to the developments of diverse types of SIM.SIM has great potential to meet the various requirements of living cells imaging.Here,we review the recent developments of SIM and its combination with other imaging techniques.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)achieves super-resolution(SR)by modulating the high-frequency information of the sample into the passband of the optical system and subsequent image reconstruction.The traditiona...Structured illumination microscopy(SIM)achieves super-resolution(SR)by modulating the high-frequency information of the sample into the passband of the optical system and subsequent image reconstruction.The traditional Wiener-filtering-based reconstruction algorithm operates in the Fourier domain,it requires prior knowledge of the sinusoidal illumination patterns which makes the time-consuming procedure of parameter estimation to raw datasets necessary,besides,the parameter estimation is sensitive to noise or aberration-induced pattern distortion which leads to reconstruction artifacts.Here,we propose a spatial-domain image reconstruction method that does not require parameter estimation but calculates patterns from raw datasets,and a reconstructed image can be obtained just by calculating the spatial covariance of differential calculated patterns and differential filtered datasets(the notch filtering operation is performed to the raw datasets for attenuating and compensating the optical transfer function(OTF)).Experiments on reconstructing raw datasets including nonbiological,biological,and simulated samples demonstrate that our method has SR capability,high reconstruction speed,and high robustness to aberration and noise.展开更多
Traditionally,optical microscopy is used to visualize the morphological features of pathogenic bacteria,of which the features are further used for the detection and ident ification of the bacteria.However,due to the r...Traditionally,optical microscopy is used to visualize the morphological features of pathogenic bacteria,of which the features are further used for the detection and ident ification of the bacteria.However,due to the resolution limitation of conventional optical microscopy as well as the lack of standard pattern library for bacteria identification,the ffectiveness of this optical microscopy-based method is limited.Here,we reported a pilot study on a combined use of Structured Illumination Microscopy(SIM)with machine learning for rapid bacteria identification.After applying machine learning to the SIM image datasets from three model bacteria(including Escherichia coli,Mycobacterium smegmatis,and Pseudomonas aeruginosa),we obtained a classifcation accuracy of up to 98%.This study points out a promising possibility for rapid bacterial identification by morphological features.展开更多
Scanning tunneling microscopy （STM） and scanning tunneling spectroscopy （STS） were performed on monolayer film of NiTPP supported on Au（111） under ultrahigh vacuum （UHV） conditions. The constant current STM im...Scanning tunneling microscopy （STM） and scanning tunneling spectroscopy （STS） were performed on monolayer film of NiTPP supported on Au（111） under ultrahigh vacuum （UHV） conditions. The constant current STM images show remarkable bias dependence. High resolution STM data clearly show the individual NiTPP molecules and allow easy differentiation between NiTPP and CoTPP reported before. Scanning tunneling spectra, as a function of molecule-tip separation, were acquired over a range of tip motion of 0.42 nm. Spectra do not show the variation in band splitting with tip distance. It appears for molecules such as NiTPP that the average potential at the molecule is essentially the same at the same metal substrate. For molecules of the height of NiTPP, the scanning tunneling spectra should give reliable occupied and unoccupied orbital energies over a wide range of tip-molecule distances.展开更多
Structured illumination microscopy(SIM)has been widely used in live-cell superresolution(SR)imaging.However,conventional physical model-based SIM SR reconstruction algorithms are prone to artifacts in handling raw ima...Structured illumination microscopy(SIM)has been widely used in live-cell superresolution(SR)imaging.However,conventional physical model-based SIM SR reconstruction algorithms are prone to artifacts in handling raw images with low signal-to-noise ratios(SNRs).Deep-learning(DL)-based methods can address this challenge but may lead to degradation and hallucinations.By combining the physical inversion model with a total deep variation(TDV)regularization,we propose a hybrid restoration method(TDV-SIM)that outperforms conventional or DL methods in suppressing artifacts and hallucinations while maintaining resolutions.We demonstrate the performance superiority of TDV-SIM in restoring actin filaments,endoplasmic reticulum,and mitochondrial cristae from extremely low SNR raw images.Thus TDV-SIM represents the ideal method for prolonged live-cell SR imaging with minimal exposure and photodamage.Overall,TDV-SIM proves the power of integrating model-based reconstruction methods with DL ones,possibly leading to the rapid exploration of similar strategies in high-fidelity reconstructions of other microscopy methods.展开更多
In recent years,notable progress has been achieved in both the hardware and algorithms of structured illumination microscopy(SIM).Nevertheless,the advancement of three-dimensional structured illumination microscopy(3D...In recent years,notable progress has been achieved in both the hardware and algorithms of structured illumination microscopy(SIM).Nevertheless,the advancement of three-dimensional structured illumination microscopy(3DSIM)has been impeded by challenges arising from the speed and intricacy of polarization modulation.We introduce a high-speed modulation 3DSIM system,leveraging the polarizationmaintaining and modulation capabilities of a digital micromirror device(DMD)in conjunction with an electrooptic modulator.The DMD-3DSIM system yields a twofold enhancement in both lateral(133 nm)and axial(300 nm)resolution compared to wide-field imaging and can acquire a data set comprising 29 sections of 1024 pixels×1024 pixels,with 15 ms exposure time and 6.75 s per volume.The versatility of the DMD-3DSIM approach was exemplified through the imaging of various specimens,including fluorescent beads,nuclear pores,microtubules,actin filaments,and mitochondria within cells,as well as plant and animal tissues.Notably,polarized 3DSIM elucidated the orientation of actin filaments.Furthermore,the implementation of diverse deconvolution algorithms further enhances 3D resolution.The DMD-based 3DSIM system presents a rapid and reliable methodology for investigating biomedical phenomena,boasting capabilities encompassing 3D superresolution,fast temporal resolution,and polarization imaging.展开更多
Structured illumination microscopy(SIM)has been widely applied in the superresolution imaging of subcellular dynamics in live cells.Higher spatial resolution is expected for the observation of finer structures.However...Structured illumination microscopy(SIM)has been widely applied in the superresolution imaging of subcellular dynamics in live cells.Higher spatial resolution is expected for the observation of finer structures.However,further increasing spatial resolution in SIM under the condition of strong background and noise levels remains challenging.Here,we report a method to achieve deep resolution enhancement of SIM by combining an untrained neural network with an alternating direction method of multipliers(ADMM)framework,i.e.,ADMM-DRE-SIM.By exploiting the implicit image priors in the neural network and the Hessian prior in the ADMM framework associated with the optical transfer model of SIM,ADMM-DRE-SIM can further realize the spatial frequency extension without the requirement of training datasets.Moreover,an image degradation model containing the convolution with equivalent point spread function of SIM and additional background map is utilized to suppress the strong background while keeping the structure fidelity.Experimental results by imaging tubulins and actins show that ADMM-DRE-SIM can obtain the resolution enhancement by a factor of∼1.6 compared to conventional SIM,evidencing the promising applications of ADMM-DRE-SIM in superresolution biomedical imaging.展开更多
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
Scanning tunnel microscopy （STM） is performed to verify if an Rh ＇nails＇ structure is formed accompanying the graphene growing during chemical vapor deposition. A structure of a graphene island in an Rh vacancy is...Scanning tunnel microscopy （STM） is performed to verify if an Rh ＇nails＇ structure is formed accompanying the graphene growing during chemical vapor deposition. A structure of a graphene island in an Rh vacancy island is used as the start. While the graphene island is removed by oxygenation, the variations of the Rh vacancy island are imaged with an in-situ high-temperature STM. By fitting with our model and calculations, we conclude that the best fit is obtained for 0% Rh, i.e., for the complete absence of nails below graphene on Rh（111）. That is, when graphene is formed on Rh（111）, the substrate remains fiat and does not develop a SUPPorting nail structure.展开更多
The microphase separation extent of biomedical segmented polyetherurethanes were greatly enhanced due to the presence of sulfoalkyl pendant groups contained in the hard segments,and the hard segments were more orderly...The microphase separation extent of biomedical segmented polyetherurethanes were greatly enhanced due to the presence of sulfoalkyl pendant groups contained in the hard segments,and the hard segments were more orderly aggregated through ionic interaction.展开更多
We report a comprehensive numerical study of super resolution (SR) structured illumination microscopy (SIM) utilizing the classic Heintzmann-Cremer SIM process and algorithm. In particular, we investigated the impact ...We report a comprehensive numerical study of super resolution (SR) structured illumination microscopy (SIM) utilizing the classic Heintzmann-Cremer SIM process and algorithm. In particular, we investigated the impact of the diffraction limit of the underlying imaging system on the optimal SIM grating frequency that can be used to obtain the highest SR enhancement with non-continuous spatial frequency support. Besides confirming the previous theoretical and experimental work that SR-SIM can achieve an enhancement close to 3 times the diffraction limit with grating pattern illuminations, we also observe and report a series of more subtle effects of SR-SIM with non-continuous spatial frequency support. Our simulations show that when the SIM grating frequency exceeds twice that of the diffraction limit, the higher SIM grating frequency can help achieve a higher SR enhancement for the underlying imaging systems whose diffraction limit is low, though this enhancement is obtained at the cost of losing resolution at some lower resolution targets. Our simulations also show that, for underlying imaging systems with high diffraction limits, however, SR-SIM grating frequencies above twice the diffraction limits tend to bring no significant extra enhancement. Furthermore, we observed that there exists a limit grating frequency above which the SR enhancement effect is lost, and the reconstructed images essentially have the same resolution as the one obtained directly from the underlying imaging system without using the SIM process.展开更多
Biochars are known for their heterogeneity, especially in pore and surface structure associated with pyrolysis processes and sources of feedstocks. The surface area of biochar is likely to be an important determinant ...Biochars are known for their heterogeneity, especially in pore and surface structure associated with pyrolysis processes and sources of feedstocks. The surface area of biochar is likely to be an important determinant of the extent of soil microbial attachment, whereas the porous structure of biochar is expected to provide protection for soil microorganisms. Potential interactions between biochars from different sources and with different particle sizes were investigated in relation to soil microbial properties in a short-term incubation study. Three particle size （sieved） fractions （0.5-1.0, 1.0-2.0 and 2.0-4.0 mm） from three woody biochars produced from jarrah wood, jarrah and wandoo wood and Australian wattle branches, respectively, were incubated in soil at 25 ℃ for 56 d. Observation by scanning electron microscopy （SEM） and characterisation of pore and surface area showed that all three woody biochars provided potential habitats for soil microorganisms due to their high porosity and surface areas. The biochars were structurally heterogeneous, varying in porosity and surface structure both within and between the biochar sources. After the 56-d incubation, hyphal colonisation was observed on biochar surfaces and in larger biochar pores. Soil clumping occurred on biochar particles, cementing and covering exposed biochar pores. This may have altered surface area and pore availability for microbial colonisation. Transient changes in soil microbial biomass, without a consistent trend, were observed among biochars during the 56-d incubation.展开更多
Theranostic visualization of dextran at the nanoscale is beneficial for understanding the bioregulatory mechanisms of this molecule. In this study, we applied structured illumination microscopy(SIM) to capture the dis...Theranostic visualization of dextran at the nanoscale is beneficial for understanding the bioregulatory mechanisms of this molecule. In this study, we applied structured illumination microscopy(SIM) to capture the distribution of Cy5-Dextran at different incubation periods in living cells. The results showed that Cy5-Dextran could be absorbed by He La cells. In addition, we clarified that Cy5-Dextran exhibited differential organelle distribution(lysosomal or mitochondrial) in a time-dependent manner. Moreover,lysosomal Cy5-Dextran localization was found to be independent of the autophagy process, while Cy5-Dextran localized to the mitochondria triggered a pro-apoptotic event, upregulating the levels of reactive oxygen species(ROS) to accelerate mitochondrial fragmentation. This work uses a visualized strategy to reveal the anti-tumor bioactivity of dextran, which was achieved by regulating apoptosis and autophagy.展开更多
The time domain entombment of bacteria by intratubular mineralization following orthograde canal obturation with mineral trioxide aggregate（MTA） was studied by scanning electron microscopy（SEM）. Single-rooted huma...The time domain entombment of bacteria by intratubular mineralization following orthograde canal obturation with mineral trioxide aggregate（MTA） was studied by scanning electron microscopy（SEM）. Single-rooted human premolars（n560） were instrumented to an apical size #50/0.06 using ProF ile and treated as follows： Group 1（n510） was filled with phosphate buffered saline（PBS）; Group 2（n510） was incubated with Enterococcus faecalis for 3 weeks, and then filled with PBS; Group 3（n520） was obturated orthograde with a paste of OrthoM TA（BioM TA, Seoul, Korea） and PBS; and Group 4（n520） was incubated with E. faecalis for 3 weeks and then obturated with OrthoM TA–PBS paste. Following their treatments, the coronal openings were sealed with PBS-soaked cotton and intermediate restorative material（IRM）, and the roots were then stored in PBS for 1, 2, 4, 8 or 16 weeks. After each incubation period, the roots were split and their dentin/MTA interfaces examined in both longitudinal and horizontal directions by SEM. There appeared to be an increase in intratubular mineralization over time in the OrthoM TA-filled roots（Groups 3 and 4）. Furthermore, there was a gradual entombment of bacteria within the dentinal tubules in the E. faecalis inoculated MTA-filled roots（Group 4）. Therefore, the orthograde obturation of root canals with OrthoM TA mixed with PBS may create a favorable environment for bacterial entombment by intratubular mineralization.展开更多
Hematologic malignancies are one of the most common malignant tumors caused by the clonal proliferation and differentiation of hematopoietic and lymphoid stem cells.The examination of bone marrow cells combined with i...Hematologic malignancies are one of the most common malignant tumors caused by the clonal proliferation and differentiation of hematopoietic and lymphoid stem cells.The examination of bone marrow cells combined with immunodeficiency typing is of great significance to the diagnostic type,treatment and prognosis of hematologic malignancies.Super-resolution fluorescence microscopy(SRM)is a special kind of optical microscopy technology,which breaks the resolution limit and was awarded the Nobel Prize in Chemistry in 2014.With the development of SRM,many related technologies have been applied to the diagnosis and treatment of clinical diseases.It was reported that a major type of SRM technique,single molecule localization microscopy(SMLM),is more sensitive than flow cytometry(FC)in detecting cell membrane antigens'expression,thus enabling better chances in detecting antigens on hematopoietic cells than traditional analytic tools.Furthermore,SRM may be applied to clinical pathology and may guide precision medicine and personalized medicine for clone hematopoietic cell diseases.In this paper,we mainly discuss the application of SRM in clone hematological malignancies.展开更多
Functional amyloid has been increasingly applied as self-assembling nanostructures to construct multifunctional biomaterials.However,little has been known how different side domains,varied fusion positions and subunit...Functional amyloid has been increasingly applied as self-assembling nanostructures to construct multifunctional biomaterials.However,little has been known how different side domains,varied fusion positions and subunits affect self-assembly and morphologies of amyloid fibrils.Here,we constructed three groups of two-component amyloid proteins based on CsgA,the major protein components of Escherichia coli biofilms,to bridge these gaps.We showed that all fusion proteins have amyloid features,as indicated by Congo red assay.Atomic force microscopy（AFM） indeed reveals that these fusion proteins are able to self-assemble into fibrils,with an average diameter of 0.5-2 nm and length of hundreds of nanometers to several micrometers.The diameter of fibrils increases with the increase of the molecular weight of fusion domains,while the dynamic assembly of recombinant proteins was delayed as a result of the introduction of fusion domains.Moreover,fusion of the same functional domains but at intermediate position seems to cause the most interference on fibril assembly compared with those fused at C or Nterminus,as mainly short and irregular fibrils were detected.This phenomenon appears more pronounced for randomly coiled mussel foot proteins（Mfps） than for rigid chitin-binding domain（CBD）.Finally,increase of the molecular weight of tandem repeats in protein monomer seemed to increase the fibril diameter of the resultant fibrils,but either reduction of the tandem repeats of CsgA to one single belta-sheet loop or increase in the number of tandem repeats of CsgAs from one to four produced shorter and intermittent fibrils compared with CsgA control protein.These studies therefore provide insights into self-assembly of two-component amyloid proteins and lay the foundation for rational design of multifunctional molecular biomaterials.展开更多
基金supported by the National Natural Science Foundation of China[Grant Nos.62205367 and 62141506]the Suzhou Basic Research Pilot Project[Grant Nos.SSD2023006 and SJC2021013]the National Key Research and Development Program of China[Grant No.2023YFF1205700].
文摘Maintaining the s-polarization state of laser beams is important to achieve high modulation depth in a laser-interference-based super-resolution structured illumination microscope(SR-SIM).However,the imperfect optical components can depolarize the laser beams hence degenerating the modulation depth.Here,we first presented a direct measurement method designed to estimate the modulation depth more precisely by shifting illumination patterns with equal phase steps.This measurement method greatly reduces the dependence of modulation depths on the samples,and then developed a polarization optimization method to achieve high modulation depth at all orientations by actively and quantitatively compensating for the additional phase difference using a combination of waveplate and a liquid crystal variable retarder(LCVR).Experimental results demonstrate that our method can achieve illumination patterns with modulation depth higher than 0.94 at three orientations with only one LCVR voltage,which enables isotropic resolution improvement.
基金supported by the National Natural Science Foundation of China[Grant Nos.62205367 and 62141506]Suzhou Basic Research Pilot Project[Grant Nos.SSD2023006 and SJC2021013]Jiangsu Provincial Key Research and Development Program[Grant No.BE2020664].
文摘Super-resolution structured illumination microscopy(SR-SIM)relies heavily on post-processing reconstruction to obtain high-quality SR images from raw data.Although many SIM reconstruction algorithms have been developed to recover fine cellular structures with high fidelity even from the noisy data,whether the pixel intensities of reconstructed SR images are still proportional to the original fluorescence intensity has been less explored.The linearity between the intensity before and after reconstruction is de fined as the intensity fidelity.Here,we proposed a method to evaluate the reconstructed SR image intensity fidelity at different spatial frequencies.With the proposed metric,we systematically investigated the impact of the key factors on the intensity fidelity in the standard Wiener-SIM reconstructions with simulated data,then evaluated the intensity fidelity of the SR images reconstructed by representative open-source packages.Our work provides a reference for SR-SIM image intensity fidelity improvement.
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 study was partly supported by the National Key Basic Research Program of China (973 Program)under Grant No.2015CB352006the National Natural Science Foundation of China under Grants Nos.61335011 and 61405035Program for Changjiang Scholars and Innovative Research Team in University under Grant No.IRT 15R10.
文摘Optical microscopy allows us to observe the biological structures and processes within living cells.However,the spatial resolution of the optical microscopy is limited to about half of the wavelength by the light di®raction.Structured illumination microscopy(SIM),a type of new emerging super-resolution microscopy,doubles the spatial resolution by illuminating the specimen with a patterned light,and the sample and light source requirements of SIM are not as strict as the other super-resolution microscopy.In addition,SIM is easier to combine with the other imaging techniques to improve their imaging resolution,leading to the developments of diverse types of SIM.SIM has great potential to meet the various requirements of living cells imaging.Here,we review the recent developments of SIM and its combination with other imaging techniques.
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funded by the National Natural Science Foundation of China(62125504,61827825,and 31901059)Zhejiang Provincial Ten Thousand Plan for Young Top Talents(2020R52001)Open Project Program of Wuhan National Laboratory for Optoelectronics(2021WNLOKF007).
文摘Structured illumination microscopy(SIM)achieves super-resolution(SR)by modulating the high-frequency information of the sample into the passband of the optical system and subsequent image reconstruction.The traditional Wiener-filtering-based reconstruction algorithm operates in the Fourier domain,it requires prior knowledge of the sinusoidal illumination patterns which makes the time-consuming procedure of parameter estimation to raw datasets necessary,besides,the parameter estimation is sensitive to noise or aberration-induced pattern distortion which leads to reconstruction artifacts.Here,we propose a spatial-domain image reconstruction method that does not require parameter estimation but calculates patterns from raw datasets,and a reconstructed image can be obtained just by calculating the spatial covariance of differential calculated patterns and differential filtered datasets(the notch filtering operation is performed to the raw datasets for attenuating and compensating the optical transfer function(OTF)).Experiments on reconstructing raw datasets including nonbiological,biological,and simulated samples demonstrate that our method has SR capability,high reconstruction speed,and high robustness to aberration and noise.
基金supported by the National Key Research and Development Program of China(Grant No.2017-YFD0500303)the National Natural Science Foundation of China(Grant Nos.31371106,91640105)+1 种基金the China Agriculture Research System(No.CARS-36)the Huazhong Agricultural University Scienti¯c and Technological Self-innovation Foundation(Program No.52204-13002).
文摘Traditionally,optical microscopy is used to visualize the morphological features of pathogenic bacteria,of which the features are further used for the detection and ident ification of the bacteria.However,due to the resolution limitation of conventional optical microscopy as well as the lack of standard pattern library for bacteria identification,the ffectiveness of this optical microscopy-based method is limited.Here,we reported a pilot study on a combined use of Structured Illumination Microscopy(SIM)with machine learning for rapid bacteria identification.After applying machine learning to the SIM image datasets from three model bacteria(including Escherichia coli,Mycobacterium smegmatis,and Pseudomonas aeruginosa),we obtained a classifcation accuracy of up to 98%.This study points out a promising possibility for rapid bacterial identification by morphological features.
基金This work was supported by the Excellent Scientist Program of South China University of Technology (324-D60090), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry and the National Natural Science Foundation of China (20643001).
文摘Scanning tunneling microscopy （STM） and scanning tunneling spectroscopy （STS） were performed on monolayer film of NiTPP supported on Au（111） under ultrahigh vacuum （UHV） conditions. The constant current STM images show remarkable bias dependence. High resolution STM data clearly show the individual NiTPP molecules and allow easy differentiation between NiTPP and CoTPP reported before. Scanning tunneling spectra, as a function of molecule-tip separation, were acquired over a range of tip motion of 0.42 nm. Spectra do not show the variation in band splitting with tip distance. It appears for molecules such as NiTPP that the average potential at the molecule is essentially the same at the same metal substrate. For molecules of the height of NiTPP, the scanning tunneling spectra should give reliable occupied and unoccupied orbital energies over a wide range of tip-molecule distances.
基金support by grants from the National Science and Technology Major Project Program(Grant Nos.2021YFA1100201,2022YFF0712500,and 2022YFC3400600)the National Natural Science Foundation of China(Grant Nos.92054301,81925022,92150301,32170691,62103071,and 31901061)+5 种基金the Beijing Natural Science Foundation(Grant No.Z20J00059)the Lingang Laboratory(Grant No.LG-QS-202206-06)Clinical Medicine Plus X-Young Scholars Project,Peking University,the Fundamental Research Funds for the Central Universities,the Natural Science Foundation of Chongqing(Grant No.cstc2021jcyj-msxmX0526)the Science and Technology Research Program of Chongqing Municipal Education Commission(Grant No.KJQN202100630)the Strategic Priority Research Program of Chinese Academy of Sciences(Grant No.XDA16021200)the High-Performance Computing Platform of Peking University.
文摘Structured illumination microscopy(SIM)has been widely used in live-cell superresolution(SR)imaging.However,conventional physical model-based SIM SR reconstruction algorithms are prone to artifacts in handling raw images with low signal-to-noise ratios(SNRs).Deep-learning(DL)-based methods can address this challenge but may lead to degradation and hallucinations.By combining the physical inversion model with a total deep variation(TDV)regularization,we propose a hybrid restoration method(TDV-SIM)that outperforms conventional or DL methods in suppressing artifacts and hallucinations while maintaining resolutions.We demonstrate the performance superiority of TDV-SIM in restoring actin filaments,endoplasmic reticulum,and mitochondrial cristae from extremely low SNR raw images.Thus TDV-SIM represents the ideal method for prolonged live-cell SR imaging with minimal exposure and photodamage.Overall,TDV-SIM proves the power of integrating model-based reconstruction methods with DL ones,possibly leading to the rapid exploration of similar strategies in high-fidelity reconstructions of other microscopy methods.
基金supported by the National Key R&D Program of China (Award No.2022YFC3401100)the National Natural Science Foundation of China。
文摘In recent years,notable progress has been achieved in both the hardware and algorithms of structured illumination microscopy(SIM).Nevertheless,the advancement of three-dimensional structured illumination microscopy(3DSIM)has been impeded by challenges arising from the speed and intricacy of polarization modulation.We introduce a high-speed modulation 3DSIM system,leveraging the polarizationmaintaining and modulation capabilities of a digital micromirror device(DMD)in conjunction with an electrooptic modulator.The DMD-3DSIM system yields a twofold enhancement in both lateral(133 nm)and axial(300 nm)resolution compared to wide-field imaging and can acquire a data set comprising 29 sections of 1024 pixels×1024 pixels,with 15 ms exposure time and 6.75 s per volume.The versatility of the DMD-3DSIM approach was exemplified through the imaging of various specimens,including fluorescent beads,nuclear pores,microtubules,actin filaments,and mitochondria within cells,as well as plant and animal tissues.Notably,polarized 3DSIM elucidated the orientation of actin filaments.Furthermore,the implementation of diverse deconvolution algorithms further enhances 3D resolution.The DMD-based 3DSIM system presents a rapid and reliable methodology for investigating biomedical phenomena,boasting capabilities encompassing 3D superresolution,fast temporal resolution,and polarization imaging.
基金supported by the National Natural Science Foundation of China(Grant Nos.12274129,12274139,12074121,92150301,62105101,62175066,and 12034008)the Science and Technology Commission of Shanghai Municipality(Grant Nos.21XD1400900,20ZR1417100,and 21JM0010700).
文摘Structured illumination microscopy(SIM)has been widely applied in the superresolution imaging of subcellular dynamics in live cells.Higher spatial resolution is expected for the observation of finer structures.However,further increasing spatial resolution in SIM under the condition of strong background and noise levels remains challenging.Here,we report a method to achieve deep resolution enhancement of SIM by combining an untrained neural network with an alternating direction method of multipliers(ADMM)framework,i.e.,ADMM-DRE-SIM.By exploiting the implicit image priors in the neural network and the Hessian prior in the ADMM framework associated with the optical transfer model of SIM,ADMM-DRE-SIM can further realize the spatial frequency extension without the requirement of training datasets.Moreover,an image degradation model containing the convolution with equivalent point spread function of SIM and additional background map is utilized to suppress the strong background while keeping the structure fidelity.Experimental results by imaging tubulins and actins show that ADMM-DRE-SIM can obtain the resolution enhancement by a factor of∼1.6 compared to conventional SIM,evidencing the promising applications of ADMM-DRE-SIM in superresolution biomedical imaging.
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 National Natural Science Foundation of China under Grant No 51402026the Basic Research Program of Jiangsu Province under Grant No BK20130236the High Technology Research Key Laboratory of Changzhou under Grant No CM20133007
文摘Scanning tunnel microscopy （STM） is performed to verify if an Rh ＇nails＇ structure is formed accompanying the graphene growing during chemical vapor deposition. A structure of a graphene island in an Rh vacancy island is used as the start. While the graphene island is removed by oxygenation, the variations of the Rh vacancy island are imaged with an in-situ high-temperature STM. By fitting with our model and calculations, we conclude that the best fit is obtained for 0% Rh, i.e., for the complete absence of nails below graphene on Rh（111）. That is, when graphene is formed on Rh（111）, the substrate remains fiat and does not develop a SUPPorting nail structure.
文摘The microphase separation extent of biomedical segmented polyetherurethanes were greatly enhanced due to the presence of sulfoalkyl pendant groups contained in the hard segments,and the hard segments were more orderly aggregated through ionic interaction.
文摘We report a comprehensive numerical study of super resolution (SR) structured illumination microscopy (SIM) utilizing the classic Heintzmann-Cremer SIM process and algorithm. In particular, we investigated the impact of the diffraction limit of the underlying imaging system on the optimal SIM grating frequency that can be used to obtain the highest SR enhancement with non-continuous spatial frequency support. Besides confirming the previous theoretical and experimental work that SR-SIM can achieve an enhancement close to 3 times the diffraction limit with grating pattern illuminations, we also observe and report a series of more subtle effects of SR-SIM with non-continuous spatial frequency support. Our simulations show that when the SIM grating frequency exceeds twice that of the diffraction limit, the higher SIM grating frequency can help achieve a higher SR enhancement for the underlying imaging systems whose diffraction limit is low, though this enhancement is obtained at the cost of losing resolution at some lower resolution targets. Our simulations also show that, for underlying imaging systems with high diffraction limits, however, SR-SIM grating frequencies above twice the diffraction limits tend to bring no significant extra enhancement. Furthermore, we observed that there exists a limit grating frequency above which the SR enhancement effect is lost, and the reconstructed images essentially have the same resolution as the one obtained directly from the underlying imaging system without using the SIM process.
文摘Biochars are known for their heterogeneity, especially in pore and surface structure associated with pyrolysis processes and sources of feedstocks. The surface area of biochar is likely to be an important determinant of the extent of soil microbial attachment, whereas the porous structure of biochar is expected to provide protection for soil microorganisms. Potential interactions between biochars from different sources and with different particle sizes were investigated in relation to soil microbial properties in a short-term incubation study. Three particle size （sieved） fractions （0.5-1.0, 1.0-2.0 and 2.0-4.0 mm） from three woody biochars produced from jarrah wood, jarrah and wandoo wood and Australian wattle branches, respectively, were incubated in soil at 25 ℃ for 56 d. Observation by scanning electron microscopy （SEM） and characterisation of pore and surface area showed that all three woody biochars provided potential habitats for soil microorganisms due to their high porosity and surface areas. The biochars were structurally heterogeneous, varying in porosity and surface structure both within and between the biochar sources. After the 56-d incubation, hyphal colonisation was observed on biochar surfaces and in larger biochar pores. Soil clumping occurred on biochar particles, cementing and covering exposed biochar pores. This may have altered surface area and pore availability for microbial colonisation. Transient changes in soil microbial biomass, without a consistent trend, were observed among biochars during the 56-d incubation.
基金supported by National Natural Science Foundation of China (Nos. 22107059, 21801158, 81870283, 82070382)Program of Taishan Scholars Programme (No. 20190979)+3 种基金Academic Promotion Programme of Shandong First Medical University (No.2019LJ003)National Postdoctoral Program for Innovative Talents(No. BX2021123)The China Postdoctoral Science Foundation (No.2021M691505)the Jiangsu Postdoctoral Research Funding Program (No. 2021K125B)。
文摘Theranostic visualization of dextran at the nanoscale is beneficial for understanding the bioregulatory mechanisms of this molecule. In this study, we applied structured illumination microscopy(SIM) to capture the distribution of Cy5-Dextran at different incubation periods in living cells. The results showed that Cy5-Dextran could be absorbed by He La cells. In addition, we clarified that Cy5-Dextran exhibited differential organelle distribution(lysosomal or mitochondrial) in a time-dependent manner. Moreover,lysosomal Cy5-Dextran localization was found to be independent of the autophagy process, while Cy5-Dextran localized to the mitochondria triggered a pro-apoptotic event, upregulating the levels of reactive oxygen species(ROS) to accelerate mitochondrial fragmentation. This work uses a visualized strategy to reveal the anti-tumor bioactivity of dextran, which was achieved by regulating apoptosis and autophagy.
基金supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0086835: Dr K Y Kum)the Ministry of Science, ICT and Future Planning (2011-0014231: Dr S W Chang)supported by a grant from the Kyung Hee University in 2013 (KHU-20131045)
文摘The time domain entombment of bacteria by intratubular mineralization following orthograde canal obturation with mineral trioxide aggregate（MTA） was studied by scanning electron microscopy（SEM）. Single-rooted human premolars（n560） were instrumented to an apical size #50/0.06 using ProF ile and treated as follows： Group 1（n510） was filled with phosphate buffered saline（PBS）; Group 2（n510） was incubated with Enterococcus faecalis for 3 weeks, and then filled with PBS; Group 3（n520） was obturated orthograde with a paste of OrthoM TA（BioM TA, Seoul, Korea） and PBS; and Group 4（n520） was incubated with E. faecalis for 3 weeks and then obturated with OrthoM TA–PBS paste. Following their treatments, the coronal openings were sealed with PBS-soaked cotton and intermediate restorative material（IRM）, and the roots were then stored in PBS for 1, 2, 4, 8 or 16 weeks. After each incubation period, the roots were split and their dentin/MTA interfaces examined in both longitudinal and horizontal directions by SEM. There appeared to be an increase in intratubular mineralization over time in the OrthoM TA-filled roots（Groups 3 and 4）. Furthermore, there was a gradual entombment of bacteria within the dentinal tubules in the E. faecalis inoculated MTA-filled roots（Group 4）. Therefore, the orthograde obturation of root canals with OrthoM TA mixed with PBS may create a favorable environment for bacterial entombment by intratubular mineralization.
基金This work was supported by the Innovation Fund of WNLO(2018WNLOKF023)the Start-up Fund of Hainan University(KYQD(ZR)-20077).
文摘Hematologic malignancies are one of the most common malignant tumors caused by the clonal proliferation and differentiation of hematopoietic and lymphoid stem cells.The examination of bone marrow cells combined with immunodeficiency typing is of great significance to the diagnostic type,treatment and prognosis of hematologic malignancies.Super-resolution fluorescence microscopy(SRM)is a special kind of optical microscopy technology,which breaks the resolution limit and was awarded the Nobel Prize in Chemistry in 2014.With the development of SRM,many related technologies have been applied to the diagnosis and treatment of clinical diseases.It was reported that a major type of SRM technique,single molecule localization microscopy(SMLM),is more sensitive than flow cytometry(FC)in detecting cell membrane antigens'expression,thus enabling better chances in detecting antigens on hematopoietic cells than traditional analytic tools.Furthermore,SRM may be applied to clinical pathology and may guide precision medicine and personalized medicine for clone hematopoietic cell diseases.In this paper,we mainly discuss the application of SRM in clone hematological malignancies.
基金supported by the Joint Funds of the National Natural Science Foundation of China(No.U1532127)supported in part by National Natural Science Foundation of China(No.31570972)+1 种基金support from Shanghai Tech University and 1000 Youth Talents Programthe Chinese Central Government
文摘Functional amyloid has been increasingly applied as self-assembling nanostructures to construct multifunctional biomaterials.However,little has been known how different side domains,varied fusion positions and subunits affect self-assembly and morphologies of amyloid fibrils.Here,we constructed three groups of two-component amyloid proteins based on CsgA,the major protein components of Escherichia coli biofilms,to bridge these gaps.We showed that all fusion proteins have amyloid features,as indicated by Congo red assay.Atomic force microscopy（AFM） indeed reveals that these fusion proteins are able to self-assemble into fibrils,with an average diameter of 0.5-2 nm and length of hundreds of nanometers to several micrometers.The diameter of fibrils increases with the increase of the molecular weight of fusion domains,while the dynamic assembly of recombinant proteins was delayed as a result of the introduction of fusion domains.Moreover,fusion of the same functional domains but at intermediate position seems to cause the most interference on fibril assembly compared with those fused at C or Nterminus,as mainly short and irregular fibrils were detected.This phenomenon appears more pronounced for randomly coiled mussel foot proteins（Mfps） than for rigid chitin-binding domain（CBD）.Finally,increase of the molecular weight of tandem repeats in protein monomer seemed to increase the fibril diameter of the resultant fibrils,but either reduction of the tandem repeats of CsgA to one single belta-sheet loop or increase in the number of tandem repeats of CsgAs from one to four produced shorter and intermittent fibrils compared with CsgA control protein.These studies therefore provide insights into self-assembly of two-component amyloid proteins and lay the foundation for rational design of multifunctional molecular biomaterials.
