Light-sheet fluorescence microscopy(LSFM)has been widely used to image the three-dimensional(3D)structures and functions of various millimeter-size bio-specimen such as zebrafish.However,the sample adsorption and scat...Light-sheet fluorescence microscopy(LSFM)has been widely used to image the three-dimensional(3D)structures and functions of various millimeter-size bio-specimen such as zebrafish.However,the sample adsorption and scattering cause shading of the light-sheet illumination,preventing the even 3D image of thick samples.Herein,we report a continuous-rotational light-sheet microscope(CR-LSM)that enables simultaneous 3D bright-field and fluorescence imaging.With a high-accuracy rotational stage,CR-LSM records the outline projections and the fluorescent images of the sample at multiple rotation angles.Then,3D morphology and fluorescent structure were reconstructed with a developed algorithm.Using CR-LSM,zebrafish’s whole-fish contour and blood vessel structures were obtained simultaneously.展开更多
In light-sheet fluorescence microscopy,the axial resolution and field of view are mutually constrained.Axially swept light-sheet microscopy(ASLM)can decouple the trade-off,but the confocal detection scheme using a rol...In light-sheet fluorescence microscopy,the axial resolution and field of view are mutually constrained.Axially swept light-sheet microscopy(ASLM)can decouple the trade-off,but the confocal detection scheme using a rolling shutter also rejects fluorescence signals from the specimen in the field of interest,which sacrifices the photon efficiency.Here,we report a laterally swept light-sheet microscopy(LSLM)scheme in which the focused beam is first scanned along the axial direction and subsequently laterally swept with the rolling shutter.We show that LSLM can obtain a higher photon efficiency when similar axial resolution and field of view can be achieved.Moreover,based on the principle of image scanning microscopy,applying the pixel reassignment to the LSLM images,hereby named iLSLM,improves the optical sectioning.Both simulation and experimental results demonstrate the higher photon efficiency with similar axial resolution and optical sectioning.Our proposed scheme is suitable for volumetric imaging of specimens that are susceptible to photobleaching or phototoxicity.展开更多
One-dimensional Airy beams allow the generation of thin light-sheets without scanning,simplifying the complex optical arrangements of light-sheet microscopes(LSMs)with an extended field of view(FOV).However,their unia...One-dimensional Airy beams allow the generation of thin light-sheets without scanning,simplifying the complex optical arrangements of light-sheet microscopes(LSMs)with an extended field of view(FOV).However,their uniaxial acceleration limits the maximum numerical aperture of the detection objective in order to keep both the active and inactive axes within the depth of field.This problem is particularly pronounced in miniaturized LSM implementations,such as those for endomicroscopy or multi-photon neural imaging in freely moving animals using head-mounted miniscopes.We propose a new method to generate a static Airy light-sheet with biaxial acceleration,based on a novel phase profile.This light-sheet has the geometry of a spherical shell whose radius of curvature can be designed to match the field curvature of the micro-objective.We present an analytical model for the analysis of the light-sheet parameters and verify it by numerical simulations in the paraxial regime.We also discuss a micro-optical experimental implementation combining gradient-index optics with a 3D-nanoprinted,fully refractive phase plate.The results confirm that we are able to match detection curvatures with radii in the range of 1.5 to 2 mm.展开更多
Fluorescent nanodiamonds(FNDs)with nitrogen-vacancy centers are promising candidates for long-term biolabeling and biosensing applications due to their biocompatibility and unique optomagnetic properties.The employmen...Fluorescent nanodiamonds(FNDs)with nitrogen-vacancy centers are promising candidates for long-term biolabeling and biosensing applications due to their biocompatibility and unique optomagnetic properties.The employment of nanomaterials in cancer therapy and diagnostics requires a deep understanding of how nanoparticles(NPs)interact with the three-dimensional(3D)tumor environment.We developed the“Tumor-in-a-Tube”platform,using 4D light-sheet microscopy to explore the spatiotemporal dynamics of FNDs with 3D tumor spheroids.By monitoring the real-time NP sedimentation,spheroid penetration,and cellular uptake of FNDs and polystyrene nanoparticles(PNPs),we marked the impact of the NP mass density on their spheroid interaction.Unlike PNPs,higher-density FNDs underwent rapid sedimentation,which minimized their effective concentration and hindered the FND−spheroid interactions.This results in constrained intratumoral accumulation and size-independent uptake and penetration.Longer FND effectiveexposure time promotes size-dependent cell uptake,verified by FND treatment on 2D monolayers.Nonetheless,FNDs exhibited good biocompatibility and long-term spheroid labeling,allowing for cell isolation from different spheroid layers.Our results suggest the need for NP effective-exposure-time calibration in comparative NP assays,in 3D static models.Overall,our platform provides a valuable tool for bridging the gap between 2D and 3D static models in NP assessment,drug delivery,toxicology profiling,and translational research.展开更多
A key challenge when imaging whole biomedical specimens is how to quickly obtain massive cellular information over a large field of view(FOV).We report a subvoxel light-sheet microscopy(SLSM)method enabling high-throu...A key challenge when imaging whole biomedical specimens is how to quickly obtain massive cellular information over a large field of view(FOV).We report a subvoxel light-sheet microscopy(SLSM)method enabling high-throughput volumetric imaging of mesoscale specimens at cellular resolution.A nonaxial,continuous scanning strategy is developed to rapidly acquire a stack of large-FOV images with three-dimensional(3-D)nanoscale shifts encoded.Then,by adopting a subvoxel-resolving procedure,the SLSM method models these low-resolution,cross-correlated images in the spatial domain and can iteratively recover a 3-D image with improved resolution throughout the sample.This technique can surpass the optical limit of a conventional light-sheet microscope by more than three times,with high acquisition speeds of gigavoxels per minute.By fast reconstruction of 3-D cultured cells,intact organs,and live embryos,SLSM method presents a convenient way to circumvent the trade-off between mapping large-scale tissue(>100 mm3)and observing single cell(∼1-μm resolution).It also eliminates the need of complicated mechanical stitching or modulated illumination,using a simple light-sheet setup and fast graphics processing unit-based computation to achieve high-throughput,high-resolution 3-D microscopy,which could be tailored for a wide range of biomedical applications in pathology,histology,neuroscience,etc.展开更多
The fluorescence from the out-of-focus region excited by the sidelobes of a Bessel beam is the major concern for light-sheet fluorescence microscopy （LSFM） with Bessel beam plane illumination. Here, we propose a met...The fluorescence from the out-of-focus region excited by the sidelobes of a Bessel beam is the major concern for light-sheet fluorescence microscopy （LSFM） with Bessel beam plane illumination. Here, we propose a method of applying the subtractive imaging to overcome the limitation of the conventional LSFM with Bessel beam plane illumination. In the proposed method, the sample is imaged twice by line scanning using the extended solid Bessel beam and the ring-like Bessel beam. By subtracting between the two images with similar out-of-focus blur, the improved image quality with the suppression of the Bessel beam sidelobes and enhanced sectioning ability with improved contrast are demonstrated.展开更多
基金the National Natural Science Foundation of China(62205368)the Key Research and Development Program of Jiangsu Province(BE2020664).
文摘Light-sheet fluorescence microscopy(LSFM)has been widely used to image the three-dimensional(3D)structures and functions of various millimeter-size bio-specimen such as zebrafish.However,the sample adsorption and scattering cause shading of the light-sheet illumination,preventing the even 3D image of thick samples.Herein,we report a continuous-rotational light-sheet microscope(CR-LSM)that enables simultaneous 3D bright-field and fluorescence imaging.With a high-accuracy rotational stage,CR-LSM records the outline projections and the fluorescent images of the sample at multiple rotation angles.Then,3D morphology and fluorescent structure were reconstructed with a developed algorithm.Using CR-LSM,zebrafish’s whole-fish contour and blood vessel structures were obtained simultaneously.
基金supported by the National Natural Science Foundation of China(62005116 and 51720105015)the Science and Technology Innovation Commission of Shenzhen(KQTD20170810110913065 and 20200925174735005).
文摘In light-sheet fluorescence microscopy,the axial resolution and field of view are mutually constrained.Axially swept light-sheet microscopy(ASLM)can decouple the trade-off,but the confocal detection scheme using a rolling shutter also rejects fluorescence signals from the specimen in the field of interest,which sacrifices the photon efficiency.Here,we report a laterally swept light-sheet microscopy(LSLM)scheme in which the focused beam is first scanned along the axial direction and subsequently laterally swept with the rolling shutter.We show that LSLM can obtain a higher photon efficiency when similar axial resolution and field of view can be achieved.Moreover,based on the principle of image scanning microscopy,applying the pixel reassignment to the LSLM images,hereby named iLSLM,improves the optical sectioning.Both simulation and experimental results demonstrate the higher photon efficiency with similar axial resolution and optical sectioning.Our proposed scheme is suitable for volumetric imaging of specimens that are susceptible to photobleaching or phototoxicity.
基金This project has received funding from the European Union’s Horizon 2020 Research and Innovation Program under Grant Agreement No.871212.
文摘One-dimensional Airy beams allow the generation of thin light-sheets without scanning,simplifying the complex optical arrangements of light-sheet microscopes(LSMs)with an extended field of view(FOV).However,their uniaxial acceleration limits the maximum numerical aperture of the detection objective in order to keep both the active and inactive axes within the depth of field.This problem is particularly pronounced in miniaturized LSM implementations,such as those for endomicroscopy or multi-photon neural imaging in freely moving animals using head-mounted miniscopes.We propose a new method to generate a static Airy light-sheet with biaxial acceleration,based on a novel phase profile.This light-sheet has the geometry of a spherical shell whose radius of curvature can be designed to match the field curvature of the micro-objective.We present an analytical model for the analysis of the light-sheet parameters and verify it by numerical simulations in the paraxial regime.We also discuss a micro-optical experimental implementation combining gradient-index optics with a 3D-nanoprinted,fully refractive phase plate.The results confirm that we are able to match detection curvatures with radii in the range of 1.5 to 2 mm.
基金supported by Independent Research Fund Denmark(grant no.0135-00142B)the Novo Nordisk Foundation(grant no.NNF20OC0061673)the Danish National Research Council(grant no.DNRF116).
文摘Fluorescent nanodiamonds(FNDs)with nitrogen-vacancy centers are promising candidates for long-term biolabeling and biosensing applications due to their biocompatibility and unique optomagnetic properties.The employment of nanomaterials in cancer therapy and diagnostics requires a deep understanding of how nanoparticles(NPs)interact with the three-dimensional(3D)tumor environment.We developed the“Tumor-in-a-Tube”platform,using 4D light-sheet microscopy to explore the spatiotemporal dynamics of FNDs with 3D tumor spheroids.By monitoring the real-time NP sedimentation,spheroid penetration,and cellular uptake of FNDs and polystyrene nanoparticles(PNPs),we marked the impact of the NP mass density on their spheroid interaction.Unlike PNPs,higher-density FNDs underwent rapid sedimentation,which minimized their effective concentration and hindered the FND−spheroid interactions.This results in constrained intratumoral accumulation and size-independent uptake and penetration.Longer FND effectiveexposure time promotes size-dependent cell uptake,verified by FND treatment on 2D monolayers.Nonetheless,FNDs exhibited good biocompatibility and long-term spheroid labeling,allowing for cell isolation from different spheroid layers.Our results suggest the need for NP effective-exposure-time calibration in comparative NP assays,in 3D static models.Overall,our platform provides a valuable tool for bridging the gap between 2D and 3D static models in NP assessment,drug delivery,toxicology profiling,and translational research.
基金This research has received funding support from the 1000 Youth Talents Plan of China(P.F.)the Fundamental Research Program of Shenzhen(P.F.,JCYJ20160429182424047)+2 种基金and the National Heart Lung and Blood Institute[R01HL111437(T.K.H.)R01HL083015(T.K.H.),R01HL118650(T.K.H.)and EB U54 EB0220002(T.K.H.)].
文摘A key challenge when imaging whole biomedical specimens is how to quickly obtain massive cellular information over a large field of view(FOV).We report a subvoxel light-sheet microscopy(SLSM)method enabling high-throughput volumetric imaging of mesoscale specimens at cellular resolution.A nonaxial,continuous scanning strategy is developed to rapidly acquire a stack of large-FOV images with three-dimensional(3-D)nanoscale shifts encoded.Then,by adopting a subvoxel-resolving procedure,the SLSM method models these low-resolution,cross-correlated images in the spatial domain and can iteratively recover a 3-D image with improved resolution throughout the sample.This technique can surpass the optical limit of a conventional light-sheet microscope by more than three times,with high acquisition speeds of gigavoxels per minute.By fast reconstruction of 3-D cultured cells,intact organs,and live embryos,SLSM method presents a convenient way to circumvent the trade-off between mapping large-scale tissue(>100 mm3)and observing single cell(∼1-μm resolution).It also eliminates the need of complicated mechanical stitching or modulated illumination,using a simple light-sheet setup and fast graphics processing unit-based computation to achieve high-throughput,high-resolution 3-D microscopy,which could be tailored for a wide range of biomedical applications in pathology,histology,neuroscience,etc.
基金supported by the National Natural Science Foundation of China(Nos.61665006,61661028,61565012,and 61378062)the Natural Science Foundation of Jiangxi Province(Nos.20161BAB212041,20162BCB23012,and 20171ACB21018)
文摘The fluorescence from the out-of-focus region excited by the sidelobes of a Bessel beam is the major concern for light-sheet fluorescence microscopy （LSFM） with Bessel beam plane illumination. Here, we propose a method of applying the subtractive imaging to overcome the limitation of the conventional LSFM with Bessel beam plane illumination. In the proposed method, the sample is imaged twice by line scanning using the extended solid Bessel beam and the ring-like Bessel beam. By subtracting between the two images with similar out-of-focus blur, the improved image quality with the suppression of the Bessel beam sidelobes and enhanced sectioning ability with improved contrast are demonstrated.
