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multiphoton microscopy:A NEW APPROACH IN PHYSIOLOGICAL STUDIES AND PATHOLOGICAL DIAGNOSIS FOR OPHTHALMOLOGY

Journal of Innovative Optical Health Sciences 2009年第1期2卷 45-60页

作者： CHIU-MEI HSUEH WEN LO SUNG-JAN LIN TSUNG-JEN WANG FUNG-RUNG HU HSIN-YUAN TAN CHEN-YUAN DONG Department of Physics National Taiwan University Taipei 106 Taiwan Institute of Biomedical Engineering National Taiwan University Taipei 106 Taiwan Department of Dermatology National Taiwan University Hospital Taipei 100 Taiwan Department of Ophthalmology Taipei Medical University Hospital Taipei 100 Taiwan Department of Ophthalmology National Taiwan University College of Medicine and Hospital Taipei 100Taiwan Department of Ophthalmology Chang-Gung University Linko 333 Taiwan Institute of Biomedical Engineering National Taiwan University Taipei 100 Taiwan

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.

关键词： multiphoton microscopy fluorescence second harmonic generation cornea sclera

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Aberration correction for multiphoton microscopy using covariance matrix adaptation evolution strategy

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Chinese Optics Letters 2023年第5期21卷 88-93页

作者：王科郑磊秦梦圆张万剑邓想全仝申程慧黄杰钟金成张颖娴邱娉 Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province College of Physics and Optoelectronic EngineeringShenzhen UniversityShenzhen 518060China

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.

关键词： multiphoton microscopy 1700-nm window adaptive optics covariance matrix adaptation evolution strategy

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Quantitative analysis of collagen morphology in breast cancer from millimeter scale using multiphoton microscopy

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创新光学健康科学杂志(英文) 2023年第4期16卷 48-57页

作者： Yulan Liu Shunwu Xu Deyong Kang Xingxin Huang Shuoyu Xu Lianhuang Li Liqin Zheng Lida Qiu Zhenlin Zhan Xiahui Han Jianxin Chen Key Laboratory of OptoElectronic Science and Technology for Medicine of Ministry of Education Fujian Provincial Key Laboratory of Photonics Technology Fujian Normal University Fuzhou 350007P.R.China School of Electronic and Mechanical Engineering Fujian Polytechnic Normal University Fuqing Fujian 350300P.R.China Department of Pathology Fujian Medical University Union Hospital Fuzhou 350001 P.R.China Department of General Surgery Nanfang Hospital Southern Medical University Guangzhou 510515P.R.China College of Physics and Electronic Information Engineering Minjiang University Fuzhou 350108P.R.China

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.

关键词： Breast cancer tumor microenvironment collagen¯ber morphology multiphoton microscopy

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DIAGNOSTIC APPLICATION OF multiphoton microscopy IN EPITHELIAL TISSUES

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Journal of Innovative Optical Health Sciences 2011年第2期4卷 159-163页

作者： XINGSHAN JIANG SHUANGMU ZHUO JIANXIN CHEN Institute of Laser and Optoelectronics Technology Fujian Provincial Key Laboratory for Photonics Technology Key Laboratory of OptoElectronic Science and Technology for Medicine of Ministry of Education Fujian Normal University Fuzhou 350007 China

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.

关键词： Epithelial tissue multiphoton microscopy two-photon excitedfluorescence secondharmonic generation

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Scanless multitarget-matching multiphoton excitation fluorescence microscopy

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Journal of Innovative Optical Health Sciences 2018年第2期11卷 1-7页

作者： Junpeng Qiu Lei Wang Bruce Zhi Gao Junle Qu Yonghong Shao College of Optoelectronics Engineering Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province Shenzhen University Shenzhen 518060P.R.China Department of Bioengineering and COMSET Clemson University ClemsonSC 29634USA

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.

关键词： Multitarget-matching multiphoton microscopy SLM temporal focusing

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Nonlinear optical microscopy for skin in vivo:Basics,development and applications

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Journal of Innovative Optical Health Sciences 2023年第1期16卷 95-113页

作者： Jianhua Zhao Yuan Zhao Zhenguo Wu Yunxian Tian Haishan Zeng Integrative Oncology Department–Imaging Unit BC Cancer Research Institute VancouverBCCanada Department of Dermatology and Skin Science University of British Columbia and Vancouver Coastal Health Research Institute VancouverBCCanada

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.

关键词： Nonlinear microscopy multiphoton microscopy coherent anti-Stokes Raman scattering microscopy skin skin cancer multiphoton therapy

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PROBING THE IMPACT OF GAMMA-IRRADIATION ON THE METABOLIC STATE OF NEURAL STEM AND PRECURSOR CELLS USING DUAL-WAVELENGTH INTRINSIC SIGNAL TWO-PHOTON EXCITED FLUORESCENCE

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Journal of Innovative Optical Health Sciences 2011年第3期4卷 289-300页

作者： TATIANA B.KRASIEVA ERICH GIEDZINSKI KATHERINE TRAN MARY LAN CHARLES L.LIMOLI BRUCE J.TROMBERG Laser Microbeam and Medical Program Beckman Laser Institute University of California Irvine1002 Health Sciences Road EastIrvineCA 92612USA Department of Radiation Oncology University of California IrvineCalifornia 92697USA

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.

关键词： multiphoton microscopy cellular metabolic index gamma radiation brain tumors NAD(P)H flavoproteins

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Live-cell imaging:new avenues to investigate retinal regeneration

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Neural Regeneration Research 2017年第8期12卷 1210-1219页

作者： Manuela Lahne David R.Hyde Department of Biological Sciences University of Notre Dame Notre Dame IN USA

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.

关键词： multiphoton microscopy live-cell imaging zebrafish interkinetic nuclear migration tissue culture retinal regeneration Miiller glia neuronal progenitor cell differentiation phagocytosis

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Video-rate multimodal multiphoton imaging and three-dimensional characterization of cellular dynamics in wounded skin

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Journal of Innovative Optical Health Sciences 2020年第2期13卷 70-80页

作者： Joanne Li Madison N.Wilson Andrew J.Bower Marina Marjanovic Eric J.Chaney Ronit Barkalifa Stephen A.Boppart Department of Bioengineering University of Illinois at Urbana-Champaign Urbana ILU.S.A Department of Electrical and Computer Engineering University of Illinois at Urbana-Champaign Urbana ILU.S.A Beckman Institute for Advanced Science and Technology University of Illinois at Urbana-Champaign Urbana ILU.S.A Carle Illinois College of Medicine University of Illinois at Urbana-Champaign Urbana ILU.S.A

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.

关键词： Cellular dynamics multimodal imaging multiphoton microscopy

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Wide-field three-photon excitation in biological samples

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Light(Science & Applications) 2016年第1期5卷 65-73页

作者： Christopher J Rowlands Demian Park Oliver T Bruns Kiryl D Piatkevich Dai Fukumura Rakesh K Jain Moungi G Bawendi Edward S Boyden Peter TC So Department of Biological Engineering Massachusetts Institute of TechnologyCambridgeMA 02139USA Media Lab Massachusetts Institute of TechnologyCambridgeMA 02139USA Department of Chemistry Massachusetts Institute of TechnologyCambridgeMA 02139USA Edwin L.Steele Laboratories Department of Radiation OncologyMassachusetts General Hospital and Harvard Medical SchoolBostonMA 02114USA Department of Brain and Cognitive Sciences McGovern Institute and MIT Center for Neurobiological EngineeringMassachusetts Institute of TechnologyCambridgeMA 02139-4307USA Department of Mechanical Engineering Massachusetts Institute of TechnologyCambridgeMA 02139USA

Three-photon wide-field depth-resolved excitation is used to overcome some of the limitations in conventional point-scanning two-and three-photon microscopy.Excitation of chromophores as diverse as channelrhodopsins and quantum dots is shown,and a penetration depth of more than 700μm into fixed scattering brain tissue is achieved,approximately twice as deep as that achieved using two-photon wide-field excitation.Compatibility with live animal experiments is confirmed by imaging the cerebral vasculature of an anesthetized mouse;a complete focal stack was obtained without any evidence of photodamage.As an additional validation of the utility of wide-field three-photon excitation,functional excitation is demonstrated by performing threephoton optogenetic stimulation of cultured mouse hippocampal neurons expressing a channelrhodopsin;action potentials could reliably be excited without causing photodamage.

关键词： biophotonics multiphoton microscopy optogenetics temporal focusing three-photon

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