Surface Electromyography (EMG) is a standard method used in clinical practice and research to assess motor function in order to help with the diagnosis of neuromuscular pathology in human and animal models. EMG record...Surface Electromyography (EMG) is a standard method used in clinical practice and research to assess motor function in order to help with the diagnosis of neuromuscular pathology in human and animal models. EMG recorded from trunk muscles involved in the activity of breathing can be used as a direct measure of respiratory motor function in patients with spinal cord injury (SCI) or other disorders associated with motor control deficits. However, EMG potentials recorded from these muscles are often contaminated with heart-induced electrocardiographic (ECG) signals. Elimination of these artifacts plays a critical role in the precise measure of the respiratory muscle electrical activity. This study was undertaken to find an optimal approach to eliminate the ECG artifacts from EMG recordings. Conventional global filtering can be used to decrease the ECG-induced artifact. However, this method can alter the EMG signal and changes physiologically relevant information. We hypothesize that, unlike global filtering, localized removal of ECG artifacts will not change the original EMG signals. We develop an approach to remove the ECG artifacts without altering the amplitude and frequency components of the EMG signal by using an externally recorded ECG signal as a mask to locate areas of the ECG spikes within EMG data. These segments containing ECG spikes were decomposed into 128 sub-wavelets by a custom-scaled Morlet Wavelet Transform. The ECG-related subwavelets at the ECG spike location were removed and a de-noised EMG signal was reconstructed. Validity of the proposed method was proven using mathematical simulated synthetic signals and EMG obtained from SCI patients. We compare the Rootmean Square Error and the Relative Change in Variance between this method, global, notch and adaptive filters. The results show that the localized wavelet-based filtering has the benefit of not introducing error in the native EMG signal and accurately removing ECG artifacts from EMG signals.展开更多
This article gives an overview of the development and applications of the surfaceenhanced Raman scattering(SERS)techniques in biomedicine.We first introduce the fundamental principles of the SERS mechanisms.We also pr...This article gives an overview of the development and applications of the surfaceenhanced Raman scattering(SERS)techniques in biomedicine.We first introduce the fundamental principles of the SERS mechanisms.We also present the different fabrication techniques of SERS nanostructures and substrates.Finally,the importance and potential roles of the SERS nanostructures and substrates in biomedical applications are summarized.展开更多
Laser diodes(LDs)have been considered as cost-effective and compact excitation sources to overcome the requirement of costly and bulky pulsed laser sources that are commonly used in photoacoustic microscopy(PAM).Howev...Laser diodes(LDs)have been considered as cost-effective and compact excitation sources to overcome the requirement of costly and bulky pulsed laser sources that are commonly used in photoacoustic microscopy(PAM).However,the spatial resolution and/or imaging speed of previously reported LD-based PAM systems have not been optimized simultaneously.In this paper,we developed a high-speed and high-resolution LD-based PAM system using a continuous wave LD,operating at a pulsed mode,with a repetition rate of 30 kHz,as an excitation source.A hybrid scanning mechanism that synchronizes a one-dimensional galvanometer mirror and a two-dimensional motorized stage is applied to achieve a fast imaging capability without signal averaging due to the high signal-to-noise ratio.By optimizing the optical system,a high lateral resolution of 4.8μm has been achieved.In vivo microvasculature imaging of a mouse ear has been demonstrated to show the high performance of our LD-based PAM system.展开更多
A new framework for early diagnosis of prostate cancer using Diffusion-Weighted Imaging (DWI) is proposed. The proposed diagnostic approach consists of the following four steps to detect locations that are suspicious ...A new framework for early diagnosis of prostate cancer using Diffusion-Weighted Imaging (DWI) is proposed. The proposed diagnostic approach consists of the following four steps to detect locations that are suspicious for prostate cancer: 1) In the first step, we isolate the prostate from the surrounding anatomical structures based on a Maximum A Posteriori (MAP) estimate of a new log-likelihood function that accounts for the shape priori, the spatial interaction, and the current appearance of prostate tissues and its background (surrounding anatomical structures);2) In order to take into account any local deformation between the segmented prostates at different b-values that could occur during the scanning process due to local motion, a non-rigid registration algorithm is employed;3) A KNN-based classifier is used to classify the prostate into benign or malignant based on three appearance features extracted from registered images;and 4) The tumor boundaries are determined using a level set deformable model controlled by the diffusion information and the spatial interactions between the prostate voxels. Preliminary experiments on 28 patients (17 malignant and 11 benign) resulted in 100% correct classification, showing that the proposed method is a promising supplement to current technologies (biopsy-based diagnostic systems) for the early diagnosis of prostate cancer.展开更多
Minimal photon fluxes(MINFLUX)nanoscopy has emerged as a transformative advancement in superresolution imaging,enabling unprecedented nanoscale observations across diverse biological scenarios.In this work,we propose,...Minimal photon fluxes(MINFLUX)nanoscopy has emerged as a transformative advancement in superresolution imaging,enabling unprecedented nanoscale observations across diverse biological scenarios.In this work,we propose,for the first time,that employing high-order vortex beams can significantly enhance the performance of MINFLUX,surpassing the limitations of the conventional MINFLUX using the first-order vortex beam.Our theoretical analysis indicates that,for standard MINFLUX,high-order vortex beams can improve the maximum localization precision by a factor corresponding to their order,which can approach a sub-nanometer scale under optimal conditions,and for raster scan MINFLUX,high-order vortex beams allow for a wider field of view while maintaining enhanced precision.These findings underscore the potential of high-order vortex beams to elevate the performance of MINFLUX,paving the way towards ultra-high resolution imaging for a broad range of applications.展开更多
Three-dimensional(3D)imaging is essential for understanding intricate biological and biomedical systems,yet live cell and tissue imaging applications still face challenges due to constrained imaging speed and strong s...Three-dimensional(3D)imaging is essential for understanding intricate biological and biomedical systems,yet live cell and tissue imaging applications still face challenges due to constrained imaging speed and strong scattering in turbid media.Here,we present a unique phase-modulated stimulated Raman scattering tomography(PM-SRST)technique to achieve rapid label-free 3D chemical imaging in cells and tissue.To accomplish PM-SRST,we utilize a spatial light modulator to electronically manipulate the focused Stokes beam along the needle Bessel pump beam for SRS tomography without the need for mechanical z scanning.We demonstrate the rapid 3D imaging capability of PM-SRST by real-time monitoring of 3D Brownian motion of polystyrene beads in water with 8.5 Hz volume rate,as well as the instant biochemical responses to acetic acid stimulants in MCF-7 cells.Further,combining the Bessel pump beam with a longer wavelength Stokes beam(NIR-II window)provides a superior scattering resilient ability in PM-SRST,enabling rapid tomography in deeper tissue areas.The PM-SRST technique providestwofold enhancement in imaging depth in highly scattering media(e.g.,polymer beads phantom and biotissue like porcine skin and brain tissue)compared with conventional point-scan SRS.We also demonstrate the rapid 3D imaging ability of PM-SRST by observing the dynamic diffusion and uptake processes of deuterium oxide molecules into plant roots.The rapid PM-SRST developed can be used to facilitate label-free 3D chemical imaging of metabolic activities and functional dynamic processes of drug delivery and therapeutics in live cells and tissue.展开更多
Optical-resolution photoacoustic microscopy(OR-PAM)has demonstrated high-spatial-resolution imaging of optical absorption in biological tissue.To date,most OR-PAM systems rely on mechanical scanning with confocally al...Optical-resolution photoacoustic microscopy(OR-PAM)has demonstrated high-spatial-resolution imaging of optical absorption in biological tissue.To date,most OR-PAM systems rely on mechanical scanning with confocally aligned optical excitation and ultrasonic detection,limiting the wide-field imaging speed of these systems.Although several multifocal OR-PA(MFOR-PA)systems have attempted to address this limitation,they are hindered by the complex design in a constrained physical space.Here,we present a two-dimensional(2D)MFOR-PAM system that utilizes a 2D microlens array and an acoustic ergodic relay.Using a single-element ultrasonic transducer,this system can detect PA signals generated from 400 optical foci in parallel and then raster scan the optical foci patterns to form an MFOR-PAM image.This system improves the imaging resolution of an acoustic ergodic relay system from 220 to 13μm and enables 400-folds shorter scanning time than that of a conventional OR-PAM system at the same resolution and laser repetition rate.We demonstrated the imaging ability of the system with both in vitro and in vivo experiments.展开更多
We report a novel stimulated Raman scattering(SRS)microscopy technique featuring phase-controlled light focusing and aberration corrections for rapid,deep tissue 3D chemical imaging with subcellular resolution.To acco...We report a novel stimulated Raman scattering(SRS)microscopy technique featuring phase-controlled light focusing and aberration corrections for rapid,deep tissue 3D chemical imaging with subcellular resolution.To accomplish phasecontrolled SRS(PC-SRS),we utilize a single spatial light modulator to electronically tune the axial positioning of both the shortened-length Bessel pump and the focused Gaussian Stokes beams,enabling z-scanning-free optical sectioning in the sample.By incorporating Zernike polynomials into the phase patterns,we simultaneously correct the system aberrations at two separate wavelengths(~240 nm difference),achieving a~3-fold enhancement in signal-to-noise ratio over the uncorrected imaging system.PC-SRS provides>2-fold improvement in imaging depth in various samples(e.g.,polystyrene bead phantoms,porcine brain tissue)as well as achieves SRS 3D imaging speed of~13 Hz per volume for real-time monitoring of Brownian motion of polymer beads in water,superior to conventional point-scanning SRS 3D imaging.We further utilize PC-SRS to observe the metabolic activities of the entire tumor liver in living zebrafish in cellsilent region,unraveling the upregulated metabolism in liver tumor compared to normal liver.This work shows that PCSRS provides unprecedented insights into morpho-chemistry,metabolic and dynamic functioning of live cells and tissue in real-time at the subcellular level.展开更多
We present a novel time-of-flight resolved Bessel light bullet-enabled stimulated Raman scattering(B2-SRS)microscopy for deeper tissue 3D chemical imaging with high resolution without a need for mechanical z-scanning....We present a novel time-of-flight resolved Bessel light bullet-enabled stimulated Raman scattering(B2-SRS)microscopy for deeper tissue 3D chemical imaging with high resolution without a need for mechanical z-scanning.To accomplish the tasks,we conceive a unique method to enable optical sectioning by generating the counterpropagating pump and Stokes Bessel light bullets in the sample,in which the group velocities of the Bessel light bullets are made ultraslow(e.g.,vg≈0.1c)and tunable by introducing programmable angular dispersions with a spatial light modulator.We theoretically analyze the working principle of the collinear multicolor Bessel light bullet generations and velocity controls with the relative time-of-flight resolved detection for SRS 3D deep tissue imaging.We have also built the B2-SRS imaging system and present the first demonstration of B2-SRS microscopy with Bessel light bullets for 3D chemical imaging in a variety of samples(e.g.,polymer bead phantoms,biological samples such as spring onion tissue and porcine brain)with high resolution.The B2-SRS technique provides a>2-fold improvement in imaging depth in porcine brain tissue compared to conventional SRS microscopy.The method of optical sectioning in tissue using counter-propagating ultraslow Bessel light bullets developed in B2-SRS is generic and easy to perform and can be readily extended to other nonlinear optical imaging modalities to advance 3D microscopic imaging in biological and biomedical systems and beyond.展开更多
Microscopy with ultraviolet surface excitation(MUSE)is a promising slide-free imaging technique to improve the time-consuming histopathology workflow.However,since the penetration depth of the excitation light is tiss...Microscopy with ultraviolet surface excitation(MUSE)is a promising slide-free imaging technique to improve the time-consuming histopathology workflow.However,since the penetration depth of the excitation light is tissue dependent,the image contrast could be significantly degraded when the depth of field of the imaging system is shallower than the penetration depth.High-resolution cellular imaging normally comes with a shallow depth of field,which also restricts the tolerance of surface roughness in biological specimens.Here we propose the incorporation of MUSE with speckle illumination(termed MUSES),which can achieve sharp imaging on thick and rough specimens.Our experimental results demonstrate the potential of MUSES in providing histological images with~1μm spatial resolution and improved contrast,within 10 minutes for a field of view of 1.7 mm×1.2 mm.With the extended depth of field feature,MUSES also relieves the constraint of tissue flatness.Furthermore,with a color transformation assisted by deep learning,a virtually stained histological image can be generated without manual tuning,improving the applicability of MUSES in clinical settings.展开更多
3D imaging technology is pivotal in monitoring the functional dynamics and morphological alterations in living cells and tissues.However,conventional volumetric imaging associated with mechanical z-scanning encounters...3D imaging technology is pivotal in monitoring the functional dynamics and morphological alterations in living cells and tissues.However,conventional volumetric imaging associated with mechanical z-scanning encounters challenges in limited 3D imaging speed with inertial artifact.Here,we present a unique phase-modulated multifoci microscopy (PM^(3)) technique to achieve snapshot 3D imaging with the advantages of extended imaging depths and adjustable imaging intervals between each focus in a rapid fashion.To accomplish the tasks,we utilize a spatial light modulator (SLM) to encode the phases of the scattered or fluorescence light emanating from a volumetric sample and then project the multiple-depth images of the sample onto a single charge-coupled device camera for rapid 3D imaging.We demonstrate that the PM^(3)technique provides~55-fold improvement in imaging depth in polystyrene beads phantom compared to the depth of field of the objective lens used.PM^(3)also enables the real-time monitoring of Brownian motion of fluorescent beads in water at a 15 Hz volume rate.By precisely manipulating the phase of scattered light on the SLM,PM^(3)can pinpoint the specific depth information in living zebrafish and rapidly observe the 3D dynamic processes of blood flow in the zebrafish trunk.This work shows that the PM^(3)technique developed is robust and versatile for fast 3D dynamic imaging in biological and biomedical systems.展开更多
Fluorescence imaging,a key technique in biological research,frequently utilizes fluorogenic probes for precise imaging in living systems.Tetrazine is an effective emission quencher in fluorogenic probe designs,which c...Fluorescence imaging,a key technique in biological research,frequently utilizes fluorogenic probes for precise imaging in living systems.Tetrazine is an effective emission quencher in fluorogenic probe designs,which can be selectively damaged upon bioorthogonal click reactions,leading to considerable emission enhancement.Despite significant efforts to increase the emission enhancement ratio(I_(AC)/I_(BC))of tetrazine-functionalized fluorogenic probes,the influence of molecular aggregation on the emission properties has been largely overlooked in these probe designs.In this study,we reveal that an ultrahigh I_(AC)/I_(BC)can be realized in the aggregate system when tetrazine is paired with aggregation-induced emission(AIE)luminogens.Tetrazine amplifies its quenching efficiency upon aggregation and drastically reduce background emissions.Subsequent click reactions damage tetrazine and trigger significant AIE,leading to considerably enhanced I_(AC)/I_(BC).We further showcase the capability of these ultra-fluorogenic systems in selective imaging of multiple organelles in living cells.We term this unique fluorogenicity of AIE luminogen-quencher complexes with amplified dark-bright states as“Matthew effect”in aggregate emission,potentially providing a universal approach to attain ultrahigh I_(AC)/I_(BC)in diverse fluorogenic systems.展开更多
文摘Surface Electromyography (EMG) is a standard method used in clinical practice and research to assess motor function in order to help with the diagnosis of neuromuscular pathology in human and animal models. EMG recorded from trunk muscles involved in the activity of breathing can be used as a direct measure of respiratory motor function in patients with spinal cord injury (SCI) or other disorders associated with motor control deficits. However, EMG potentials recorded from these muscles are often contaminated with heart-induced electrocardiographic (ECG) signals. Elimination of these artifacts plays a critical role in the precise measure of the respiratory muscle electrical activity. This study was undertaken to find an optimal approach to eliminate the ECG artifacts from EMG recordings. Conventional global filtering can be used to decrease the ECG-induced artifact. However, this method can alter the EMG signal and changes physiologically relevant information. We hypothesize that, unlike global filtering, localized removal of ECG artifacts will not change the original EMG signals. We develop an approach to remove the ECG artifacts without altering the amplitude and frequency components of the EMG signal by using an externally recorded ECG signal as a mask to locate areas of the ECG spikes within EMG data. These segments containing ECG spikes were decomposed into 128 sub-wavelets by a custom-scaled Morlet Wavelet Transform. The ECG-related subwavelets at the ECG spike location were removed and a de-noised EMG signal was reconstructed. Validity of the proposed method was proven using mathematical simulated synthetic signals and EMG obtained from SCI patients. We compare the Rootmean Square Error and the Relative Change in Variance between this method, global, notch and adaptive filters. The results show that the localized wavelet-based filtering has the benefit of not introducing error in the native EMG signal and accurately removing ECG artifacts from EMG signals.
基金supported by the Academic Research Fund from the Ministry of Education,the Biomedical Research Council,the National Medical Research Council,and the Faculty Research Fund from the National University of Singapore.
文摘This article gives an overview of the development and applications of the surfaceenhanced Raman scattering(SERS)techniques in biomedicine.We first introduce the fundamental principles of the SERS mechanisms.We also present the different fabrication techniques of SERS nanostructures and substrates.Finally,the importance and potential roles of the SERS nanostructures and substrates in biomedical applications are summarized.
基金Hong Kong Innovation and Technology Commission,No.ITS/036/19Research Grants Council of the Hong Kong Special Administrative Region,No.26203619.
文摘Laser diodes(LDs)have been considered as cost-effective and compact excitation sources to overcome the requirement of costly and bulky pulsed laser sources that are commonly used in photoacoustic microscopy(PAM).However,the spatial resolution and/or imaging speed of previously reported LD-based PAM systems have not been optimized simultaneously.In this paper,we developed a high-speed and high-resolution LD-based PAM system using a continuous wave LD,operating at a pulsed mode,with a repetition rate of 30 kHz,as an excitation source.A hybrid scanning mechanism that synchronizes a one-dimensional galvanometer mirror and a two-dimensional motorized stage is applied to achieve a fast imaging capability without signal averaging due to the high signal-to-noise ratio.By optimizing the optical system,a high lateral resolution of 4.8μm has been achieved.In vivo microvasculature imaging of a mouse ear has been demonstrated to show the high performance of our LD-based PAM system.
文摘A new framework for early diagnosis of prostate cancer using Diffusion-Weighted Imaging (DWI) is proposed. The proposed diagnostic approach consists of the following four steps to detect locations that are suspicious for prostate cancer: 1) In the first step, we isolate the prostate from the surrounding anatomical structures based on a Maximum A Posteriori (MAP) estimate of a new log-likelihood function that accounts for the shape priori, the spatial interaction, and the current appearance of prostate tissues and its background (surrounding anatomical structures);2) In order to take into account any local deformation between the segmented prostates at different b-values that could occur during the scanning process due to local motion, a non-rigid registration algorithm is employed;3) A KNN-based classifier is used to classify the prostate into benign or malignant based on three appearance features extracted from registered images;and 4) The tumor boundaries are determined using a level set deformable model controlled by the diffusion information and the spatial interactions between the prostate voxels. Preliminary experiments on 28 patients (17 malignant and 11 benign) resulted in 100% correct classification, showing that the proposed method is a promising supplement to current technologies (biopsy-based diagnostic systems) for the early diagnosis of prostate cancer.
基金supported in part by the Academic Research Fund(AcRF)-Tier 2(A-8000117-01-00)and Tier 1(A-8003279-00-00)from the Ministry of Education(MOE)of Singapore,Science and Technology Project of Jiangsu Province(BZ2022056),NUS(Suzhou)Research Institute/Biomedical and Health Technology Platform,2024 Tsinghua-NUS Joint Research Initiative Fund(A-8002557-00-00)the National Medical Research Council(NMRC)(A-0009502-01-00,and A-8001143-00-00),Singapore.
文摘Minimal photon fluxes(MINFLUX)nanoscopy has emerged as a transformative advancement in superresolution imaging,enabling unprecedented nanoscale observations across diverse biological scenarios.In this work,we propose,for the first time,that employing high-order vortex beams can significantly enhance the performance of MINFLUX,surpassing the limitations of the conventional MINFLUX using the first-order vortex beam.Our theoretical analysis indicates that,for standard MINFLUX,high-order vortex beams can improve the maximum localization precision by a factor corresponding to their order,which can approach a sub-nanometer scale under optimal conditions,and for raster scan MINFLUX,high-order vortex beams allow for a wider field of view while maintaining enhanced precision.These findings underscore the potential of high-order vortex beams to elevate the performance of MINFLUX,paving the way towards ultra-high resolution imaging for a broad range of applications.
基金supported by the Academic Research Fund(AcRF)-Tier 2(A-8000117-01-00)and Tier 1(R397-000334-114,R397-000-371-114,and R397-000-378-114)from the Ministry of Education(MOE)the Merlion Fund(WBS R-397-000-356-133)the National Medical Research Council(NMRC)(A-0009502-01-00 and A-8001143-00-00),Singapore
文摘Three-dimensional(3D)imaging is essential for understanding intricate biological and biomedical systems,yet live cell and tissue imaging applications still face challenges due to constrained imaging speed and strong scattering in turbid media.Here,we present a unique phase-modulated stimulated Raman scattering tomography(PM-SRST)technique to achieve rapid label-free 3D chemical imaging in cells and tissue.To accomplish PM-SRST,we utilize a spatial light modulator to electronically manipulate the focused Stokes beam along the needle Bessel pump beam for SRS tomography without the need for mechanical z scanning.We demonstrate the rapid 3D imaging capability of PM-SRST by real-time monitoring of 3D Brownian motion of polystyrene beads in water with 8.5 Hz volume rate,as well as the instant biochemical responses to acetic acid stimulants in MCF-7 cells.Further,combining the Bessel pump beam with a longer wavelength Stokes beam(NIR-II window)provides a superior scattering resilient ability in PM-SRST,enabling rapid tomography in deeper tissue areas.The PM-SRST technique providestwofold enhancement in imaging depth in highly scattering media(e.g.,polymer beads phantom and biotissue like porcine skin and brain tissue)compared with conventional point-scan SRS.We also demonstrate the rapid 3D imaging ability of PM-SRST by observing the dynamic diffusion and uptake processes of deuterium oxide molecules into plant roots.The rapid PM-SRST developed can be used to facilitate label-free 3D chemical imaging of metabolic activities and functional dynamic processes of drug delivery and therapeutics in live cells and tissue.
基金supported in part by National Institutes of Health grants DP1 EB016986(NIH Director’s Pioneer Award),R01 CA186567(NIH Director’s Transformative Research Award),U01 NS090579(BRAIN1 Initiative)and U01 NS099717(BRAIN Initiative).
文摘Optical-resolution photoacoustic microscopy(OR-PAM)has demonstrated high-spatial-resolution imaging of optical absorption in biological tissue.To date,most OR-PAM systems rely on mechanical scanning with confocally aligned optical excitation and ultrasonic detection,limiting the wide-field imaging speed of these systems.Although several multifocal OR-PA(MFOR-PA)systems have attempted to address this limitation,they are hindered by the complex design in a constrained physical space.Here,we present a two-dimensional(2D)MFOR-PAM system that utilizes a 2D microlens array and an acoustic ergodic relay.Using a single-element ultrasonic transducer,this system can detect PA signals generated from 400 optical foci in parallel and then raster scan the optical foci patterns to form an MFOR-PAM image.This system improves the imaging resolution of an acoustic ergodic relay system from 220 to 13μm and enables 400-folds shorter scanning time than that of a conventional OR-PAM system at the same resolution and laser repetition rate.We demonstrated the imaging ability of the system with both in vitro and in vivo experiments.
基金supported by the Academic Research Fund(AcRF)from the Ministry of Education(MOE)(Tier 2(A-8000117-01-00)Tier 1(R397-000-334-114,R397-000-371-114,and R397-000-378-114)2024 Tsinghua-NUS Joint Research Initiative Fund,and the National Medical Research Council(NMRC)(A-0009502-01-00,and A-8001143-00-00),Singapore.
文摘We report a novel stimulated Raman scattering(SRS)microscopy technique featuring phase-controlled light focusing and aberration corrections for rapid,deep tissue 3D chemical imaging with subcellular resolution.To accomplish phasecontrolled SRS(PC-SRS),we utilize a single spatial light modulator to electronically tune the axial positioning of both the shortened-length Bessel pump and the focused Gaussian Stokes beams,enabling z-scanning-free optical sectioning in the sample.By incorporating Zernike polynomials into the phase patterns,we simultaneously correct the system aberrations at two separate wavelengths(~240 nm difference),achieving a~3-fold enhancement in signal-to-noise ratio over the uncorrected imaging system.PC-SRS provides>2-fold improvement in imaging depth in various samples(e.g.,polystyrene bead phantoms,porcine brain tissue)as well as achieves SRS 3D imaging speed of~13 Hz per volume for real-time monitoring of Brownian motion of polymer beads in water,superior to conventional point-scanning SRS 3D imaging.We further utilize PC-SRS to observe the metabolic activities of the entire tumor liver in living zebrafish in cellsilent region,unraveling the upregulated metabolism in liver tumor compared to normal liver.This work shows that PCSRS provides unprecedented insights into morpho-chemistry,metabolic and dynamic functioning of live cells and tissue in real-time at the subcellular level.
基金the Academic Research Fund(AcRF)from the Ministry of Education(MOE)(Tier 2(A-8000117-01-00)Tier 1(R397-000-334-114,R397-000-371-114,and R397-000-378-114)the Merlion Fund(R397-000-356-133),2024 Tsinghua-NUS Joint Research Initiative Fund,and the National Medical Research Council(NMRC)(A-0009502-01-00 and A-8001143-00-00),Singapore.
文摘We present a novel time-of-flight resolved Bessel light bullet-enabled stimulated Raman scattering(B2-SRS)microscopy for deeper tissue 3D chemical imaging with high resolution without a need for mechanical z-scanning.To accomplish the tasks,we conceive a unique method to enable optical sectioning by generating the counterpropagating pump and Stokes Bessel light bullets in the sample,in which the group velocities of the Bessel light bullets are made ultraslow(e.g.,vg≈0.1c)and tunable by introducing programmable angular dispersions with a spatial light modulator.We theoretically analyze the working principle of the collinear multicolor Bessel light bullet generations and velocity controls with the relative time-of-flight resolved detection for SRS 3D deep tissue imaging.We have also built the B2-SRS imaging system and present the first demonstration of B2-SRS microscopy with Bessel light bullets for 3D chemical imaging in a variety of samples(e.g.,polymer bead phantoms,biological samples such as spring onion tissue and porcine brain)with high resolution.The B2-SRS technique provides a>2-fold improvement in imaging depth in porcine brain tissue compared to conventional SRS microscopy.The method of optical sectioning in tissue using counter-propagating ultraslow Bessel light bullets developed in B2-SRS is generic and easy to perform and can be readily extended to other nonlinear optical imaging modalities to advance 3D microscopic imaging in biological and biomedical systems and beyond.
基金Research Grants Council of the Hong Kong Special Administrative Region(26203619,16208620)Hong Kong Innovation and Technology Commission(ITS/036/19)。
文摘Microscopy with ultraviolet surface excitation(MUSE)is a promising slide-free imaging technique to improve the time-consuming histopathology workflow.However,since the penetration depth of the excitation light is tissue dependent,the image contrast could be significantly degraded when the depth of field of the imaging system is shallower than the penetration depth.High-resolution cellular imaging normally comes with a shallow depth of field,which also restricts the tolerance of surface roughness in biological specimens.Here we propose the incorporation of MUSE with speckle illumination(termed MUSES),which can achieve sharp imaging on thick and rough specimens.Our experimental results demonstrate the potential of MUSES in providing histological images with~1μm spatial resolution and improved contrast,within 10 minutes for a field of view of 1.7 mm×1.2 mm.With the extended depth of field feature,MUSES also relieves the constraint of tissue flatness.Furthermore,with a color transformation assisted by deep learning,a virtually stained histological image can be generated without manual tuning,improving the applicability of MUSES in clinical settings.
基金National Medical Research Council (NMRC),Singapore (A-0009502-01-00, A-8001143-00-00)Merlion Fund (R397-000-356-133)Academic Research Fund(AcRF) from the Ministry of Education (MOE),Singapore(Tier 1 (R397-000-334-114),Tier 1 (R397-000-371-114,R397-000-378-114),Tier 2 (A-8000117-01-00))。
文摘3D imaging technology is pivotal in monitoring the functional dynamics and morphological alterations in living cells and tissues.However,conventional volumetric imaging associated with mechanical z-scanning encounters challenges in limited 3D imaging speed with inertial artifact.Here,we present a unique phase-modulated multifoci microscopy (PM^(3)) technique to achieve snapshot 3D imaging with the advantages of extended imaging depths and adjustable imaging intervals between each focus in a rapid fashion.To accomplish the tasks,we utilize a spatial light modulator (SLM) to encode the phases of the scattered or fluorescence light emanating from a volumetric sample and then project the multiple-depth images of the sample onto a single charge-coupled device camera for rapid 3D imaging.We demonstrate that the PM^(3)technique provides~55-fold improvement in imaging depth in polystyrene beads phantom compared to the depth of field of the objective lens used.PM^(3)also enables the real-time monitoring of Brownian motion of fluorescent beads in water at a 15 Hz volume rate.By precisely manipulating the phase of scattered light on the SLM,PM^(3)can pinpoint the specific depth information in living zebrafish and rapidly observe the 3D dynamic processes of blood flow in the zebrafish trunk.This work shows that the PM^(3)technique developed is robust and versatile for fast 3D dynamic imaging in biological and biomedical systems.
基金Hong Kong PhD Fellowship Scheme,Grant/Award Number:PF18-15484National Natural Science Foundation of China,Grant/Award Numbers:21788102,22274106+4 种基金Research Grants Council of Hong Kong,Grant/Award Numbers:16306620,16303221,N_HKUST609/19,C6014-20WInnovation and Technology Commission,Grant/Award Number:ITC-CNERC14SC01Shenzhen Science and Technology Innovation CommitteeJSPS KAKENHI,Grant/Award Numbers:JP23H01977,JP23H04631JST the establishment of university fellowships towards the creation of science technology innovation,Grant/Award Number:JPMJFS2132。
文摘Fluorescence imaging,a key technique in biological research,frequently utilizes fluorogenic probes for precise imaging in living systems.Tetrazine is an effective emission quencher in fluorogenic probe designs,which can be selectively damaged upon bioorthogonal click reactions,leading to considerable emission enhancement.Despite significant efforts to increase the emission enhancement ratio(I_(AC)/I_(BC))of tetrazine-functionalized fluorogenic probes,the influence of molecular aggregation on the emission properties has been largely overlooked in these probe designs.In this study,we reveal that an ultrahigh I_(AC)/I_(BC)can be realized in the aggregate system when tetrazine is paired with aggregation-induced emission(AIE)luminogens.Tetrazine amplifies its quenching efficiency upon aggregation and drastically reduce background emissions.Subsequent click reactions damage tetrazine and trigger significant AIE,leading to considerably enhanced I_(AC)/I_(BC).We further showcase the capability of these ultra-fluorogenic systems in selective imaging of multiple organelles in living cells.We term this unique fluorogenicity of AIE luminogen-quencher complexes with amplified dark-bright states as“Matthew effect”in aggregate emission,potentially providing a universal approach to attain ultrahigh I_(AC)/I_(BC)in diverse fluorogenic systems.
