Traumatic penumbra(TP)is a region with recoverable potential around the primary lesion of brain injury.Rapid and accurate imaging for identifying TP is essential for treating traumatic brain injury(TBI).In this study,...Traumatic penumbra(TP)is a region with recoverable potential around the primary lesion of brain injury.Rapid and accurate imaging for identifying TP is essential for treating traumatic brain injury(TBI).In this study,we first established traumatic brain injuries(TBIs)in rats using a modified Feeney method,followed by label-free imaging of brain tissue sections with multiphoton fluorescence microscopy.The results showed that the technique effectively imaged normal and traumatic brain tissues,and revealed pathological features such as extracellular matrix changes,vascular cell proliferation,and intracellular edema in the traumatic penumbra.Compared with normal brain tissue,the extracellular matrix in the TP was sparse,cells were disorganized,and hyperplastic vascular cells emitted higher two-photon excited fluorescence(TPEF)signals.Our research demonstrates the potential of multiphoton fluorescence technology in the rapid diagnosis and therapeutic evaluation of TBI.展开更多
INSULIN secretion was traditionally measured with biochemical and immunological methods such as enzyme linked immunosorbant assay and radioimmunoassay. However, these methods can only tell the amount of insulin secret...INSULIN secretion was traditionally measured with biochemical and immunological methods such as enzyme linked immunosorbant assay and radioimmunoassay. However, these methods can only tell the amount of insulin secreted; they give no information about the secretion process or mechanism of exocytosis. In recent years, an imaging technique known as total internal reflection fluorescence （TIRF） microscopy has been employed to study insulin secretion.展开更多
An ultimate goal of neuroscience is to decipher the principles underlying neuronal information processing at the molecular,cellular,circuit,and system levels.The advent of miniature fluorescence microscopy has further...An ultimate goal of neuroscience is to decipher the principles underlying neuronal information processing at the molecular,cellular,circuit,and system levels.The advent of miniature fluorescence microscopy has furthered the quest by visualizing brain activities and structural dynamics in animals engaged in self-determined behaviors.In this brief review,we summarize recent advances in miniature fluorescence microscopy for neuroscience,focusing mostly on two mainstream solutions-miniature single-photon microscopy,and miniature two-photon microscopy.We discuss their technical advantages and limitations as well as unmet challenges for future improvement.Examples of preliminary applications are also presented to reflect on a new trend of brain imaging in experimental paradigms involving body movements,long and complex protocols,and even disease progression and aging.展开更多
Single-particle microbeam as a powerful tool can open a research field to find answers to many enigmas in radiobiology. A single-particle microbeam facility has been constructed at the Key Laboratory of Ion Beam Bioen...Single-particle microbeam as a powerful tool can open a research field to find answers to many enigmas in radiobiology. A single-particle microbeam facility has been constructed at the Key Laboratory of Ion Beam Bioengineering （LIBB）, Chinese Academy of Sciences （CAS）, China. However there has been less research activities in this field concerning the original process of the interaction between low-energy ions and complicated organisms. To address this challenge, an in situ multi-dimensional quantitative fluorescence microscopy system combined with the CAS-LIBB single-particle microbeam II endstation is proposed. In this article, the rationale, logistics and development of many aspects of the proposed system are discussed.展开更多
Background:Fluorescence microscopy has increasingly promising applications in life science.This bibliometrics-based review focuses on deep learning assisted fluorescence microscopy imaging techniques.Methods:Papers on...Background:Fluorescence microscopy has increasingly promising applications in life science.This bibliometrics-based review focuses on deep learning assisted fluorescence microscopy imaging techniques.Methods:Papers on this topic retrieved by Core Collection on Web of Science between 2017 and July 2022 were used for the analysis.In addition to presenting the representative papers that have received the most attention,the process of development of the topic,the structure of authors and institutions,the selection of journals,and the keywords are analyzed in detail in this review.Results:The analysis found that this topic gained immediate popularity among scholars from its emergence in 2017,gaining explosive growth within three years.This phenomenon is because deep learning techniques that have been well established in other fields can be migrated to the analysis of fluorescence micrographs.From 2020 onwards,this topic tapers off but has attracted a few stable research groups to tackle the remaining challenges.Although this topic has been very popular,it has not attracted scientists from all over the world.The USA,China,Germany,and the UK are the key players in this topic.Keyword analysis and clustering are applied to understand the different focuses on this topic.Conclusion:Based on the bibliometric analysis,the current state of this topic to date and future perspectives are summarized at the end.展开更多
Fluorescence microscopy is indispensable in life science research,yet denoising remains challenging due to varied biological samples and imaging conditions.We introduce a wavelet-enhanced transformer based on DnCNN th...Fluorescence microscopy is indispensable in life science research,yet denoising remains challenging due to varied biological samples and imaging conditions.We introduce a wavelet-enhanced transformer based on DnCNN that fuses wavelet preprocessing with a dual-branch transformer-convolutional neural network(CNN)architecture.Wavelet decomposition separates highand low-frequency components for targeted noise reduction;the CNN branch restores local details,whereas the transformer branch captures global context;and an adaptive loss balances quantitative fidelity with perceptual quality.On the fluorescence microscopy denoising benchmark,our method surpasses leading CNNand transformer-based approaches,improving peak signal-to-noise ratio by 2.34%and 0.88%and structural similarity index measure by 0.53%and 1.07%,respectively.This framework offers enhanced generalization and practical gains for fluorescence image denoising.展开更多
Time-resolved volumetric fluorescence imaging over an extended duration with high spatial/temporal resolution is a key driving force in biomedical research for investigating spatial-temporal dynamics at organism-level...Time-resolved volumetric fluorescence imaging over an extended duration with high spatial/temporal resolution is a key driving force in biomedical research for investigating spatial-temporal dynamics at organism-level systems,yet it remains a major challenge due to the trade-off among imaging speed,light exposure,illumination power,and image quality.Here,we present a deep-learning enhanced light sheet fluorescence microscopy(LSFM)approach that addresses the restoration of rapid volumetric time-lapse imaging with less than 0.03%light exposure and 3.3%acquisition time compared to a typical standard acquisition.We demonstrate that the convolutional neural network(CNN)-transformer network developed here,namely U-net integrated transformer(UI-Trans),successfully achieves the mitigation of complex noise-scattering-coupled degradation and outperforms state-of-the-art deep learning networks,due to its capability of faithfully learning fine details while comprehending complex global features.With the fast generation of appropriate training data via flexible switching between confocal line-scanning LSFM(LS-LSFM)and conventional LSFM,this method achieves a three-to five-fold signal-to-noise ratio(SNR)improvement and~1.8 times contrast improvement in ex vivo zebrafish heart imaging and long-term in vivo 4D(3D morphology+time)imaging of heartbeat dynamics at different developmental stages with ultra-economical acquisitions in terms of light dosage and acquisition time.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
Light-field microscopy(LFM)enables rapid volumetric imaging through single-frame acquisition and fast 3D reconstruction algorithms.The high speed and low phototoxicity of LFM make it highly suitable for real-time 3D f...Light-field microscopy(LFM)enables rapid volumetric imaging through single-frame acquisition and fast 3D reconstruction algorithms.The high speed and low phototoxicity of LFM make it highly suitable for real-time 3D fluorescence imaging,such as studies of neural activity monitoring and blood flow analysis.However,in in vivo fluorescence imaging scenarios,the light intensity needs to be reduced as much as possible to achieve longer-term observations.The resulting low signal-to-noise ratio(SNR)caused by reduced light intensity significantly degrades the quality of 3D reconstruction in LFM.Existing deep-learning-based methods struggle to incorporate the structured intensity distribution and noise characteristics inherent to LFM data,often leading to artifacts and uneven energy distributions.To address these challenges,we propose the denoise-weighted view-channel-depth(DNW-VCD)network,integrating a two-step noise model and energy weight matrix into an LFM reconstruction framework.Additionally,we developed an attenuator-induced imaging system for dual-SNR image acquisition to validate DNW-VCD’s performance.Experimental results show that our method achieves artifact-reduced,realtime 3D imaging with isotropic resolution and lower phototoxicity,as verified through imaging of fluorescent beads,algae,and zebrafish heart.展开更多
Resolution is undoubtedly the most important parameter in optical microscopy by providing an estimation on the maximum resolving power of a certain optical microscope. For centuries, the resolution of an optical micro...Resolution is undoubtedly the most important parameter in optical microscopy by providing an estimation on the maximum resolving power of a certain optical microscope. For centuries, the resolution of an optical microscope is generally considered to be limited only by the numerical aperture of the optical system and the wavelength of light. However, since the invention and popularity of various advanced fluorescence microscopy techniques, especially super-resolution fluorescence microscopy, many new methods have been proposed for estimating the resolution, leading to confusions for researchers who need to quantify the resolution of their fluorescence microscopes. In this paper, we firstly summarize the early concepts and criteria for predicting the resolution limit of an ideal optical system. Then, we discuss some important influence factors that deteriorate the resolution of a certain fluorescence microscope. Finally, we provide methods and examples on how to measure the resolution of a fluorescence microscope from captured fluorescence images. This paper aims to answer as best as possible the theoretical and practical issues regarding the resolution estimation in fluorescence microscopy.展开更多
Fluorescent studies of living plant cells such as confocal microscopy and fluorescence lifetime imaging often suffer from a strong autofluorescent background contribution that significantly reduces the dynamic image c...Fluorescent studies of living plant cells such as confocal microscopy and fluorescence lifetime imaging often suffer from a strong autofluorescent background contribution that significantly reduces the dynamic image contrast and the quantitative access to sub-cellular processes at high spatial resolution. Here, we present a novel technique--fluorescence intensity decay shape analysis microscopy （FIDSAM） to enhance the dynamic contrast of a fluorescence image of at least one order of magnitude. The method is based on the analysis of the shape of the fluorescence intensity decay （fluorescence lifetime curve） and benefits from the fact that the decay patterns of typical fluorescence label dyes strongly differ from emission decay curves of autofluorescent sample areas. Using FIDSAM, we investigated Arabidopsis thaliana hypocotyl cells in their tissue environment, which accumulate an eGFP fusion of the plasma membrane marker protein LTI6b （LTI6b-eGFP） to low level. Whereas in conventional confocal fluorescence images, the membranes of neighboring cells can hardly be optically resolved due to the strong autofluorescence of the cell wall, FIDSAM allows for imaging of single, isolated membranes at high spatial resolution. Thus, FIDSAM will enable the sub-cellular analysis of even low-expressed fluorophoretagged proteins in living plant cells. Furthermore, the combination of FIDSAM with fluorescence lifetime imaging provides the basis to study the local physico-chemical environment of fluorophore-tagged biomolecules in living plant cells.展开更多
To maximize signal collection in nonlinear optical microscopy,non-descanned epi-detection is generally adopted for in vivo imaging.However,because of severe scattering in biological samples,most of the emitted fluores...To maximize signal collection in nonlinear optical microscopy,non-descanned epi-detection is generally adopted for in vivo imaging.However,because of severe scattering in biological samples,most of the emitted fluorescence photons go beyond the collection angles of objectives and thus cannot be detected.Here,we propose an extended detection scheme to enhance the collection of scattered photons in nonlinear fluorescence microscopy using a silicon photomultiplier array ahead of the front apertures of objectives.We perform numerical simulations to demonstrate the enhanced fluorescence collection via extended epi-detection in the multi-photon fluorescence imaging of human skin and mouse brain through craniotomy windows and intact skulls.For example,with red fluorescence emission at a depth of 600μm in human skin,the increased collection can be as much as about 150%with a 10×,0.6-NA objective.We show that extended epi-detection is a generally applicable,feasible technique for use in nonlinear fluorescence microscopy to enhance signal detection.展开更多
Dear EditorProbing protein-protein interaction has become a routine practice in the post genomic era. Multiple in vitro or in vivo techniques have been developed to detect or report direct or indirect interactions of ...Dear EditorProbing protein-protein interaction has become a routine practice in the post genomic era. Multiple in vitro or in vivo techniques have been developed to detect or report direct or indirect interactions of functionally related proteins （Lalonde et al., 2008）. These techniques sometimes are technically challenging, however, because the readout would demand sophisticated detectors and/or complicated calculations. Besides, a common drawback of many of these techniques is they can render inherent false positives to various degrees so that an interaction often cannot be judged unambiguously.展开更多
Plasmonic nanostructures have been proved effective not only in catalyzing chemical reactions,but also in improving the activity of non-plasmonic photocatalysts.It is essential to reveal the synergy between the plasmo...Plasmonic nanostructures have been proved effective not only in catalyzing chemical reactions,but also in improving the activity of non-plasmonic photocatalysts.It is essential to reveal the synergy between the plasmonic structure and the non-plasmonic metal photocatalyst for expounding the underlying mechanism of plasmon-enhanced catalysis.Herein,the enhancement of resazurin reduction at the heterostructure of silver nanowire(AgNW)and palladium nanoparticles(PdNPs)is observed in situ by single-molecule fluorescence microscopy.The catalysis mapping results around single AgNW suggest that the catalytic activity of PdNPs is enhanced for~20 times due to the excitation of localized surface plasmon resonance(LSPR)in the vicinity of the AgNW.This catalysis enhancement is also highly related to the wavelength and polarization of the excitation light.In addition,the palladium catalysis is further enhanced by~10 times in the vicinity of a roughened AgNW or a AgNW-AgNW nanogap because of the improvement of catalytic hotspots.These findings clarify the contribution of plasmon excitation in palladium catalysis at microscopic scale,which will help to deepen the understanding of the plasmon-enhanced photocatalysis and provide a guideline for developing highly efficient plasmon-based photocatalysts.展开更多
Super-resolution optical imaging is crucial to the study of cellular processes.Current super-resolution fluorescence microscopy is restricted by the need of special fluorophores or sophisticated optical systems,or lon...Super-resolution optical imaging is crucial to the study of cellular processes.Current super-resolution fluorescence microscopy is restricted by the need of special fluorophores or sophisticated optical systems,or long acquisition and computational times.In this work,we present a deep-learning-based super-resolution technique of confocal microscopy.We devise a two-channel attention network(TCAN),which takes advantage of both spatial representations and frequency contents to learn a more precise mapping from low-resolution images to high-resolution ones.This scheme is robust against changes in the pixel size and the imaging setup,enabling the optimal model to generalize to different fluorescence microscopy modalities unseen in the training set.Our algorithm is validated on diverse biological structures and dual-color confocal images of actin-microtubules,improving the resolution from~230 nm to~110 nm.Last but not least,we demonstrate live-cell super-resolution imaging by revealing the detailed structures and dynamic instability of microtubules.展开更多
With super-resolution microscopy,we attempt to visualize(biological)structures and processes at the sub-cellular level(i.e.,nanoscale).To obtain this information,the samples are labeled with fluorophores that have a s...With super-resolution microscopy,we attempt to visualize(biological)structures and processes at the sub-cellular level(i.e.,nanoscale).To obtain this information,the samples are labeled with fluorophores that have a stochastic on/off switching of their emissions,which help to overcome the optical diffraction limit of around 250 nm,related to the use of optical micro-scopes.However,nowadays,research focuses on the imaging of live cells and thicker samples.These investigations require a high amount of simultaneously active fluorophores(i.e.,high-density imaging)and are challenging due to the collapse of the single-molecule localization techniques and the increased background in the image.Therefore,recent efforts have shifted towards the development of new ways to process the data.This publication gives an introduction to wide-field super-resolution fluorescence microscopy,explaining the concepts of the technique,and then gives an overview of the recently developed methods to provide super-resolution images for high-density data of live cells and ways to overcome the issues related to the imaging of these samples.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
Understanding the heterogeneous catalytic properties of nanoparticles is of great significance for the development of high efficient nanocatalysts, but the intrinsic heterogeneities of nanocatalysts were always covere...Understanding the heterogeneous catalytic properties of nanoparticles is of great significance for the development of high efficient nanocatalysts, but the intrinsic heterogeneities of nanocatalysts were always covered in traditional ensemble studies. This issue can be overcome if one can follow the catalysis of individual nanoparticles in real time. This paper mainly summarizes recent developments in single- molecule nanocatalysis at single particle level in Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. These developments include the revealing of catalytic kinetics of different types （plane ＆ edge） of surface atoms on individual Pd nanocubes, the observing of in situ deactivation of indi- vidual carbon-supported Pt nanoparticles during the electrocatalytic hydrogen-oxidation reaction, and the measurement of catalytic activation energies on single nanocatalysts for both product formation process and dissociation process, etc. These studies further indicate the advantages or unique abilities of single-molecule methods in the studies of nanocatalvsis or even chemical reactions.展开更多
Titanium dioxide （TiO2） nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA d...Titanium dioxide （TiO2） nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical patholog36 to detect TiO2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods （transmission electron microscopy, X-ray fluorescence microscop~ etc.） have low throughput and technical and operational complications. Herein, we describe two in situ post- treatment labeling approaches to stain TiO2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO2 nanoparticles with alkyne- conjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy （XFM） at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Therefore, future experiments with TiO2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.展开更多
Fluorescence microscopic imaging is essentially a convolution process distorted by random noise,limiting critical parameters such as imaging speed,duration,and resolution.Though algorithmic compensation has shown grea...Fluorescence microscopic imaging is essentially a convolution process distorted by random noise,limiting critical parameters such as imaging speed,duration,and resolution.Though algorithmic compensation has shown great potential to enhance these pivotal aspects,its fidelity remains questioned.Here we develop a physics-rooted computational resolution extension and denoising method with ensured fidelity.Our approach employs a multi-resolution analysis(MRA)framework to extract the two main characteristics of fluorescence images against noise:across-edge contrast,and along-edge continuity.By constraining the two features in a model-solution framework using framelet and curvelet,we develop MRA deconvolution algorithms,which improve the signal-to-noise ratio(SNR)up to 10 dB higher than spatial derivative based penalties,and can provide up to two-fold fidelity-ensured resolution improvement rather than the artifact-prone Richardson-Lucy inference.We demonstrate our methods can improve the performance of various diffraction-limited and super-resolution microscopies with ensured fidelity,enabling accomplishments of more challenging imaging tasks.展开更多
基金funded by the Science and Technology Research Program of Chongqing Municipal Education Commission(KJZD-K202301105,KJQN202201107)the Scienti¯c and Technological Transformative Program of Chongqing Banan District(KY202208161124020).
文摘Traumatic penumbra(TP)is a region with recoverable potential around the primary lesion of brain injury.Rapid and accurate imaging for identifying TP is essential for treating traumatic brain injury(TBI).In this study,we first established traumatic brain injuries(TBIs)in rats using a modified Feeney method,followed by label-free imaging of brain tissue sections with multiphoton fluorescence microscopy.The results showed that the technique effectively imaged normal and traumatic brain tissues,and revealed pathological features such as extracellular matrix changes,vascular cell proliferation,and intracellular edema in the traumatic penumbra.Compared with normal brain tissue,the extracellular matrix in the TP was sparse,cells were disorganized,and hyperplastic vascular cells emitted higher two-photon excited fluorescence(TPEF)signals.Our research demonstrates the potential of multiphoton fluorescence technology in the rapid diagnosis and therapeutic evaluation of TBI.
文摘INSULIN secretion was traditionally measured with biochemical and immunological methods such as enzyme linked immunosorbant assay and radioimmunoassay. However, these methods can only tell the amount of insulin secreted; they give no information about the secretion process or mechanism of exocytosis. In recent years, an imaging technique known as total internal reflection fluorescence （TIRF） microscopy has been employed to study insulin secretion.
基金We thank Dr.Zhe Zhao and Dr.Haitao Wu for helping with the experiments for Fig.2D,and Dr.Weijian Zong for discussion.This work was supported by grants from the National Natural Science Foundation of China(31327901,31570839,61975002,31830036,31821091,and 8182780030)the Major State Basic Research Program of China(2016 YFA0500400 and 2016YFA0500403)and the National Postdoctoral Program for Innovative Talents of China(BX20190011).
文摘An ultimate goal of neuroscience is to decipher the principles underlying neuronal information processing at the molecular,cellular,circuit,and system levels.The advent of miniature fluorescence microscopy has furthered the quest by visualizing brain activities and structural dynamics in animals engaged in self-determined behaviors.In this brief review,we summarize recent advances in miniature fluorescence microscopy for neuroscience,focusing mostly on two mainstream solutions-miniature single-photon microscopy,and miniature two-photon microscopy.We discuss their technical advantages and limitations as well as unmet challenges for future improvement.Examples of preliminary applications are also presented to reflect on a new trend of brain imaging in experimental paradigms involving body movements,long and complex protocols,and even disease progression and aging.
文摘Single-particle microbeam as a powerful tool can open a research field to find answers to many enigmas in radiobiology. A single-particle microbeam facility has been constructed at the Key Laboratory of Ion Beam Bioengineering （LIBB）, Chinese Academy of Sciences （CAS）, China. However there has been less research activities in this field concerning the original process of the interaction between low-energy ions and complicated organisms. To address this challenge, an in situ multi-dimensional quantitative fluorescence microscopy system combined with the CAS-LIBB single-particle microbeam II endstation is proposed. In this article, the rationale, logistics and development of many aspects of the proposed system are discussed.
文摘Background:Fluorescence microscopy has increasingly promising applications in life science.This bibliometrics-based review focuses on deep learning assisted fluorescence microscopy imaging techniques.Methods:Papers on this topic retrieved by Core Collection on Web of Science between 2017 and July 2022 were used for the analysis.In addition to presenting the representative papers that have received the most attention,the process of development of the topic,the structure of authors and institutions,the selection of journals,and the keywords are analyzed in detail in this review.Results:The analysis found that this topic gained immediate popularity among scholars from its emergence in 2017,gaining explosive growth within three years.This phenomenon is because deep learning techniques that have been well established in other fields can be migrated to the analysis of fluorescence micrographs.From 2020 onwards,this topic tapers off but has attracted a few stable research groups to tackle the remaining challenges.Although this topic has been very popular,it has not attracted scientists from all over the world.The USA,China,Germany,and the UK are the key players in this topic.Keyword analysis and clustering are applied to understand the different focuses on this topic.Conclusion:Based on the bibliometric analysis,the current state of this topic to date and future perspectives are summarized at the end.
基金supported by the National Natural Science Foundation of China(Grant No.62275210)the National Leading Talent Program,the National Young Talent Program,the Key Research and Development Program of Shaanxi(Grant No.2024SF2-GJHX-25)+5 种基金the Scientific Research Program Funded by the Education Department of Shaanxi Provincial Government(Grant No.24JS016)the Xidian University Specially Funded Project for Interdisciplinary Exploration(Grant No.TZJHF202523)the Fundamental Research Funds for Central Universities(Grant No.YJSJ25014)the Guangdong Provincial General Colleges and Universities Young Innovative Talents Research Project(Grant No.2024KQNCX172)the Shenzhen Science and Technology Program(Grant No.GJHZ20210705141805015)the Key Research Areas Support Science and Technology Project of Shenzhen Institute of Information Technology(Grant No.SZIIT2024KJ056).
文摘Fluorescence microscopy is indispensable in life science research,yet denoising remains challenging due to varied biological samples and imaging conditions.We introduce a wavelet-enhanced transformer based on DnCNN that fuses wavelet preprocessing with a dual-branch transformer-convolutional neural network(CNN)architecture.Wavelet decomposition separates highand low-frequency components for targeted noise reduction;the CNN branch restores local details,whereas the transformer branch captures global context;and an adaptive loss balances quantitative fidelity with perceptual quality.On the fluorescence microscopy denoising benchmark,our method surpasses leading CNNand transformer-based approaches,improving peak signal-to-noise ratio by 2.34%and 0.88%and structural similarity index measure by 0.53%and 1.07%,respectively.This framework offers enhanced generalization and practical gains for fluorescence image denoising.
基金supported by National Natural Science Foundation of China(52122008,52270008,52370003,62025502)Guangdong Introducing Innovative and Entrepreneurial Teams of“The Pearl River Talent Recruitment Program”(2021ZT09X044)Shenzhen Technology University under Grant JSZZ202301010.
文摘Time-resolved volumetric fluorescence imaging over an extended duration with high spatial/temporal resolution is a key driving force in biomedical research for investigating spatial-temporal dynamics at organism-level systems,yet it remains a major challenge due to the trade-off among imaging speed,light exposure,illumination power,and image quality.Here,we present a deep-learning enhanced light sheet fluorescence microscopy(LSFM)approach that addresses the restoration of rapid volumetric time-lapse imaging with less than 0.03%light exposure and 3.3%acquisition time compared to a typical standard acquisition.We demonstrate that the convolutional neural network(CNN)-transformer network developed here,namely U-net integrated transformer(UI-Trans),successfully achieves the mitigation of complex noise-scattering-coupled degradation and outperforms state-of-the-art deep learning networks,due to its capability of faithfully learning fine details while comprehending complex global features.With the fast generation of appropriate training data via flexible switching between confocal line-scanning LSFM(LS-LSFM)and conventional LSFM,this method achieves a three-to five-fold signal-to-noise ratio(SNR)improvement and~1.8 times contrast improvement in ex vivo zebrafish heart imaging and long-term in vivo 4D(3D morphology+time)imaging of heartbeat dynamics at different developmental stages with ultra-economical acquisitions in terms of light dosage and acquisition time.
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.
基金National Natural Science Foundation of China(T2221003,62025108,62071219,62371006)China Postdoctoral Science Foundation(GZC20230057)。
文摘Light-field microscopy(LFM)enables rapid volumetric imaging through single-frame acquisition and fast 3D reconstruction algorithms.The high speed and low phototoxicity of LFM make it highly suitable for real-time 3D fluorescence imaging,such as studies of neural activity monitoring and blood flow analysis.However,in in vivo fluorescence imaging scenarios,the light intensity needs to be reduced as much as possible to achieve longer-term observations.The resulting low signal-to-noise ratio(SNR)caused by reduced light intensity significantly degrades the quality of 3D reconstruction in LFM.Existing deep-learning-based methods struggle to incorporate the structured intensity distribution and noise characteristics inherent to LFM data,often leading to artifacts and uneven energy distributions.To address these challenges,we propose the denoise-weighted view-channel-depth(DNW-VCD)network,integrating a two-step noise model and energy weight matrix into an LFM reconstruction framework.Additionally,we developed an attenuator-induced imaging system for dual-SNR image acquisition to validate DNW-VCD’s performance.Experimental results show that our method achieves artifact-reduced,realtime 3D imaging with isotropic resolution and lower phototoxicity,as verified through imaging of fluorescent beads,algae,and zebrafish heart.
基金supported by the National Natural Science Foundation of China (81427801, 81827901)National Basic Research Program of China (2015CB352003)+2 种基金Science Fund for Creative Research Groups (61721092)Fundamental Research Funds for the Central Universities (2018KFYXKJC039)Director Fund of WNLO。
文摘Resolution is undoubtedly the most important parameter in optical microscopy by providing an estimation on the maximum resolving power of a certain optical microscope. For centuries, the resolution of an optical microscope is generally considered to be limited only by the numerical aperture of the optical system and the wavelength of light. However, since the invention and popularity of various advanced fluorescence microscopy techniques, especially super-resolution fluorescence microscopy, many new methods have been proposed for estimating the resolution, leading to confusions for researchers who need to quantify the resolution of their fluorescence microscopes. In this paper, we firstly summarize the early concepts and criteria for predicting the resolution limit of an ideal optical system. Then, we discuss some important influence factors that deteriorate the resolution of a certain fluorescence microscope. Finally, we provide methods and examples on how to measure the resolution of a fluorescence microscope from captured fluorescence images. This paper aims to answer as best as possible the theoretical and practical issues regarding the resolution estimation in fluorescence microscopy.
文摘Fluorescent studies of living plant cells such as confocal microscopy and fluorescence lifetime imaging often suffer from a strong autofluorescent background contribution that significantly reduces the dynamic image contrast and the quantitative access to sub-cellular processes at high spatial resolution. Here, we present a novel technique--fluorescence intensity decay shape analysis microscopy （FIDSAM） to enhance the dynamic contrast of a fluorescence image of at least one order of magnitude. The method is based on the analysis of the shape of the fluorescence intensity decay （fluorescence lifetime curve） and benefits from the fact that the decay patterns of typical fluorescence label dyes strongly differ from emission decay curves of autofluorescent sample areas. Using FIDSAM, we investigated Arabidopsis thaliana hypocotyl cells in their tissue environment, which accumulate an eGFP fusion of the plasma membrane marker protein LTI6b （LTI6b-eGFP） to low level. Whereas in conventional confocal fluorescence images, the membranes of neighboring cells can hardly be optically resolved due to the strong autofluorescence of the cell wall, FIDSAM allows for imaging of single, isolated membranes at high spatial resolution. Thus, FIDSAM will enable the sub-cellular analysis of even low-expressed fluorophoretagged proteins in living plant cells. Furthermore, the combination of FIDSAM with fluorescence lifetime imaging provides the basis to study the local physico-chemical environment of fluorophore-tagged biomolecules in living plant cells.
基金Project supported by the National Natural Science Foundation of China(Nos.61831014 and 61771287)the Tsinghua University Initiative Scientific Research Program,China(No.20193080076)the Graduate Education Innovation Grants,Tsinghua University,China(No.201905J003)。
文摘To maximize signal collection in nonlinear optical microscopy,non-descanned epi-detection is generally adopted for in vivo imaging.However,because of severe scattering in biological samples,most of the emitted fluorescence photons go beyond the collection angles of objectives and thus cannot be detected.Here,we propose an extended detection scheme to enhance the collection of scattered photons in nonlinear fluorescence microscopy using a silicon photomultiplier array ahead of the front apertures of objectives.We perform numerical simulations to demonstrate the enhanced fluorescence collection via extended epi-detection in the multi-photon fluorescence imaging of human skin and mouse brain through craniotomy windows and intact skulls.For example,with red fluorescence emission at a depth of 600μm in human skin,the increased collection can be as much as about 150%with a 10×,0.6-NA objective.We show that extended epi-detection is a generally applicable,feasible technique for use in nonlinear fluorescence microscopy to enhance signal detection.
文摘Dear EditorProbing protein-protein interaction has become a routine practice in the post genomic era. Multiple in vitro or in vivo techniques have been developed to detect or report direct or indirect interactions of functionally related proteins （Lalonde et al., 2008）. These techniques sometimes are technically challenging, however, because the readout would demand sophisticated detectors and/or complicated calculations. Besides, a common drawback of many of these techniques is they can render inherent false positives to various degrees so that an interaction often cannot be judged unambiguously.
基金This work was supported by the National Natural Science Foundation of China(No.11974180)the Postgraduate Research and Practice Innovation Program of Jiangsu Province(Nos.KYCX21_1095 and SJCX21_0472).
文摘Plasmonic nanostructures have been proved effective not only in catalyzing chemical reactions,but also in improving the activity of non-plasmonic photocatalysts.It is essential to reveal the synergy between the plasmonic structure and the non-plasmonic metal photocatalyst for expounding the underlying mechanism of plasmon-enhanced catalysis.Herein,the enhancement of resazurin reduction at the heterostructure of silver nanowire(AgNW)and palladium nanoparticles(PdNPs)is observed in situ by single-molecule fluorescence microscopy.The catalysis mapping results around single AgNW suggest that the catalytic activity of PdNPs is enhanced for~20 times due to the excitation of localized surface plasmon resonance(LSPR)in the vicinity of the AgNW.This catalysis enhancement is also highly related to the wavelength and polarization of the excitation light.In addition,the palladium catalysis is further enhanced by~10 times in the vicinity of a roughened AgNW or a AgNW-AgNW nanogap because of the improvement of catalytic hotspots.These findings clarify the contribution of plasmon excitation in palladium catalysis at microscopic scale,which will help to deepen the understanding of the plasmon-enhanced photocatalysis and provide a guideline for developing highly efficient plasmon-based photocatalysts.
基金The National Key R&D Program of China(2021YFF0502900)National Natural Science Foundation of China(61835009,62127819,61620106016,62005171,61975127)+3 种基金Natural Science Foundation of Guangdong Province(2020A1515010679)Key Project of Guangdong Provincial Department of Education(2021ZDZX2013)Shenzhen Science and Technology R&D and Innovation Foundation(JCYJ20220531102807017)Shenzhen International Cooperation Research Project(GJHZ20190822095420249).
文摘Super-resolution optical imaging is crucial to the study of cellular processes.Current super-resolution fluorescence microscopy is restricted by the need of special fluorophores or sophisticated optical systems,or long acquisition and computational times.In this work,we present a deep-learning-based super-resolution technique of confocal microscopy.We devise a two-channel attention network(TCAN),which takes advantage of both spatial representations and frequency contents to learn a more precise mapping from low-resolution images to high-resolution ones.This scheme is robust against changes in the pixel size and the imaging setup,enabling the optimal model to generalize to different fluorescence microscopy modalities unseen in the training set.Our algorithm is validated on diverse biological structures and dual-color confocal images of actin-microtubules,improving the resolution from~230 nm to~110 nm.Last but not least,we demonstrate live-cell super-resolution imaging by revealing the detailed structures and dynamic instability of microtubules.
基金C.R.and M.S acknowledge the financial support of the Agence National de la Recherche(ANR-14-CE08-0015-01 Ultrafast Nanoscopy).
文摘With super-resolution microscopy,we attempt to visualize(biological)structures and processes at the sub-cellular level(i.e.,nanoscale).To obtain this information,the samples are labeled with fluorophores that have a stochastic on/off switching of their emissions,which help to overcome the optical diffraction limit of around 250 nm,related to the use of optical micro-scopes.However,nowadays,research focuses on the imaging of live cells and thicker samples.These investigations require a high amount of simultaneously active fluorophores(i.e.,high-density imaging)and are challenging due to the collapse of the single-molecule localization techniques and the increased background in the image.Therefore,recent efforts have shifted towards the development of new ways to process the data.This publication gives an introduction to wide-field super-resolution fluorescence microscopy,explaining the concepts of the technique,and then gives an overview of the recently developed methods to provide super-resolution images for high-density data of live cells and ways to overcome the issues related to the imaging of these samples.
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.
基金supported by the National Basic Research Program of China(2014CB932700)the National Natural Science Foundation of China(21422307,21303180,21433003,21573215,21503212,and 21503211)+1 种基金‘‘the Recruitment Program of Global youth Experts”of China,Science and Technology Innovation Foundation of Jilin Province for Talents Cultivation(20160519005JH)Jilin Youth foundation(20160520137JH)
文摘Understanding the heterogeneous catalytic properties of nanoparticles is of great significance for the development of high efficient nanocatalysts, but the intrinsic heterogeneities of nanocatalysts were always covered in traditional ensemble studies. This issue can be overcome if one can follow the catalysis of individual nanoparticles in real time. This paper mainly summarizes recent developments in single- molecule nanocatalysis at single particle level in Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. These developments include the revealing of catalytic kinetics of different types （plane ＆ edge） of surface atoms on individual Pd nanocubes, the observing of in situ deactivation of indi- vidual carbon-supported Pt nanoparticles during the electrocatalytic hydrogen-oxidation reaction, and the measurement of catalytic activation energies on single nanocatalysts for both product formation process and dissociation process, etc. These studies further indicate the advantages or unique abilities of single-molecule methods in the studies of nanocatalvsis or even chemical reactions.
文摘Titanium dioxide （TiO2） nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical patholog36 to detect TiO2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods （transmission electron microscopy, X-ray fluorescence microscop~ etc.） have low throughput and technical and operational complications. Herein, we describe two in situ post- treatment labeling approaches to stain TiO2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO2 nanoparticles with alkyne- conjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy （XFM） at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Therefore, future experiments with TiO2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.
基金supported by the National Key R&D Program of China(2022YFC3401100)the National Natural Science Foundation of China(62025501,31971376,92150301,62335008).
文摘Fluorescence microscopic imaging is essentially a convolution process distorted by random noise,limiting critical parameters such as imaging speed,duration,and resolution.Though algorithmic compensation has shown great potential to enhance these pivotal aspects,its fidelity remains questioned.Here we develop a physics-rooted computational resolution extension and denoising method with ensured fidelity.Our approach employs a multi-resolution analysis(MRA)framework to extract the two main characteristics of fluorescence images against noise:across-edge contrast,and along-edge continuity.By constraining the two features in a model-solution framework using framelet and curvelet,we develop MRA deconvolution algorithms,which improve the signal-to-noise ratio(SNR)up to 10 dB higher than spatial derivative based penalties,and can provide up to two-fold fidelity-ensured resolution improvement rather than the artifact-prone Richardson-Lucy inference.We demonstrate our methods can improve the performance of various diffraction-limited and super-resolution microscopies with ensured fidelity,enabling accomplishments of more challenging imaging tasks.
