Multiphoton microscopy(MPM),with the advantages of improved penetration depth,decreased photo-damage,and optical sectioning capability,has become an indispensable tool for biomedical imaging.The combination of multiph...Multiphoton microscopy(MPM),with the advantages of improved penetration depth,decreased photo-damage,and optical sectioning capability,has become an indispensable tool for biomedical imaging.The combination of multiphoton fluorescence(MF)and second-harmonic generation(SHG)microscopy is particularly effective in imaging tissue structures of the ocular surface.This work is intended to be a review of advances that MPM has made in ophthalmic imaging.The MPM not only can be used for the label-free imaging of ocular structures,it can also be applied for investigating the morphological alterations in corneal pathologies,such as keratoconus,infected keratitis,and corneal scar.Furthermore,the corneal wound healing process after refractive surgical procedures such as conductive keratoplasty(CK)can also be studied with MPM.Finally,qualitative and quantitative SHG microscopy is effective for characterizing corneal thermal denaturation.With additional development,multiphoton imaging has the potential to be developed into an effective imaging technique for in vivo studies and clinical diagnosis in ophthalmology.展开更多
Multiphoton microscopy, relying on the simultaneous absorption of two or more photons by a fluorophore, has come to occupy a prominent place in modern biomedical research with its ability to allow real-time observatio...Multiphoton microscopy, relying on the simultaneous absorption of two or more photons by a fluorophore, has come to occupy a prominent place in modern biomedical research with its ability to allow real-time observation of a single cell and molecules in intact tissues. Multiphoton microscopy exhibits nonlinear optical contrast properties, which can make it possible to provide an exceptionally large depth penetration with less phototoxicity. This system becomes more and more an inspiring tool for a non-invasive imaging system to realize "optical biopsy" and to examine the functions of living cells. In this review, we briefly present the physical principles and properties of multiphoton microscopy as well as the current applications in biological fields. In addition, we address what we see as the future potential of multiphoton microscopy for gastroenterologic research.展开更多
Multifocal multiphoton microscopy(MMM)has recently become an important tool in biomedicine for performing three-dimensional fastfluorescence imaging.Using various beamsplitting techniques,MMM splits the near-infrared ...Multifocal multiphoton microscopy(MMM)has recently become an important tool in biomedicine for performing three-dimensional fastfluorescence imaging.Using various beamsplitting techniques,MMM splits the near-infrared laser beam into multiple beamlets and produces a multifocal array on the sample for parallel multiphoton excitation and then recordsfluorescence signal from all foci simultaneously with an area array detector,which significantly improves the imaging speed of multiphoton microscopy and allows for high efficiency in use of the excitation light.In this paper,we discuss the features of several MMM setups using different beamsplitting devices,including a Nipkow spinning disk,a microlens array,a set of beamsplitting mirrors,or a diffractive optical element(DOE).In particular,we present our recent work on the development of an MMM using a spatial light modulator(SLM).展开更多
The tumor microenvironment(TME)is now recognized as an important participant of tumor progression.As the most abundant extracellular matrix component in TME,collagen plays an important role in tumor development.The im...The tumor microenvironment(TME)is now recognized as an important participant of tumor progression.As the most abundant extracellular matrix component in TME,collagen plays an important role in tumor development.The imaging study of collagen morphological feature in TME is of great significance for understanding the state of tumor.Multiphoton microscopy(MPM),based on second harmonic generation(SHG)and two-photon excitation fluorescence(TPEF),can be used to monitor the morphological changes of biological tissues without labeling.In this study,we used MPM for large-scale imaging of early invasive breast cancer from the tumor center to normal tissues far from the tumor.We found that there were signi¯cant di®erences in collagen morphology between breast cancer tumor boundary,near tumor transition region and normal tissues far from the tumor.Furthermore,the morphological feature of eight collagen¯bers was extracted to quantify the variation trend of collagen in three regions.These results may provide a new perspective for the optimal negative margin width of breast-conserving surgery and the understanding of tumor metastasis.展开更多
Epithelial cancer comprises more than 85%of human cancers.The detection and treatment at the early stage has been demonstrated to apparently improve patient survival.In this review,we summarize our recent research wor...Epithelial cancer comprises more than 85%of human cancers.The detection and treatment at the early stage has been demonstrated to apparently improve patient survival.In this review,we summarize our recent research works on the diagnostic application of epithelial tissue based on multiphoton microscopy(MPM),including identification of the layered structures of esophagus,oral cavity,skin and bronchus tissues,establishment of the diagnostic features for distinguishing gastric normal tissue from cancerous tissue,linking collagen alteration and ectocervical epithelial tumor progression for evaluating epithelial tumor progression,and differentiating normal,inflammatory,and dysplastic ectocervical epithelial tissues.These results provide the groundwork for developing MPM into clinical multiphoton endoscopy.展开更多
This review summarizes the historical and more recent developments of multiphoton microscopy,as applied to dermatology.Multiphoton microscopy offers several advantages over competingmicroscopy techniques:there is an i...This review summarizes the historical and more recent developments of multiphoton microscopy,as applied to dermatology.Multiphoton microscopy offers several advantages over competingmicroscopy techniques:there is an inherent axial sectioning,penetration depths that competewll with confocal microscopy on account of the use of near-infrared light,and many t wo-pho ton contrast mechanisms,such as second-harmonic generation,have no analogue in one-photonmicroscopy.While the penetration depths of photons into tissue are typically limited on the orderof hundreds of microns,this is of les concern in dermatology,as the skin is thin and readlily.accessible.As a result,multiphoton microscopy in dermatology has generated a great deal ofmterest,much of which is summarized here.The review covers the interaction of light and tissue,as well as the various considerations that must be made when designing an instrument.The stateof multiphoton microscopy in imaging skin cancer and various other diseases is also discussed,along with the investigation of aging and regeneration phenomena,and finally,the use of mul-tiphoton microscopy to analyze the transdermal transport of drugs,cosmetics and other agents issummarized.The review concludes with a look at potential future research directions,especiallythose that are necessary to push these techniques into widespread clinical acceptance.展开更多
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
We developed an imaging technique combining two-photon computed super-resolution microscopy and suction-based stabilization to achieve the resolution of the single-cell level and organelles in vivo.To accomplish this,...We developed an imaging technique combining two-photon computed super-resolution microscopy and suction-based stabilization to achieve the resolution of the single-cell level and organelles in vivo.To accomplish this,a conventional two-photon microscope was equipped with a 3D-printed holders,which stabilize the tissue surface within the focal plane of immersion objectives.Further computational image stabilization and noise reduction were applied,followed by superresolution radial fluctuations(SRRF)analysis,doubling image resolution,and enhancing signal-to-noise ratios for in vivo subcellular process investigation.Stabilization of<1μm was obtained by suction,and<25 nm were achieved by subsequent algorithmic image stabilization.A Mito-Dendra2 mouse model,expressing green fluorescent protein(GFP)in mitochondria,demonstrated the potential of long-term intravital subcellular imaging.In vivo mitochondrial fission and fusion,mitochondrial status migration,and the effects of alcohol consumption(modeled as an alcoholic liver disease)and berberine treatment on hepatocyte mitochondrial dynamics are directly observed intravitally.Suction-based stabilization in two-photon intravital imaging,coupled with computational super-resolution holds promise for advancing in vivo subcellular imaging studies.展开更多
Multiphoton microscopy is the enabling tool for biomedical research,but the aberrations of biological tissues have limited its imaging performance.Adaptive optics(AO)has been developed to partially overcome aberration...Multiphoton microscopy is the enabling tool for biomedical research,but the aberrations of biological tissues have limited its imaging performance.Adaptive optics(AO)has been developed to partially overcome aberration to restore imaging performance.For indirect AO,algorithm is the key to its successful implementation.Here,based on the fact that indirect AO has an analogy to the black-box optimization problem,we successfully apply the covariance matrix adaptation evolution strategy(CMA-ES)used in the latter,to indirect AO in multiphoton microscopy(MPM).Compared with the traditional genetic algorithm(GA),our algorithm has a greater improvement in convergence speed and convergence accuracy,which provides the possibility of realizing real-time dynamic aberration correction for deep in vivo biological tissues.展开更多
Using the combination of a refective blazed grating and a reflective phase-only difractive spatiallight modulator(SLM),scanless multitarget-matching multiphoton excitation fuorescence mi.croscopy(SMTM-MP M)was achieve...Using the combination of a refective blazed grating and a reflective phase-only difractive spatiallight modulator(SLM),scanless multitarget-matching multiphoton excitation fuorescence mi.croscopy(SMTM-MP M)was achieved.The SLM shaped an incoming mode-locked,near-infraredTi:sapphire laser beam into an excitation pattern with addressable shapes and sizes that matchedthe samples of interest in the field of view.Temporal and spatial focusing were simultaneouslyrealized by combining an objective lens and a blazed grating.The fluorescence signal fromilluminated areas was recorded by a two-dimensional sCMOS camera.Compared with a conventional temporal focusing multiphoton microscope,our microscope achieved effective use of thelaser power and decreased photodamage with higher axial resolution.展开更多
To date,numerous studies have been performed to elucidate the complex cellular dynamics in skin diseases,but few have attempted to characterize these cellular events under conditions similar to the native environment....To date,numerous studies have been performed to elucidate the complex cellular dynamics in skin diseases,but few have attempted to characterize these cellular events under conditions similar to the native environment.To address this challenge,a three-dimensional(3D)multimodal analysis platform was developed for characterizing in vivo cellular dynamics in skin,which was then utilized to process in vivo wound healing data to demonstrate its applicability.Special attention is focused on in vivo biological parameters that are difficult to study with ex vivo analysis,including 3D cell tracking and techniques to connect biological information obtained from different imaging modalities.These results here open new possibilities for evaluating 3D cellular dynamics in vivo,and can potentially provide new tools for characterizing the skin microenvironment and pathologies in the future.展开更多
Multiphoton microscopy(MPM),based on two-photon excited fuorescence and second harmonic generation,enables direct noninvasive visualization of tissue architecture and cell morphology in live tissues without the admini...Multiphoton microscopy(MPM),based on two-photon excited fuorescence and second harmonic generation,enables direct noninvasive visualization of tissue architecture and cell morphology in live tissues without the administration of exogenous contrast agents.In this paper,we used MPM to image the microstructures of the mucosa in fresh,unfixed,and unstained intestinal tissue of mouse.The morphology and distribution of the main components in mucosa layer such as columnar cells,goblet cells,intestinal glands,and a little collagen fibers were clearly observed in MPM images,and then compared with standard H&:E images from paired specimens.Our results indicate that MPM combined with endoscopy and miniaturization probes has the potential application in the clinical diagnosis and in vivo monitoring of early intestinal cancer.展开更多
Multi-photon microscopy(MPM)and coherent anti-Stokes Raman scattering(CARS)are two advanced nonlinear optical imaging techniques,which provide complementary information and have great potential in combination for noni...Multi-photon microscopy(MPM)and coherent anti-Stokes Raman scattering(CARS)are two advanced nonlinear optical imaging techniques,which provide complementary information and have great potential in combination for noninvasive in vrito biomedical applications.This paper provides a detailed discussion of the basics,development and applications of these technologies for in vrivo skin research,covering the following topics:The principle and advantage of MPM and CARS,instrumentation development for in vino applications,MPM and CARS of normal skin,application of MPM and CARS in skin cancer and disease diagnosis;application of MPM in skin disease intervention,ie.,imaging guided two-photon photothermolysis.展开更多
Simultaneous metabolic and oxygen imaging is promising to follow up therapy response,dis-ease development and to determine prognostic factors.FLIM of metabolic coenzymes is now widely accepted to be the most reliable ...Simultaneous metabolic and oxygen imaging is promising to follow up therapy response,dis-ease development and to determine prognostic factors.FLIM of metabolic coenzymes is now widely accepted to be the most reliable method to determine cellular bioenergetics.Also,oxygen consumption has to be taken into account to understand treatment responses.The phosphorescence lifetimne of oxygen sensors is able to indicate local oxygen changes.For phosphorescence lifetime imaging(PLIM)dyes based on ruthenium(I)coordination com-plexes are useful,in detaill TLD1433 which possesses a variety of different triplet states,enables complex photochemistry and redox reactions.PLIM is usally reached by two photon exci-tation of the drug with a femtosecond(fs)pulsed Ti:Sapphire laser working at 80 MHz repe-tition rate and(time-correlated single photon counting)(TCSPC)detection electronics.The interesting question was whether it is possible to follow up PLIM 1using faster repetition rates.Faster repetition rates could be advantageous for the induction of specific photochemical reactions because of similar light doses used normally in standard CW light treatments.For this,a default 2p-FLIM-PLIM system was expanded by adding a second fs pulsed laser("helixx")which provides 50 fs pulses at a repetition rate of 250 MHz,more than 2.3 w average power and tunable from 720 nm to 920 nm.The laser beam was coupled into the AOM instead of the default 80 MHz laser.We demonstrated siuccessful applications of the 250 MHz laser for PLIM which correlates well with measurements done by excitation with the conventional 80MHx laser source.展开更多
Optical microscopy has become an indispensable tool for visualizing sub-cellular structures andbiological processes.However,scattering in biological tissues is a major obstacle that preventshigh-resolution images from...Optical microscopy has become an indispensable tool for visualizing sub-cellular structures andbiological processes.However,scattering in biological tissues is a major obstacle that preventshigh-resolution images from being obtained from deep regions of tissue.We review commontechniques,such as multiphoton microscopy(MPM)and optical coherence microscopy(OCM),for diffraction limited imaging beyond an imaging depth of 0.5 mm.Novel implementations havebeen emerging in recent years giving higher imaging speed,deeper penetration,and better imagequality.Focal modulation microscopy(FMM)is a novel method that combines confocal spatialfltering with focal modulation to reject out-of-focus background.FMM has demonstrated animaging depth comparable to those of MPM and OCM,near-real-time image acquisition,and thecapability for multiple contrast mechanisms.展开更多
Broad and safe access to ultrafast laser technology has been hindered by the absence of optical fiber-delivered pulses with tunable central wavelength,pulse repetition rate,and pulse width in the picosecond–femtoseco...Broad and safe access to ultrafast laser technology has been hindered by the absence of optical fiber-delivered pulses with tunable central wavelength,pulse repetition rate,and pulse width in the picosecond–femtosecond regime.To address this long-standing obstacle,we developed a reliable accessory for femtosecond ytterbium fiber chirped pulse amplifiers,termed a fiber-optic nonlinear wavelength converter(FNWC),as an adaptive optical source for the emergent field of femtosecond biophotonics.This accessory empowers the fixed-wavelength laser to produce fiber-delivered∼20 nJ pulses with central wavelength across 950 to 1150 nm,repetition rate across 1 to 10 MHz,and pulse width across 40 to 400 fs,with a long-term stability of>2000 h.As a prototypical label-free application in biology and medicine,we demonstrate the utility of FNWC in real-time intravital imaging synergistically integrated with modern machine learning and largescale fluorescence lifetime imaging microscopy.展开更多
Multiphoton absorption of femtosecond laser pulses focused through an objective with high numerical aperture(NA)can be used to image and manipulate cellular and intracellular objects.This review highlights recent adva...Multiphoton absorption of femtosecond laser pulses focused through an objective with high numerical aperture(NA)can be used to image and manipulate cellular and intracellular objects.This review highlights recent advances in intracellular manipulation,including nanosurgery and labeling in living cells with femtosecond lasers.展开更多
Sensing and responding to our environment requires functional neurons that act in concert. Neuronal cell loss resulting from degenerative diseases cannot be replaced in humans, causing a functional impairment to integ...Sensing and responding to our environment requires functional neurons that act in concert. Neuronal cell loss resulting from degenerative diseases cannot be replaced in humans, causing a functional impairment to integrate and/or respond to sensory cues. In contrast, zebrafish（Danio rerio） possess an endogenous capacity to regenerate lost neurons. Here, we will focus on the processes that lead to neuronal regeneration in the zebrafish retina. Dying retinal neurons release a damage signal, tumor necrosis factor α, which induces the resident radial glia, the Müller glia, to reprogram and re-enter the cell cycle. The Müller glia divide asymmetrically to produce a Müller glia that exits the cell cycle and a neuronal progenitor cell. The arising neuronal progenitor cells undergo several rounds of cell divisions before they migrate to the site of damage to differentiate into the neuronal cell types that were lost. Molecular and immunohistochemical studies have predominantly provided insight into the mechanisms that regulate retinal regeneration. However, many processes during retinal regeneration are dynamic and require live-cell imaging to fully discern the underlying mechanisms. Recently, a multiphoton imaging approach of adult zebrafish retinal cultures was developed. We will discuss the use of live-cell imaging, the currently available tools and those that need to be developed to advance our knowledge on major open questions in the field of retinal regeneration.展开更多
Two-photon excitedfluorescence(TPEF)spectroscopy and imaging were used to investigate the effects of gamma-irradiation on neural stem and precursor cells(NSPCs).While the observed signal from reduced nicotinamide aden...Two-photon excitedfluorescence(TPEF)spectroscopy and imaging were used to investigate the effects of gamma-irradiation on neural stem and precursor cells(NSPCs).While the observed signal from reduced nicotinamide adenine dinucleotide(NADH)was localized to the mitochondria,the signal typically associated with oxidizedflavoproteins(Fp)was distributed diffusely throughout the cell.The measured TPEF emission and excitation spectra were similar to the established spectra of NAD(P)H and Fp.Fpfluorescence intensity was markedly increased by addition of the electron transport chain(ETC)modulator menadione to the medium,along with a concomitant decrease in the NAD(P)H signal.Three-dimensional(3D)neurospheres were imaged to obtain the cellular metabolic index(CMI),calculated as the ratio of Fp to NAD(P)Hfluorescence intensity.Radiation effects were found to differ between low-dose(50 cGy)and high-dose(50 cGy)exposures.Low-dose irradiation caused a marked drop in CMI values accompanied by increased cellular proliferation.At higher doses,both NAD(P)H and Fp signals increased,leading to an overall elevation in CMI values.Thesefindings underscore the complex relationship between radiation dose,metabolic state,and proliferation status in NSPCs and highlight the ability of TPEF spectroscopy and imaging to characterize metabolism in 3D spheroids.展开更多
Elastic cartilage in the rabbit external ear is an important animal model with attractive potential value for researching the physiological and pathological states of cartilages especially during wound healing.In this...Elastic cartilage in the rabbit external ear is an important animal model with attractive potential value for researching the physiological and pathological states of cartilages especially during wound healing.In this work,nonlinear optical microscopy based on two-photon excited fluor-escence and second harmonic generation were employed for imaging and quantifying the intact elastic cartilage.The morphology and distribution of main components in elastic carti lage incuding cartilage cells,collagen and elastic fibers were clearly observed from the high-resolution two-di mensional nonlinear optical images.The areas of cell nuclei,a parameter related to the pathological changes of nornal or abnormal elastic cartilage,can be easily quantifed.Moreover,the three-dimensional structure of chondrocytes and matrix were displayed by constructing threedimensional image of cartilage tissue.At last,the enmission spectra from cartilage were obtained and analyzed.We found that the different ratio of collagen over elastic fibens can be used to locate the observed position in the elastic cartilage.The redox ratio based on the ratio of nicotinamide adenine dimucleotide(NADH)over flavin adenine dinucleotide(FAD)fuorescence can also be calculated to analyze the metabolic state of chondrocytes in different regions.Our results demonstrated that this technique has the potential to provide more accurate and comprehensive information for the physiological states of elastic cartilage.展开更多
基金support of the National Research Program of Genomic Medicine(NRPGM)of the National Science Council(NSC)in Taiwan.
文摘Multiphoton microscopy(MPM),with the advantages of improved penetration depth,decreased photo-damage,and optical sectioning capability,has become an indispensable tool for biomedical imaging.The combination of multiphoton fluorescence(MF)and second-harmonic generation(SHG)microscopy is particularly effective in imaging tissue structures of the ocular surface.This work is intended to be a review of advances that MPM has made in ophthalmic imaging.The MPM not only can be used for the label-free imaging of ocular structures,it can also be applied for investigating the morphological alterations in corneal pathologies,such as keratoconus,infected keratitis,and corneal scar.Furthermore,the corneal wound healing process after refractive surgical procedures such as conductive keratoplasty(CK)can also be studied with MPM.Finally,qualitative and quantitative SHG microscopy is effective for characterizing corneal thermal denaturation.With additional development,multiphoton imaging has the potential to be developed into an effective imaging technique for in vivo studies and clinical diagnosis in ophthalmology.
文摘Multiphoton microscopy, relying on the simultaneous absorption of two or more photons by a fluorophore, has come to occupy a prominent place in modern biomedical research with its ability to allow real-time observation of a single cell and molecules in intact tissues. Multiphoton microscopy exhibits nonlinear optical contrast properties, which can make it possible to provide an exceptionally large depth penetration with less phototoxicity. This system becomes more and more an inspiring tool for a non-invasive imaging system to realize "optical biopsy" and to examine the functions of living cells. In this review, we briefly present the physical principles and properties of multiphoton microscopy as well as the current applications in biological fields. In addition, we address what we see as the future potential of multiphoton microscopy for gastroenterologic research.
基金This work has been partially supported by NIH(SC COBRE P20RR021949 and Career Award 1k25hl088262-01)NSF(MRI CBET-0923311 and SC EPSCoR RII EPS-0903795 through SC GEAR program)+3 种基金The National Natural Science Foundation of China(31171372,61078067)Guangdong Province Science and Technology Project(2010B060300002)Shenzhen University Application Technology Development Project(201136,CXB201104220021A,JC201005250032A,200854)the Fundamental Research Funds for the Central Universities(K50510050006).
文摘Multifocal multiphoton microscopy(MMM)has recently become an important tool in biomedicine for performing three-dimensional fastfluorescence imaging.Using various beamsplitting techniques,MMM splits the near-infrared laser beam into multiple beamlets and produces a multifocal array on the sample for parallel multiphoton excitation and then recordsfluorescence signal from all foci simultaneously with an area array detector,which significantly improves the imaging speed of multiphoton microscopy and allows for high efficiency in use of the excitation light.In this paper,we discuss the features of several MMM setups using different beamsplitting devices,including a Nipkow spinning disk,a microlens array,a set of beamsplitting mirrors,or a diffractive optical element(DOE).In particular,we present our recent work on the development of an MMM using a spatial light modulator(SLM).
基金supported by the National Natural Science Foundation of China (Grant Nos.82171991 and 81700576)Natural Science Foundation of Fujian Province (Nos.2020J01154,2020J011008,2019J01269 and 2020J01839)+1 种基金Joint Funds for the Innovation of Science and Technology of Fujian Province (2017Y9038 and 2019Y9101)the special Funds of the Central Government Guiding Local Science and Technology Development (No.2020L3008).
文摘The tumor microenvironment(TME)is now recognized as an important participant of tumor progression.As the most abundant extracellular matrix component in TME,collagen plays an important role in tumor development.The imaging study of collagen morphological feature in TME is of great significance for understanding the state of tumor.Multiphoton microscopy(MPM),based on second harmonic generation(SHG)and two-photon excitation fluorescence(TPEF),can be used to monitor the morphological changes of biological tissues without labeling.In this study,we used MPM for large-scale imaging of early invasive breast cancer from the tumor center to normal tissues far from the tumor.We found that there were signi¯cant di®erences in collagen morphology between breast cancer tumor boundary,near tumor transition region and normal tissues far from the tumor.Furthermore,the morphological feature of eight collagen¯bers was extracted to quantify the variation trend of collagen in three regions.These results may provide a new perspective for the optimal negative margin width of breast-conserving surgery and the understanding of tumor metastasis.
基金The work was supported by the National Natural Science Foundation of China(No.60908043 and No.30970783)the Program for New Century Excellent Talents in University(NCET-07-0191)the Natural Science Funds for Distinguished Young Scholar in Fujian Province(2009J06031).
文摘Epithelial cancer comprises more than 85%of human cancers.The detection and treatment at the early stage has been demonstrated to apparently improve patient survival.In this review,we summarize our recent research works on the diagnostic application of epithelial tissue based on multiphoton microscopy(MPM),including identification of the layered structures of esophagus,oral cavity,skin and bronchus tissues,establishment of the diagnostic features for distinguishing gastric normal tissue from cancerous tissue,linking collagen alteration and ectocervical epithelial tumor progression for evaluating epithelial tumor progression,and differentiating normal,inflammatory,and dysplastic ectocervical epithelial tissues.These results provide the groundwork for developing MPM into clinical multiphoton endoscopy.
基金PTCSO acknowledges support from:NII 9P41EB015871-26A1,R01-EX017656,5 R01 NS051320,4R.44EB012415-02,NSF CBET-0939511,the Sin-gapore-MIT Alliance 2 and the MIT SkolTech in-itiativeEYYS issupported by the National Research Foundation Singapore through the Singapore MIT Alliance for Research and Technology's Bio Symresearch programmeCJR is funded by a Wellcome Trust,MIT Pastdoctoral Research Fellow ship 093831/Z/10/Z.
文摘This review summarizes the historical and more recent developments of multiphoton microscopy,as applied to dermatology.Multiphoton microscopy offers several advantages over competingmicroscopy techniques:there is an inherent axial sectioning,penetration depths that competewll with confocal microscopy on account of the use of near-infrared light,and many t wo-pho ton contrast mechanisms,such as second-harmonic generation,have no analogue in one-photonmicroscopy.While the penetration depths of photons into tissue are typically limited on the orderof hundreds of microns,this is of les concern in dermatology,as the skin is thin and readlily.accessible.As a result,multiphoton microscopy in dermatology has generated a great deal ofmterest,much of which is summarized here.The review covers the interaction of light and tissue,as well as the various considerations that must be made when designing an instrument.The stateof multiphoton microscopy in imaging skin cancer and various other diseases is also discussed,along with the investigation of aging and regeneration phenomena,and finally,the use of mul-tiphoton microscopy to analyze the transdermal transport of drugs,cosmetics and other agents issummarized.The review concludes with a look at potential future research directions,especiallythose that are necessary to push these techniques into widespread clinical acceptance.
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.
基金supported by the Ministry of Science,ICT and Future Planning(MSIP)through the National Research Foundation of Korea(NRF)(RS-2024-00450201)supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute(KHIDI),funded by the Ministry of Health and Welfare,Republic of Korea(HI22C1374).
文摘We developed an imaging technique combining two-photon computed super-resolution microscopy and suction-based stabilization to achieve the resolution of the single-cell level and organelles in vivo.To accomplish this,a conventional two-photon microscope was equipped with a 3D-printed holders,which stabilize the tissue surface within the focal plane of immersion objectives.Further computational image stabilization and noise reduction were applied,followed by superresolution radial fluctuations(SRRF)analysis,doubling image resolution,and enhancing signal-to-noise ratios for in vivo subcellular process investigation.Stabilization of<1μm was obtained by suction,and<25 nm were achieved by subsequent algorithmic image stabilization.A Mito-Dendra2 mouse model,expressing green fluorescent protein(GFP)in mitochondria,demonstrated the potential of long-term intravital subcellular imaging.In vivo mitochondrial fission and fusion,mitochondrial status migration,and the effects of alcohol consumption(modeled as an alcoholic liver disease)and berberine treatment on hepatocyte mitochondrial dynamics are directly observed intravitally.Suction-based stabilization in two-photon intravital imaging,coupled with computational super-resolution holds promise for advancing in vivo subcellular imaging studies.
基金supported by the National Natural Science Foundation of China(Nos.62075135 and 61975126)the Science,Technology and Innovation Commission of Shenzhen Municipality(Nos.JCYJ20190808174819083 and JCYJ20190808175201640)。
文摘Multiphoton microscopy is the enabling tool for biomedical research,but the aberrations of biological tissues have limited its imaging performance.Adaptive optics(AO)has been developed to partially overcome aberration to restore imaging performance.For indirect AO,algorithm is the key to its successful implementation.Here,based on the fact that indirect AO has an analogy to the black-box optimization problem,we successfully apply the covariance matrix adaptation evolution strategy(CMA-ES)used in the latter,to indirect AO in multiphoton microscopy(MPM).Compared with the traditional genetic algorithm(GA),our algorithm has a greater improvement in convergence speed and convergence accuracy,which provides the possibility of realizing real-time dynamic aberration correction for deep in vivo biological tissues.
基金supported by Specially Funded Program on National Key Scienti¯c Instruments and Equipment Development(61527827),Program 973(2015CB352005)the National Natural Science Foundation of China(31171372/61525503/61378091/61620106016),Guangdong Natural Science Foundation(2014A030312008/2015A020214023/2015KGJHZ002)Shenzhen Science and Technology R&D Foundation(JCYJ20160422151611496).
文摘Using the combination of a refective blazed grating and a reflective phase-only difractive spatiallight modulator(SLM),scanless multitarget-matching multiphoton excitation fuorescence mi.croscopy(SMTM-MP M)was achieved.The SLM shaped an incoming mode-locked,near-infraredTi:sapphire laser beam into an excitation pattern with addressable shapes and sizes that matchedthe samples of interest in the field of view.Temporal and spatial focusing were simultaneouslyrealized by combining an objective lens and a blazed grating.The fluorescence signal fromilluminated areas was recorded by a two-dimensional sCMOS camera.Compared with a conventional temporal focusing multiphoton microscope,our microscope achieved effective use of thelaser power and decreased photodamage with higher axial resolution.
基金funded in part by grants from the National Institutes of Health(1R01CA213149,5R01EB023232)the National Science Foundation(CBET 18-41539).
文摘To date,numerous studies have been performed to elucidate the complex cellular dynamics in skin diseases,but few have attempted to characterize these cellular events under conditions similar to the native environment.To address this challenge,a three-dimensional(3D)multimodal analysis platform was developed for characterizing in vivo cellular dynamics in skin,which was then utilized to process in vivo wound healing data to demonstrate its applicability.Special attention is focused on in vivo biological parameters that are difficult to study with ex vivo analysis,including 3D cell tracking and techniques to connect biological information obtained from different imaging modalities.These results here open new possibilities for evaluating 3D cellular dynamics in vivo,and can potentially provide new tools for characterizing the skin microenvironment and pathologies in the future.
基金supported by the Program for Changjiang Scholars and Innovative Research Team in University(Grant No.IRT1115)the National Natural Science Foundation of China(Grant Nos.81271620,61275006,81101209,30970783).
文摘Multiphoton microscopy(MPM),based on two-photon excited fuorescence and second harmonic generation,enables direct noninvasive visualization of tissue architecture and cell morphology in live tissues without the administration of exogenous contrast agents.In this paper,we used MPM to image the microstructures of the mucosa in fresh,unfixed,and unstained intestinal tissue of mouse.The morphology and distribution of the main components in mucosa layer such as columnar cells,goblet cells,intestinal glands,and a little collagen fibers were clearly observed in MPM images,and then compared with standard H&:E images from paired specimens.Our results indicate that MPM combined with endoscopy and miniaturization probes has the potential application in the clinical diagnosis and in vivo monitoring of early intestinal cancer.
文摘Multi-photon microscopy(MPM)and coherent anti-Stokes Raman scattering(CARS)are two advanced nonlinear optical imaging techniques,which provide complementary information and have great potential in combination for noninvasive in vrito biomedical applications.This paper provides a detailed discussion of the basics,development and applications of these technologies for in vrivo skin research,covering the following topics:The principle and advantage of MPM and CARS,instrumentation development for in vino applications,MPM and CARS of normal skin,application of MPM and CARS in skin cancer and disease diagnosis;application of MPM in skin disease intervention,ie.,imaging guided two-photon photothermolysis.
基金supported by the Ministry of Research and Development,FKZ order:13N14508("OMOXI")by the Ministry of Economics,ZIM-Project,FKZ:ZF4322901RE6("UFEMPU").
文摘Simultaneous metabolic and oxygen imaging is promising to follow up therapy response,dis-ease development and to determine prognostic factors.FLIM of metabolic coenzymes is now widely accepted to be the most reliable method to determine cellular bioenergetics.Also,oxygen consumption has to be taken into account to understand treatment responses.The phosphorescence lifetimne of oxygen sensors is able to indicate local oxygen changes.For phosphorescence lifetime imaging(PLIM)dyes based on ruthenium(I)coordination com-plexes are useful,in detaill TLD1433 which possesses a variety of different triplet states,enables complex photochemistry and redox reactions.PLIM is usally reached by two photon exci-tation of the drug with a femtosecond(fs)pulsed Ti:Sapphire laser working at 80 MHz repe-tition rate and(time-correlated single photon counting)(TCSPC)detection electronics.The interesting question was whether it is possible to follow up PLIM 1using faster repetition rates.Faster repetition rates could be advantageous for the induction of specific photochemical reactions because of similar light doses used normally in standard CW light treatments.For this,a default 2p-FLIM-PLIM system was expanded by adding a second fs pulsed laser("helixx")which provides 50 fs pulses at a repetition rate of 250 MHz,more than 2.3 w average power and tunable from 720 nm to 920 nm.The laser beam was coupled into the AOM instead of the default 80 MHz laser.We demonstrated siuccessful applications of the 250 MHz laser for PLIM which correlates well with measurements done by excitation with the conventional 80MHx laser source.
文摘Optical microscopy has become an indispensable tool for visualizing sub-cellular structures andbiological processes.However,scattering in biological tissues is a major obstacle that preventshigh-resolution images from being obtained from deep regions of tissue.We review commontechniques,such as multiphoton microscopy(MPM)and optical coherence microscopy(OCM),for diffraction limited imaging beyond an imaging depth of 0.5 mm.Novel implementations havebeen emerging in recent years giving higher imaging speed,deeper penetration,and better imagequality.Focal modulation microscopy(FMM)is a novel method that combines confocal spatialfltering with focal modulation to reject out-of-focus background.FMM has demonstrated animaging depth comparable to those of MPM and OCM,near-real-time image acquisition,and thecapability for multiple contrast mechanisms.
基金support from the National Institutes of Health,U.S.Department of Health and Human Services(Grant No.R01 CA241618)J.E.S.and R.R.I were supported by NIBIB/NIH(Award No.T32EB019944).
文摘Broad and safe access to ultrafast laser technology has been hindered by the absence of optical fiber-delivered pulses with tunable central wavelength,pulse repetition rate,and pulse width in the picosecond–femtosecond regime.To address this long-standing obstacle,we developed a reliable accessory for femtosecond ytterbium fiber chirped pulse amplifiers,termed a fiber-optic nonlinear wavelength converter(FNWC),as an adaptive optical source for the emergent field of femtosecond biophotonics.This accessory empowers the fixed-wavelength laser to produce fiber-delivered∼20 nJ pulses with central wavelength across 950 to 1150 nm,repetition rate across 1 to 10 MHz,and pulse width across 40 to 400 fs,with a long-term stability of>2000 h.As a prototypical label-free application in biology and medicine,we demonstrate the utility of FNWC in real-time intravital imaging synergistically integrated with modern machine learning and largescale fluorescence lifetime imaging microscopy.
文摘Multiphoton absorption of femtosecond laser pulses focused through an objective with high numerical aperture(NA)can be used to image and manipulate cellular and intracellular objects.This review highlights recent advances in intracellular manipulation,including nanosurgery and labeling in living cells with femtosecond lasers.
基金supported by NIH-NEI grants to DRH(R01-EY018417,R01-EY024519)the Center for Zebrafish Research,University of Notre Dame,USA
文摘Sensing and responding to our environment requires functional neurons that act in concert. Neuronal cell loss resulting from degenerative diseases cannot be replaced in humans, causing a functional impairment to integrate and/or respond to sensory cues. In contrast, zebrafish（Danio rerio） possess an endogenous capacity to regenerate lost neurons. Here, we will focus on the processes that lead to neuronal regeneration in the zebrafish retina. Dying retinal neurons release a damage signal, tumor necrosis factor α, which induces the resident radial glia, the Müller glia, to reprogram and re-enter the cell cycle. The Müller glia divide asymmetrically to produce a Müller glia that exits the cell cycle and a neuronal progenitor cell. The arising neuronal progenitor cells undergo several rounds of cell divisions before they migrate to the site of damage to differentiate into the neuronal cell types that were lost. Molecular and immunohistochemical studies have predominantly provided insight into the mechanisms that regulate retinal regeneration. However, many processes during retinal regeneration are dynamic and require live-cell imaging to fully discern the underlying mechanisms. Recently, a multiphoton imaging approach of adult zebrafish retinal cultures was developed. We will discuss the use of live-cell imaging, the currently available tools and those that need to be developed to advance our knowledge on major open questions in the field of retinal regeneration.
基金supported by U.S.Department of Energy,Grant No.DE-FG02-09ER64798(CLL)National Aeronautics and Space Administration Grant No.NNX09AK25G(CLL)+2 种基金American Cancer Society Grant No.RSG-00-036-04-CNE(CLL)National Institute of Health NIH LAMMP P41 Grant No.R01192(BJT,TBK)National Cancer Institute 2P30CA62203(BJT,TBK).
文摘Two-photon excitedfluorescence(TPEF)spectroscopy and imaging were used to investigate the effects of gamma-irradiation on neural stem and precursor cells(NSPCs).While the observed signal from reduced nicotinamide adenine dinucleotide(NADH)was localized to the mitochondria,the signal typically associated with oxidizedflavoproteins(Fp)was distributed diffusely throughout the cell.The measured TPEF emission and excitation spectra were similar to the established spectra of NAD(P)H and Fp.Fpfluorescence intensity was markedly increased by addition of the electron transport chain(ETC)modulator menadione to the medium,along with a concomitant decrease in the NAD(P)H signal.Three-dimensional(3D)neurospheres were imaged to obtain the cellular metabolic index(CMI),calculated as the ratio of Fp to NAD(P)Hfluorescence intensity.Radiation effects were found to differ between low-dose(50 cGy)and high-dose(50 cGy)exposures.Low-dose irradiation caused a marked drop in CMI values accompanied by increased cellular proliferation.At higher doses,both NAD(P)H and Fp signals increased,leading to an overall elevation in CMI values.Thesefindings underscore the complex relationship between radiation dose,metabolic state,and proliferation status in NSPCs and highlight the ability of TPEF spectroscopy and imaging to characterize metabolism in 3D spheroids.
基金supported by the Program for Changjiang Scholars and Innovative Research Team in University(Grant No.IRT1115)the National Natural Science Foundation of China(Grant Nos.81101209,61275006,81271620).
文摘Elastic cartilage in the rabbit external ear is an important animal model with attractive potential value for researching the physiological and pathological states of cartilages especially during wound healing.In this work,nonlinear optical microscopy based on two-photon excited fluor-escence and second harmonic generation were employed for imaging and quantifying the intact elastic cartilage.The morphology and distribution of main components in elastic carti lage incuding cartilage cells,collagen and elastic fibers were clearly observed from the high-resolution two-di mensional nonlinear optical images.The areas of cell nuclei,a parameter related to the pathological changes of nornal or abnormal elastic cartilage,can be easily quantifed.Moreover,the three-dimensional structure of chondrocytes and matrix were displayed by constructing threedimensional image of cartilage tissue.At last,the enmission spectra from cartilage were obtained and analyzed.We found that the different ratio of collagen over elastic fibens can be used to locate the observed position in the elastic cartilage.The redox ratio based on the ratio of nicotinamide adenine dimucleotide(NADH)over flavin adenine dinucleotide(FAD)fuorescence can also be calculated to analyze the metabolic state of chondrocytes in different regions.Our results demonstrated that this technique has the potential to provide more accurate and comprehensive information for the physiological states of elastic cartilage.
