Morphological analyses are key outcome assessments for nerve regeneration studies but are historically limited to tissue sections.Novel optical tissue clearing techniques enabling three-dimensional imaging of entire o...Morphological analyses are key outcome assessments for nerve regeneration studies but are historically limited to tissue sections.Novel optical tissue clearing techniques enabling three-dimensional imaging of entire organs at a subcellular resolution have revolutionized morphological studies of the brain.To extend their applicability to experimental nerve repair studies we adapted these techniques to nerves and their motor and sensory targets in rats.The solvent-based protocols rendered harvested peripheral nerves and their target organs transparent within 24 hours while preserving tissue architecture and fluorescence.The optical clearing was compatible with conventional laboratory techniques,including retrograde labeling studies,and computational image segmentation,providing fast and precise cell quantitation.Further,optically cleared organs enabled three-dimensional morphometry at an unprecedented scale including dermatome-wide innervation studies,tracing of intramuscular nerve branches or mapping of neurovascular networks.Given their wide-ranging applicability,rapid processing times,and low costs,tissue clearing techniques are likely to be a key technology for next-generation nerve repair studies.All procedures were approved by the Hospital for Sick Children’s Laboratory Animal Services Committee(49871/9)on November 9,2019.展开更多
Successful establishment of reconnection between retinal ganglion cells and retinorecipient regions in the brain is critical to optic nerve regeneration.However,morphological assessments of retinorecipient regions are...Successful establishment of reconnection between retinal ganglion cells and retinorecipient regions in the brain is critical to optic nerve regeneration.However,morphological assessments of retinorecipient regions are limited by the opacity of brain tissue.In this study,we used an innovative tissue cleaning technique combined with retrograde trans-synaptic viral tracing to observe changes in retinorecipient regions connected to retinal ganglion cells in mice after optic nerve injury.Specifically,we performed light-sheet imaging of whole brain tissue after a clearing process.We found that pseudorabies virus 724(PRV724)mostly infected retinal ganglion cells,and that we could use it to retrogradely trace the retinorecipient regions in whole tissue-cleared brains.Unexpectedly,PRV724-traced neurons were more widely distributed compared with data from previous studies.We found that optic nerve injury could selectively modify projections from retinal ganglion cells in the hypothalamic paraventricular nucleus,intergeniculate leaflet,ventral lateral geniculate nucleus,central amygdala,basolateral amygdala,Edinger-Westphal nucleus,and oculomotor nucleus,but not the superior vestibular nucleus,red nucleus,locus coeruleus,gigantocellular reticular nucleus,or facial nerve nucleus.Our findings demonstrate that the tissue clearing technique,combined with retrograde trans-synaptic viral tracing,can be used to objectively and comprehensively evaluate changes in mouse retinorecipient regions that receive projections from retinal ganglion cells after optic nerve injury.Thus,our approach may be useful for future estimations of optic nerve injury and regeneration.展开更多
Organisms are composed of a vast number of highly specialized cells with characteristic physiological functions determined by their structure.Therefore,understanding physiological and pathological mechanisms requires ...Organisms are composed of a vast number of highly specialized cells with characteristic physiological functions determined by their structure.Therefore,understanding physiological and pathological mechanisms requires obtaining 3-dimensional(3D)structural information at the cellular level across tissues,organs,and even the whole body.However,the scattering and absorption of tissue limits the penetration depth of light and thus extremely reduces the imaging performance in deep tissue.It is well known that 3D imaging outperforms 2D imaging in various scenarios for 3D reconstruction of tissue structures.This includes tracing convoluted 3D structures like vasculature,neurons,and prostate glands,as well as elucidating intricate distributions of cells or lesions.展开更多
Confocal Raman microspectroscopy(CRM)with 633-and 785-nm excitation wavelengths combined with optical clearing(OC)technique was used for ex-vivo study of porcine skin in the Raman fingerprint region.The optical cleari...Confocal Raman microspectroscopy(CRM)with 633-and 785-nm excitation wavelengths combined with optical clearing(OC)technique was used for ex-vivo study of porcine skin in the Raman fingerprint region.The optical clearing has been performed on the skin samples by applying a mixture of glycerol and distilled water and a mixture of glycerol,distilled water and chemical penetration enhancer dimethyl sulfoxide(DMSO)during 30 min and 60 min of treat-ment.It was shown that the combined use of the optical clearing technique and CRM at 633 nm allowed one to preserve the high probing depth,signal-to-noise ratio and spectral resolution simultaneously.Comparing the effect of different optical clearing agents on porcine skin showed that an optical clearing agent containing chemical penetration enhancer provides higher optical clearing efficiency.Also,an increase in treatment time allows to improve the optical clearing efficiency of both optical clearing agents.As a result of optical clearing,the detection of the amide-Ⅲ spectral region indicating well-distinguishable structural differences between the type-Ⅰ and type-Ⅳ collagens has been improved.展开更多
The current work is focused on the study of optical clearing of skeletal muscles under local compression.The experiments were performed on in vitro bovine skeletal muscle.The time dependence of optical clearing was st...The current work is focused on the study of optical clearing of skeletal muscles under local compression.The experiments were performed on in vitro bovine skeletal muscle.The time dependence of optical clearing was studied by monitoring the luminescence intensity of NaYF_(4)∶Er,Yb upconverting particles located under tissue layers.This study shows the possibility to use upconverting nanoparticles(UCNPs)both for studying the dynamics of the optical clearing of biological tissue under compression and to detect moments of cell wall damage under excessive pressure.The advantage of using UCNPs is the presence of several bands in their luminescence spectra,located both at close wavelengths and far apart.展开更多
For the first time,the changes in autofluorescence spectra of ex vivo rat skin have been experimentally investigated using the combination of fluorescence spectroscopy and optical immersion clearing.The glucose,glycer...For the first time,the changes in autofluorescence spectra of ex vivo rat skin have been experimentally investigated using the combination of fluorescence spectroscopy and optical immersion clearing.The glucose,glycerol and propylene glycol solutions were used as clearing agents.The optical clearing was performed from the dermal side of skin imitating the in vivo injection of clearing agent under the dermal layers.In this contribution,the common properties of autofluorescence variation during optical immersion clearing were determined.The tendency of autofluorescence signal to decrease with reduction of scattering in tissue was noticed and discussed in detail.However,the differences in the shape of spectral curves under application of different clearing agents showed that optical clearing affects the autofluorescence properties of tissue differently depending on the type of clearing liquid.The results obtained are useful for the understanding of tissue optical clearing mechanisms and for improving techniques such as fluorescence spectroscopy.展开更多
Three-dimensional(3D)cell cultures have contributed to a variety of biological research fields by filling the gap between monolayers and animal models.The modern optical sectioning microscopic methods make it possible...Three-dimensional(3D)cell cultures have contributed to a variety of biological research fields by filling the gap between monolayers and animal models.The modern optical sectioning microscopic methods make it possible to probe the complexity of 3D cell cultures but are limited by the inherent opaqueness.While tissue optical clearing methods have emerged as powerful tools for investigating whole-mount tissues in 3D,they often have limitations,such as being too harsh for fragile 3D cell cultures,requiring complex handling protocols,or inducing tissue deformation with shrinkage or expansion.To address this issue,we proposed a modified optical clearing method for 3D cell cultures,called MACS-W,which is simple,highly efficient,and morphology-preserving.In our evaluation of MACS-W,we found that it exhibits excellent clearing capability in just 10 min,with minimal deformation,and helps drug evaluation on tumor spheroids.In summary,MACS-W is a fast,minimally-deformative and fluorescence compatible clearing method that has the potential to be widely used in the studies of 3D cell cultures.展开更多
Dear Editor,The mammalian brain exhibits cross-scale complexity in neuronal morphology and connectivity,the study of which demands high-resolution morphological reconstruction of individual neurons across the entire b...Dear Editor,The mammalian brain exhibits cross-scale complexity in neuronal morphology and connectivity,the study of which demands high-resolution morphological reconstruction of individual neurons across the entire brain[1-4].Current commonly used approaches for such mesoscale brain mapping include two main types of three-dimensional fluorescence microscopy:the block-face methods,and the lightsheet-based methods[5,6].In general,the high imaging speed and light efficiency of light-sheet microscopy make it a suitable tool for high-throughput volumetric imaging,especially when combined with tissue-clearing techniques.However,large brain samples pose major challenges to this approach.展开更多
Three-dimensional reconstruction of tissue architecture is crucial for biomedical research.Tissue optical clearing technology overcomes light scattering limitations in biological tissues,providing an essential tool fo...Three-dimensional reconstruction of tissue architecture is crucial for biomedical research.Tissue optical clearing technology overcomes light scattering limitations in biological tissues,providing an essential tool for high-resolution three-dimensional imaging.Given the high degree of similarity between large model animals(e.g.,pigs,non-human primates)and humans in terms of anatomical structure,physiologic function,and disease mechanisms,the application of this technology in these models holds significant value for biomedical research.While well-established tissue clearing protocols exist for tissue sections,whole organs,and even entire bodies in rodents,scaling up to large animal specimens presents substantial challenges due to dimensional effects and compositional variations.This review systematically examines the methodological translation from rodent to large animals,particularly on species-specific differences in brain architecture and parenchymal organ composition that critically impact clearing efficiency.We comprehensively summarize recent applications in large animals,focusing on representative areas including neural circuit mapping,sensory organ imaging,and other related research domains,while proposing optimization strategies to overcome cross-species compatibility barriers.We hope this review will serve as a valuable reference for advancing tissue optical clearing applications in large-animal biomedical research.展开更多
As the control center of organisms, the brain remains little understood due to its complexity. Taking advantage of imaging methods, scientists have found an accessible approach to unraveling the mystery of neuroscienc...As the control center of organisms, the brain remains little understood due to its complexity. Taking advantage of imaging methods, scientists have found an accessible approach to unraveling the mystery of neuroscience. Among these methods, optical imaging techniques are widely used due to their high molecular specificity and single-molecule sensitivity. Here, we overview several optical imaging techniques in neuroscience of recent years, including brain clearing, the micro-optical sectioning tomography system, and deep tissue imaging.展开更多
Neurodegenerative disease is a brain disorder caused by the loss of structure andfunction of neurons that lowers the quality of human life. Apart from the limitedpotential for endogenous regeneration, stem cell-based ...Neurodegenerative disease is a brain disorder caused by the loss of structure andfunction of neurons that lowers the quality of human life. Apart from the limitedpotential for endogenous regeneration, stem cell-based therapies hold considerablepromise for maintaining homeostatic tissue regeneration and enhancingplasticity. Despite many studies, there remains insufficient evidence for stem celltracing and its correlation with endogenous neural cells in brain tissue with threedimensionalstructures. Recent advancements in tissue optical clearing techniqueshave been developed to overcome the existing shortcomings of cross-sectionaltissue analysis in thick and complex tissues. This review focuses on recentprogress of stem cell treatments to improve neurodegenerative disease, andintroduces tissue optical clearing techniques that can implement a threedimensionalimage as a proof of concept. This review provides a more comprehensiveunderstanding of stem cell tracing that will play an important role inevaluating therapeutic efficacy and cellular interrelationship for regeneration inneurodegenerative diseases.展开更多
Diabetes mellitus(DM)is a kind of metabolic disorder characterized by chronic hyperglycemia and glucose intolerance due to absolute or relative lack of insulin,leading to chronic damage of vasculature within various o...Diabetes mellitus(DM)is a kind of metabolic disorder characterized by chronic hyperglycemia and glucose intolerance due to absolute or relative lack of insulin,leading to chronic damage of vasculature within various organ systems.These detrimental e®ects on the vascular networks will result in the development of various diseases associated with microvascular injury.Modern optical imaging techniques provide essential tools for accurate evaluation of the structural and functional changes of blood vessels down to capillaries level,which can o®er valuable insight on understanding the development of DM-associated complications and design of targeted therapy.This review will brie°y introduce the DM-induced structural and functional alterations of vasculature within di®erent organs such as skin,cerebrum and kidneys,as well as how novel optical imaging techniques facilitate the studies focusing on exploration of these pathological changes of vasculature caused by DM both in-vivo and ex-vivo.展开更多
Coordinated contraction of skeletal muscles relies on selective connections between the muscles and multiple classes of the spinal motoneuro ns.Howeve r,current research on the spatial location of the spinal motoneuro...Coordinated contraction of skeletal muscles relies on selective connections between the muscles and multiple classes of the spinal motoneuro ns.Howeve r,current research on the spatial location of the spinal motoneurons innervating differe nt muscles is limited.In this study,we investigated the spatial distribution and relative position of different motoneurons that control the deep muscles of the mouse hindlimbs,which were innervated by the obturator nerve,femoral nerve,inferior gluteal nerve,deep pe roneal nerve,and tibial nerve.Locations were visualized by combining a multiplex retrograde tracking technique compatible with three-dimensional imaging of solvent-cleared o rgans(3DISCO)and 3-D imaging technology based on lightsheet fluorescence microscopy(LSFM).Additionally,we propose the hypothesis that"messenger zones"exist as interlaced areas between the motoneuron pools that dominate the synergistic or antagonist muscle groups.We hypothesize that these interlaced neurons may participate in muscle coordination as messenger neurons.Analysis revealed the precise mutual positional relationships among the many motoneurons that innervate different deep muscles of the mouse.Not only do these findings update and supplement our knowledge regarding the overall spatial layout of spinal motoneurons that control mouse limb muscles,but they also provide insights into the mechanisms through which muscle activity is coordinated and the architecture of motor circuits.展开更多
Three-dimensional（3D） histology utilizes tissue clearing techniques to turn intact tissues transparent,allowing rapid interrogation of tissue architecture in three dimensions.In this article,we summarized the availa...Three-dimensional（3D） histology utilizes tissue clearing techniques to turn intact tissues transparent,allowing rapid interrogation of tissue architecture in three dimensions.In this article,we summarized the available tissue clearing methods and classified them according to their physicochemical principles of operation,which provided a framework for one to choose the best techniques for various research settings.Recent attempts in addressing various questions regarding the degenerating and regenerating nervous system have been promising with the use of 3D histological techniques.展开更多
Motor endplates(MEPs) are important sites of information exchange between motor neurons and skeletal muscle, and are distributed in an organized pattern of lamellae in the muscle. Delayed repair of peripheral nerve in...Motor endplates(MEPs) are important sites of information exchange between motor neurons and skeletal muscle, and are distributed in an organized pattern of lamellae in the muscle. Delayed repair of peripheral nerve injury typically results in unsatisfactory functional recovery because of MEP degeneration. In this study, the mouse tibial nerve was transected and repaired with a biodegradable chitin conduit, immediately following or 1 or 3 months after the injury. Fluorescent α-bungarotoxin was injected to label MEPs. Tissue optical clearing combined with light-sheet microscopy revealed that MEPs were distributed in an organized pattern of lamellae in skeletal muscle after delayed repair for 1 and 3 months. However, the total number of MEPs, the number of MEPs per lamellar cluster, and the maturation of single MEPs in gastrocnemius muscle gradually decreased with increasing denervation time. These findings suggest that delayed repair can restore the spatial distribution of MEPs, but it has an adverse effect on the homogeneity of MEPs in the lamellar clusters and the total number of MEPs in the target muscle. The study procedures were approved by the Animal Ethics Committee of the Peking University People's Hospital(approval No. 2019 PHC015) on April 8, 2019.展开更多
Ischemic stroke causes long-term disability and results in motor impairments.Such impairments are associated with structural changes in the neuromuscular junction(NMJ),including detailed morphology and three-dimension...Ischemic stroke causes long-term disability and results in motor impairments.Such impairments are associated with structural changes in the neuromuscular junction(NMJ),including detailed morphology and three-dimensional(3D)distribution.However,previous studies only explored morphological changes of individual NMJs after stroke,which limits the understanding of their role in post-stroke motor impairment.Here,we examine 3D distributions and detailed morphology of NMJs in entire mouse muscles after unilateral and bilateral strokes induced by photothrombosis.The results show that 3D distributions and numbers of NMJs do not change after stroke,and severe unilateral stroke causes similar levels of NMJ fragmentation and area enlargement to bilateral stroke.This research provides structural data,deepening the understanding of neuromuscular pathophysiology after stroke.展开更多
With the objective to study the variation of optical properties of rat muscle during optical clearing,we have performed a set of optical measurements from that kind of tissue.The.measurements performed were total tr a...With the objective to study the variation of optical properties of rat muscle during optical clearing,we have performed a set of optical measurements from that kind of tissue.The.measurements performed were total tr ansmittance,ollimated transmit tance,specular reflec-tance and total reflectance.This set of measurements is suficient to determine diffuse reflectance and absorbance of the sample,also necessary to est imate the optical properties.All the per formed measurements and calculated quantities will be used later in inverse Monte Carlo(IMC)simu-lations to determine the evolution of the optical properties of muscle during treatments with ethylene glycol and glucose.The results obt ained with the measurements already provide some information about the optical c learing treatments applied to the muscle and translate the mechanisms of turning the tissue more transparent and sequence of regimes of optical clearing.展开更多
The quest for high spatial resolution in molecular identification is critical across various domains,including physiology,pathology,and pharmaceutical research.Super-resolution microscopy has made strides by surpassin...The quest for high spatial resolution in molecular identification is critical across various domains,including physiology,pathology,and pharmaceutical research.Super-resolution microscopy has made strides by surpassing the Abbe diffraction limit,but it relies on sophisticated equipment and is limited by the sample size to handle.Expansion microscopy,an emerging technique,has broadened the scope of subdiffraction imaging.It chemically preserves tissues at a large scale and physically enlarges them 4−20 times linearly,enabling superresolution observation.This review begins by exploring the foundational concepts of tissue clearing and the latest methodologies in the field.It then delves into the core tenets of expansion microscopy,covering a range of protocols.The review spotlights advancements in enhancing resolution,improving labeling efficiency,and ensuring isotropic tissue expansion.Finally,the review offers insights into the prospective evolution of expansion microscopy.It emphasizes the potential role of machine learning in refining image quality and in the autonomous extraction of data,which could revolutionize the way we visualize and understand biological tissues.展开更多
Spinal motoneurons control muscle fibers contraction and drive all motor behaviors in vertebrates.Although spinal motoneurons share the fundamental role of innervating muscle fibers,they exhibit remarkable diversity t...Spinal motoneurons control muscle fibers contraction and drive all motor behaviors in vertebrates.Although spinal motoneurons share the fundamental role of innervating muscle fibers,they exhibit remarkable diversity that reflects their specific identities.Defining the morphological changes during postnatal development is critical for elucidating this diversity.However,our understanding of the three-dimensional(3D)morphology of spinal motoneurons at these stages remains limited,largely due to the lack of high-throughput imaging tools.Using tiling light sheet microscopy combined with tissue clearing methods,we imaged motoneurons of the lateral and median motor column in the cervical and lumbar cord during postnatal development.By analyzing their soma size,we found that motoneurons innervating the upper limbs differentiate into two subpopulations with distinct soma size by postnatal day 14(P14),while differentiation of motoneurons innervating the lower limbs is delayed.Furthermore,coupling adenovirus labeling with 3D volumetric reconstruction,we traced and measured the number and lengths of dendrites of flexor and extensor motoneurons in the lumbar cord,finding that the number of dendrites initially increases and subsequently declines as dendritic order rises.Together,these findings provide a quantitative analysis of the 3D morphological changes underlying spinal motoneuron diversity.展开更多
Two-photon fluorescence microscopy(TPFM)is widely used for imaging of biological tissue due to its robustness to scattering,high resolution,and ease of multiplexing fluorescent probes.However,TPFM volumetric imaging r...Two-photon fluorescence microscopy(TPFM)is widely used for imaging of biological tissue due to its robustness to scattering,high resolution,and ease of multiplexing fluorescent probes.However,TPFM volumetric imaging rates are typically low,limiting the ability to image whole cleared tissues and large surgical specimens.While innovations in TPFM technology,such as parallel-scanning,have drastically increased imaging speed,these improvements have typically focused on high frame rate,single field-of-view imaging rather than extending the area/volume imaging rate.In this work,we bridge the gap between high imaging speed and high area and volumetric imaging throughput by combining parallel scanning with tilted-plane strip-scanning using custom silicon photomultiplier(SiPM)tiled-array detectors.We demonstrate 200 MP/s with four spectral channels(800 MSpectra/s)and an effective area imaging speed of up to 52 mm^(2)∕s using four parallel beams.Custom detectors and lens array enable non-descanned imaging with minimal crosstalk combined with light collection efficiency comparable to a conventional single-point scanning TPFM.Finally,the low-cost of the custom detectors(∼$250 per channel)and the scalability of the detection optics allow for ease of spectral multiplexing.展开更多
基金the German Research Foundation(DA 2255/1-1,to SCD).
文摘Morphological analyses are key outcome assessments for nerve regeneration studies but are historically limited to tissue sections.Novel optical tissue clearing techniques enabling three-dimensional imaging of entire organs at a subcellular resolution have revolutionized morphological studies of the brain.To extend their applicability to experimental nerve repair studies we adapted these techniques to nerves and their motor and sensory targets in rats.The solvent-based protocols rendered harvested peripheral nerves and their target organs transparent within 24 hours while preserving tissue architecture and fluorescence.The optical clearing was compatible with conventional laboratory techniques,including retrograde labeling studies,and computational image segmentation,providing fast and precise cell quantitation.Further,optically cleared organs enabled three-dimensional morphometry at an unprecedented scale including dermatome-wide innervation studies,tracing of intramuscular nerve branches or mapping of neurovascular networks.Given their wide-ranging applicability,rapid processing times,and low costs,tissue clearing techniques are likely to be a key technology for next-generation nerve repair studies.All procedures were approved by the Hospital for Sick Children’s Laboratory Animal Services Committee(49871/9)on November 9,2019.
基金supported by the National Natural Science Foundation of China,No.81870655(to MBY)。
文摘Successful establishment of reconnection between retinal ganglion cells and retinorecipient regions in the brain is critical to optic nerve regeneration.However,morphological assessments of retinorecipient regions are limited by the opacity of brain tissue.In this study,we used an innovative tissue cleaning technique combined with retrograde trans-synaptic viral tracing to observe changes in retinorecipient regions connected to retinal ganglion cells in mice after optic nerve injury.Specifically,we performed light-sheet imaging of whole brain tissue after a clearing process.We found that pseudorabies virus 724(PRV724)mostly infected retinal ganglion cells,and that we could use it to retrogradely trace the retinorecipient regions in whole tissue-cleared brains.Unexpectedly,PRV724-traced neurons were more widely distributed compared with data from previous studies.We found that optic nerve injury could selectively modify projections from retinal ganglion cells in the hypothalamic paraventricular nucleus,intergeniculate leaflet,ventral lateral geniculate nucleus,central amygdala,basolateral amygdala,Edinger-Westphal nucleus,and oculomotor nucleus,but not the superior vestibular nucleus,red nucleus,locus coeruleus,gigantocellular reticular nucleus,or facial nerve nucleus.Our findings demonstrate that the tissue clearing technique,combined with retrograde trans-synaptic viral tracing,can be used to objectively and comprehensively evaluate changes in mouse retinorecipient regions that receive projections from retinal ganglion cells after optic nerve injury.Thus,our approach may be useful for future estimations of optic nerve injury and regeneration.
基金funded by the National Nature Science Foundation of China(grant nos.62375096,61860206009,32361133552,and 82361138569)the Russian Science Foundation(grant no.24-44-00082)。
文摘Organisms are composed of a vast number of highly specialized cells with characteristic physiological functions determined by their structure.Therefore,understanding physiological and pathological mechanisms requires obtaining 3-dimensional(3D)structural information at the cellular level across tissues,organs,and even the whole body.However,the scattering and absorption of tissue limits the penetration depth of light and thus extremely reduces the imaging performance in deep tissue.It is well known that 3D imaging outperforms 2D imaging in various scenarios for 3D reconstruction of tissue structures.This includes tracing convoluted 3D structures like vasculature,neurons,and prostate glands,as well as elucidating intricate distributions of cells or lesions.
基金Ths work was supported by the VEKOP-2.3.2-16-2016-00011 Grantwhich is co-financed by the European Union and European Social FundValery V.Tuchin was supported by RFBR Grant 18-52-16025 and the Grant of the Government of the Russian Federation(Registration No.2020-220-08-2389).
文摘Confocal Raman microspectroscopy(CRM)with 633-and 785-nm excitation wavelengths combined with optical clearing(OC)technique was used for ex-vivo study of porcine skin in the Raman fingerprint region.The optical clearing has been performed on the skin samples by applying a mixture of glycerol and distilled water and a mixture of glycerol,distilled water and chemical penetration enhancer dimethyl sulfoxide(DMSO)during 30 min and 60 min of treat-ment.It was shown that the combined use of the optical clearing technique and CRM at 633 nm allowed one to preserve the high probing depth,signal-to-noise ratio and spectral resolution simultaneously.Comparing the effect of different optical clearing agents on porcine skin showed that an optical clearing agent containing chemical penetration enhancer provides higher optical clearing efficiency.Also,an increase in treatment time allows to improve the optical clearing efficiency of both optical clearing agents.As a result of optical clearing,the detection of the amide-Ⅲ spectral region indicating well-distinguishable structural differences between the type-Ⅰ and type-Ⅳ collagens has been improved.
基金This work was supported by the Russian Science Foundation,project no.19-12-00118.
文摘The current work is focused on the study of optical clearing of skeletal muscles under local compression.The experiments were performed on in vitro bovine skeletal muscle.The time dependence of optical clearing was studied by monitoring the luminescence intensity of NaYF_(4)∶Er,Yb upconverting particles located under tissue layers.This study shows the possibility to use upconverting nanoparticles(UCNPs)both for studying the dynamics of the optical clearing of biological tissue under compression and to detect moments of cell wall damage under excessive pressure.The advantage of using UCNPs is the presence of several bands in their luminescence spectra,located both at close wavelengths and far apart.
文摘For the first time,the changes in autofluorescence spectra of ex vivo rat skin have been experimentally investigated using the combination of fluorescence spectroscopy and optical immersion clearing.The glucose,glycerol and propylene glycol solutions were used as clearing agents.The optical clearing was performed from the dermal side of skin imitating the in vivo injection of clearing agent under the dermal layers.In this contribution,the common properties of autofluorescence variation during optical immersion clearing were determined.The tendency of autofluorescence signal to decrease with reduction of scattering in tissue was noticed and discussed in detail.However,the differences in the shape of spectral curves under application of different clearing agents showed that optical clearing affects the autofluorescence properties of tissue differently depending on the type of clearing liquid.The results obtained are useful for the understanding of tissue optical clearing mechanisms and for improving techniques such as fluorescence spectroscopy.
基金support from the National Key Research and Development Program of China(Grant No.2017YFA0700501),and the Innovation Fund of WNLO.
文摘Three-dimensional(3D)cell cultures have contributed to a variety of biological research fields by filling the gap between monolayers and animal models.The modern optical sectioning microscopic methods make it possible to probe the complexity of 3D cell cultures but are limited by the inherent opaqueness.While tissue optical clearing methods have emerged as powerful tools for investigating whole-mount tissues in 3D,they often have limitations,such as being too harsh for fragile 3D cell cultures,requiring complex handling protocols,or inducing tissue deformation with shrinkage or expansion.To address this issue,we proposed a modified optical clearing method for 3D cell cultures,called MACS-W,which is simple,highly efficient,and morphology-preserving.In our evaluation of MACS-W,we found that it exhibits excellent clearing capability in just 10 min,with minimal deformation,and helps drug evaluation on tumor spheroids.In summary,MACS-W is a fast,minimally-deformative and fluorescence compatible clearing method that has the potential to be widely used in the studies of 3D cell cultures.
基金supported by the STI 2030-Major Project(2021ZD0204400,2022ZD0205203,2021ZD0200104,2022ZD0211900)the Shenzhen Science and Technology Program(RCYX20210706092100003,RCBS20221008093311027)+3 种基金the Shenzhen Medical Research Funds(A2303005)the Youth Innovation Promotion Association CAS(2022367)the National Natural Science Foundation of China(32100896)NSFC-Guangdong Joint Fund(U20A6005).
文摘Dear Editor,The mammalian brain exhibits cross-scale complexity in neuronal morphology and connectivity,the study of which demands high-resolution morphological reconstruction of individual neurons across the entire brain[1-4].Current commonly used approaches for such mesoscale brain mapping include two main types of three-dimensional fluorescence microscopy:the block-face methods,and the lightsheet-based methods[5,6].In general,the high imaging speed and light efficiency of light-sheet microscopy make it a suitable tool for high-throughput volumetric imaging,especially when combined with tissue-clearing techniques.However,large brain samples pose major challenges to this approach.
基金supported by the National Natural Science Foundation of China(Grant Nos.62375096,82361138569,82372012,82402341)the Open Competition Project of Wuhan East Lake High-tech Development Zone(Grant No.2023KJB224)the Innovation Project of Optics Valley Laboratory(Grant No.OVL2025BB008).
文摘Three-dimensional reconstruction of tissue architecture is crucial for biomedical research.Tissue optical clearing technology overcomes light scattering limitations in biological tissues,providing an essential tool for high-resolution three-dimensional imaging.Given the high degree of similarity between large model animals(e.g.,pigs,non-human primates)and humans in terms of anatomical structure,physiologic function,and disease mechanisms,the application of this technology in these models holds significant value for biomedical research.While well-established tissue clearing protocols exist for tissue sections,whole organs,and even entire bodies in rodents,scaling up to large animal specimens presents substantial challenges due to dimensional effects and compositional variations.This review systematically examines the methodological translation from rodent to large animals,particularly on species-specific differences in brain architecture and parenchymal organ composition that critically impact clearing efficiency.We comprehensively summarize recent applications in large animals,focusing on representative areas including neural circuit mapping,sensory organ imaging,and other related research domains,while proposing optimization strategies to overcome cross-species compatibility barriers.We hope this review will serve as a valuable reference for advancing tissue optical clearing applications in large-animal biomedical research.
基金supported by the National Basic Research Development Program(973 Program)of China(2015CB352005)the National Natural Science Foundation of China(6142780065,81527901,and 31571110)+1 种基金Natural Science Foundation of Zhejiang Province of China(Y16F050002)Fundamental Research Funds for the Central Universities of China
文摘As the control center of organisms, the brain remains little understood due to its complexity. Taking advantage of imaging methods, scientists have found an accessible approach to unraveling the mystery of neuroscience. Among these methods, optical imaging techniques are widely used due to their high molecular specificity and single-molecule sensitivity. Here, we overview several optical imaging techniques in neuroscience of recent years, including brain clearing, the micro-optical sectioning tomography system, and deep tissue imaging.
基金the National Research Foundation of Korea(NRF)Grant funded by the Korea government(MSIT),No.NRF-2020R1C1C1013535.
文摘Neurodegenerative disease is a brain disorder caused by the loss of structure andfunction of neurons that lowers the quality of human life. Apart from the limitedpotential for endogenous regeneration, stem cell-based therapies hold considerablepromise for maintaining homeostatic tissue regeneration and enhancingplasticity. Despite many studies, there remains insufficient evidence for stem celltracing and its correlation with endogenous neural cells in brain tissue with threedimensionalstructures. Recent advancements in tissue optical clearing techniqueshave been developed to overcome the existing shortcomings of cross-sectionaltissue analysis in thick and complex tissues. This review focuses on recentprogress of stem cell treatments to improve neurodegenerative disease, andintroduces tissue optical clearing techniques that can implement a threedimensionalimage as a proof of concept. This review provides a more comprehensiveunderstanding of stem cell tracing that will play an important role inevaluating therapeutic efficacy and cellular interrelationship for regeneration inneurodegenerative diseases.
基金the National Key Research and Development Program of China(Grant No.2017YFA0700501)the National Natu-ral Science Foundation of China(Grant Nos.61860206009,81870934,62105113 and 81961138015)+1 种基金China Postdoctoral Science Foundation Funded Project(Nos.BX20200138,BX20190131,2021M691145 and 2019M662633)the Innovation Fund of WNLO.
文摘Diabetes mellitus(DM)is a kind of metabolic disorder characterized by chronic hyperglycemia and glucose intolerance due to absolute or relative lack of insulin,leading to chronic damage of vasculature within various organ systems.These detrimental e®ects on the vascular networks will result in the development of various diseases associated with microvascular injury.Modern optical imaging techniques provide essential tools for accurate evaluation of the structural and functional changes of blood vessels down to capillaries level,which can o®er valuable insight on understanding the development of DM-associated complications and design of targeted therapy.This review will brie°y introduce the DM-induced structural and functional alterations of vasculature within di®erent organs such as skin,cerebrum and kidneys,as well as how novel optical imaging techniques facilitate the studies focusing on exploration of these pathological changes of vasculature caused by DM both in-vivo and ex-vivo.
基金supported by the Chinese National General Program of the National Natural Science Foundation of China,No.82072162(to XY)。
文摘Coordinated contraction of skeletal muscles relies on selective connections between the muscles and multiple classes of the spinal motoneuro ns.Howeve r,current research on the spatial location of the spinal motoneurons innervating differe nt muscles is limited.In this study,we investigated the spatial distribution and relative position of different motoneurons that control the deep muscles of the mouse hindlimbs,which were innervated by the obturator nerve,femoral nerve,inferior gluteal nerve,deep pe roneal nerve,and tibial nerve.Locations were visualized by combining a multiplex retrograde tracking technique compatible with three-dimensional imaging of solvent-cleared o rgans(3DISCO)and 3-D imaging technology based on lightsheet fluorescence microscopy(LSFM).Additionally,we propose the hypothesis that"messenger zones"exist as interlaced areas between the motoneuron pools that dominate the synergistic or antagonist muscle groups.We hypothesize that these interlaced neurons may participate in muscle coordination as messenger neurons.Analysis revealed the precise mutual positional relationships among the many motoneurons that innervate different deep muscles of the mouse.Not only do these findings update and supplement our knowledge regarding the overall spatial layout of spinal motoneurons that control mouse limb muscles,but they also provide insights into the mechanisms through which muscle activity is coordinated and the architecture of motor circuits.
文摘Three-dimensional（3D） histology utilizes tissue clearing techniques to turn intact tissues transparent,allowing rapid interrogation of tissue architecture in three dimensions.In this article,we summarized the available tissue clearing methods and classified them according to their physicochemical principles of operation,which provided a framework for one to choose the best techniques for various research settings.Recent attempts in addressing various questions regarding the degenerating and regenerating nervous system have been promising with the use of 3D histological techniques.
基金supported by the National Natural Science Foundation of China,Nos.82072162(to XFY),81971177the Natural Science Foundation of Beijing of China,No.7192215(to XFY)。
文摘Motor endplates(MEPs) are important sites of information exchange between motor neurons and skeletal muscle, and are distributed in an organized pattern of lamellae in the muscle. Delayed repair of peripheral nerve injury typically results in unsatisfactory functional recovery because of MEP degeneration. In this study, the mouse tibial nerve was transected and repaired with a biodegradable chitin conduit, immediately following or 1 or 3 months after the injury. Fluorescent α-bungarotoxin was injected to label MEPs. Tissue optical clearing combined with light-sheet microscopy revealed that MEPs were distributed in an organized pattern of lamellae in skeletal muscle after delayed repair for 1 and 3 months. However, the total number of MEPs, the number of MEPs per lamellar cluster, and the maturation of single MEPs in gastrocnemius muscle gradually decreased with increasing denervation time. These findings suggest that delayed repair can restore the spatial distribution of MEPs, but it has an adverse effect on the homogeneity of MEPs in the lamellar clusters and the total number of MEPs in the target muscle. The study procedures were approved by the Animal Ethics Committee of the Peking University People's Hospital(approval No. 2019 PHC015) on April 8, 2019.
基金supported by the National Natural Science Foundation of China(No.61860206009)the Key Research and Development Project of Hubei Province(No.2022BCA023)the Innovation Fund of WNLO.
文摘Ischemic stroke causes long-term disability and results in motor impairments.Such impairments are associated with structural changes in the neuromuscular junction(NMJ),including detailed morphology and three-dimensional(3D)distribution.However,previous studies only explored morphological changes of individual NMJs after stroke,which limits the understanding of their role in post-stroke motor impairment.Here,we examine 3D distributions and detailed morphology of NMJs in entire mouse muscles after unilateral and bilateral strokes induced by photothrombosis.The results show that 3D distributions and numbers of NMJs do not change after stroke,and severe unilateral stroke causes similar levels of NMJ fragmentation and area enlargement to bilateral stroke.This research provides structural data,deepening the understanding of neuromuscular pathophysiology after stroke.
基金supported in part by grants:RF Governmental contracts 11.519.11.2035,14.B37.21.0728,and 14.B37.11.0563FiDiPro,TEKES Program(40111/11),Finland+1 种基金SCOPES EC,Uzb/Switz/RF,Swiss NSF,IZ74ZO 137423/1RF President's grant"Scientific Schools",1177.2012.2.
文摘With the objective to study the variation of optical properties of rat muscle during optical clearing,we have performed a set of optical measurements from that kind of tissue.The.measurements performed were total tr ansmittance,ollimated transmit tance,specular reflec-tance and total reflectance.This set of measurements is suficient to determine diffuse reflectance and absorbance of the sample,also necessary to est imate the optical properties.All the per formed measurements and calculated quantities will be used later in inverse Monte Carlo(IMC)simu-lations to determine the evolution of the optical properties of muscle during treatments with ethylene glycol and glucose.The results obt ained with the measurements already provide some information about the optical c learing treatments applied to the muscle and translate the mechanisms of turning the tissue more transparent and sequence of regimes of optical clearing.
基金the financial support from the Fundamental Research Funds for the Central Universities(Grant Nos.14380532,2024300419)Natural science foundation of Jiangsu province(Grant Nos.BK20220780,BE2022818).
文摘The quest for high spatial resolution in molecular identification is critical across various domains,including physiology,pathology,and pharmaceutical research.Super-resolution microscopy has made strides by surpassing the Abbe diffraction limit,but it relies on sophisticated equipment and is limited by the sample size to handle.Expansion microscopy,an emerging technique,has broadened the scope of subdiffraction imaging.It chemically preserves tissues at a large scale and physically enlarges them 4−20 times linearly,enabling superresolution observation.This review begins by exploring the foundational concepts of tissue clearing and the latest methodologies in the field.It then delves into the core tenets of expansion microscopy,covering a range of protocols.The review spotlights advancements in enhancing resolution,improving labeling efficiency,and ensuring isotropic tissue expansion.Finally,the review offers insights into the prospective evolution of expansion microscopy.It emphasizes the potential role of machine learning in refining image quality and in the autonomous extraction of data,which could revolutionize the way we visualize and understand biological tissues.
基金supported by Westlake Education Foundation,the National Natural Science Foundation of China(32150015)the Zhejiang Province Natural Science Foundation(LR20 C070002).
文摘Spinal motoneurons control muscle fibers contraction and drive all motor behaviors in vertebrates.Although spinal motoneurons share the fundamental role of innervating muscle fibers,they exhibit remarkable diversity that reflects their specific identities.Defining the morphological changes during postnatal development is critical for elucidating this diversity.However,our understanding of the three-dimensional(3D)morphology of spinal motoneurons at these stages remains limited,largely due to the lack of high-throughput imaging tools.Using tiling light sheet microscopy combined with tissue clearing methods,we imaged motoneurons of the lateral and median motor column in the cervical and lumbar cord during postnatal development.By analyzing their soma size,we found that motoneurons innervating the upper limbs differentiate into two subpopulations with distinct soma size by postnatal day 14(P14),while differentiation of motoneurons innervating the lower limbs is delayed.Furthermore,coupling adenovirus labeling with 3D volumetric reconstruction,we traced and measured the number and lengths of dendrites of flexor and extensor motoneurons in the lumbar cord,finding that the number of dendrites initially increases and subsequently declines as dendritic order rises.Together,these findings provide a quantitative analysis of the 3D morphological changes underlying spinal motoneuron diversity.
基金National Institute of Biomedical Imaging and Bioengineering(R21-EB032839)National Cancer Institute(R37-CA258376)。
文摘Two-photon fluorescence microscopy(TPFM)is widely used for imaging of biological tissue due to its robustness to scattering,high resolution,and ease of multiplexing fluorescent probes.However,TPFM volumetric imaging rates are typically low,limiting the ability to image whole cleared tissues and large surgical specimens.While innovations in TPFM technology,such as parallel-scanning,have drastically increased imaging speed,these improvements have typically focused on high frame rate,single field-of-view imaging rather than extending the area/volume imaging rate.In this work,we bridge the gap between high imaging speed and high area and volumetric imaging throughput by combining parallel scanning with tilted-plane strip-scanning using custom silicon photomultiplier(SiPM)tiled-array detectors.We demonstrate 200 MP/s with four spectral channels(800 MSpectra/s)and an effective area imaging speed of up to 52 mm^(2)∕s using four parallel beams.Custom detectors and lens array enable non-descanned imaging with minimal crosstalk combined with light collection efficiency comparable to a conventional single-point scanning TPFM.Finally,the low-cost of the custom detectors(∼$250 per channel)and the scalability of the detection optics allow for ease of spectral multiplexing.
