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Compact and effective photon-resolved image scanning microscope

Advanced Photonics 2024年第1期6卷 84-95页

作者： Giorgio Tortarolo Alessandro Zunino Simonluca Piazza Mattia Donato Sabrina Zappone Agnieszka Pierzyńska-Mach Marco Castello Giuseppe Vicidomini Istituto Italiano di Tecnologia Molecular Microscopy and SpectroscopyGenoaItaly Genoa Instruments GenoaItaly University of Genoa Dipartimento di InformaticaBioingegneriaRobotica e Ingegneria dei SistemiGenoaItaly Istituto Italiano di Tecnologia Nanoscopy and NIC@IITGenoaItaly École Polytechnique Fédérale de Lausanne Laboratory of Experimental BiophysicsLausanneSwitzerland

Fluorescence confocal laser-scanning microscopy(LSM)is one of the most popular tools for life science *** popularity is expected to grow thanks to single-photon array detectors tailored for *** detectors offer unique single-photon spatiotemporal information,opening new perspectives for gentle and quantitative superresolution ***,a flawless recording of this information poses significant challenges for the microscope data acquisition(DAQ)*** present a DAQ module based on the digital frequency domain principle,able to record essential spatial and temporal features of *** use this module to extend the capabilities of established imaging techniques based on single-photon avalanche diode(SPAD)array detectors,such as fluorescence lifetime image scanning ***,we use the module to introduce a robust multispecies approach encoding the fluorophore excitation spectra in the time ***,we combine time-resolved stimulated emission depletion microscopy with image scanning microscopy,boosting spatial *** results demonstrate how a conventional fluorescence laser scanning microscope can transform into a simple,information-rich,superresolved imaging system with the simple addition of a SPAD array detector with a tailored data acquisition *** expected a blooming of advanced single-photon imaging techniques,which effectively harness all the sample information encoded in each photon.

关键词： fluorescence lifetime image scanning microscopy digital frequency domain single photon

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Fast repetition rate fs pulsed lasers for advanced PLIM microscopy

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Journal of Innovative Optical Health Sciences 2019年第5期12卷 86-94页

作者： Sviatlana Kalinina Alexander Jelzow Tobias Plotzing Angelika Rück University of Ulm Core Facility Confocal and Multiphoton MicroscopyN24 Albert-Einstein-Allee 1189081 UlmGermany Becker&Hickl GmbH Nahmitzer Damm 3012277 BerlinGermany Laser Quantum GmbH Max-Stromeyer-Str.11678467 KonstanzGermany

Simultaneous metabolic and oxygen imaging is promising to follow up therapy response,dis-ease development and to determine prognostic *** of metabolic coenzymes is now widely accepted to be the most reliable method to determine cellular ***,oxygen consumption has to be taken into account to understand treatment *** phosphorescence lifetimne of oxygen sensors is able to indicate local oxygen *** 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 *** 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 *** interesting question was whether it is possible to follow up PLIM 1using faster repetition *** repetition rates could be advantageous for the induction of specific photochemical reactions because of similar light doses used normally in standard CW light *** 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 *** laser beam was coupled into the AOM instead of the default 80 MHz *** demonstrated siuccessful applications of the 250 MHz laser for PLIM which correlates well with measurements done by excitation with the conventional 80MHx laser source.

关键词： Multiphoton microscopy axygen consumption phosphorescence lifetime microscopy fast repetition rate excitation short laser pulses Ru(I)complexes

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In vivo confocal microscopy: qualitative investigation of the conjunctival and corneal surface in open angle glaucomatous patients undergoing the XEN-Gel implant, trabeculectomy or medical therapy

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Eye and Vision 2020年第1期7卷 136-149页

作者： Stefano Baiocchi Cosimo Mazzotta Arianna Sgheri Alessandro Di Maggio Simone Alex Bagaglia Matteo Posarelli Leonardo Ciompi Alessandro Meduri Gian Marco Tosi Department of Medicine Surgery and NeurosciencesOphthalmology UnitPoliclinico Santa Maria alle ScotteUniversity of SienaViale Bracci 8ZIP code 53100 SienaItaly Ophthalmology unit Department of Biomedical SciencesUniversity of MessinaMessinaItaly

Purpose:Assessing the quality of the ocular surface by in vivo scanning laser confocal microscopy(IVCM)in primary open angle glaucoma(POAG)patients treated by Xen 45 Gel Stent,medical therapy and ***:Retrospective,single-center,single-masked,comparative study including 60 eyes of 30 patients(mean age 61.16±10 years)affected by *** were divided into 3 groups:Group 1 eyes underwent the Xen 45 Gel Stent procedure,Group 2 eyes were under medical therapy,Group 3 eyes were surgically treated by *** patients underwent HRT II IVCM analysis of cornea,limbus,conjunctiva,sub-tenionian space and ***:The Xen 45 Gel stent,if properly positioned in the sub-conjunctival space preserves goblet cells and limits ocular surface *** corneal epithelial cells with micro-cysts,and normo-reflective sub-epithelial nerve plexus are documented by *** sub Tenon’s implants an alternative lamellar intra-scleral filtration is *** surgical procedures show a noticeable number of inflammatory cells with rare ***-trabeculectomy inflammatory reaction is more evident than Xen 45 Gel Stent associated surgical procedures,but less than medical therapy where a conspicuous presence of Langerhans cells,peri-neural infiltrates,marked loss of goblet cells and fibrosis is ***:Ocular surface inflammation was more notable in topical therapy than after trabeculectomy,which itself causes more inflammation than XEN Gel stents.

关键词： Glaucoma Primary open angle glaucoma POAG MIGS Xen 45 gel stent Ocular surface Inflammation confocal microscopy IVCM

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Electron microscopy:essentials for viral structure,morphogenesis and rapid diagnosis

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Science China(Life Sciences) 2013年第9期56卷 421-430页

作者： ZHANG Ying HUNG Tao SONG JingDong HE JinSheng College of Life Sciences and Bioengineering Electron Microscopy LaboratorySchool of ScienceBeijing Jiaotong University Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention

Electron microscopy(EM) should be used in the front line for detection of agents in emergencies and bioterrorism,on accounts of its speed and ***,the number of EM diagnostic laboratories has decreased considerably and an increasing number of people encounter difficulties with EM ***,the research on viral structure and morphology has become important in EM diagnostic *** has several technological advantages,and should be a fundamental tool in clinical diagnosis of viruses,particularly when agents are unknown or *** this article,we review the historical contribution of EM to virology,and its use in virus differentiation,localization of specific virus antigens,virus-cell interaction,and viral *** is essential that EM investigations are based on clinical and comprehensive pathogenesis data from light or confocal ***,avoidance of artifacts or false results is necessary to exploit fully the advantages while minimizing its limitations.

关键词： electron microscopy viral structure viral morphology viral diagnosis

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Functional analysis of tomato calmodulin gene family during fruit development and ripening

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Horticulture Research 2014年第1期1卷 31-39页

作者： Tianbao Yang Hui Peng Gary R Bauchan Food Quality Laboratory Beltsville Agricultural Research CenterAgricultural Research ServiceUnited States Department of AgricultureBeltsvilleMD 20705USA College of Life Sciences Guangxi Normal UniversityGuilin 541004China Electron and confocal microscopy unit Beltsville Agricultural Research CenterAgricultural Research ServiceUnited States Department of AgricultureBeltsvilleMD 20705USA

Calmodulin is a ubiquitous calcium sensor to recognize the different developmental and/or stimulus-triggered calcium changes and regulate plant growth and ***,the function of calmodulin remains elusive for fleshy fruit *** performed expression studies of a family of six calmodulin genes(SlCaMs)in tomato *** calmodulins showed a double peak expression *** first flat peak appeared at 10–30 days after anthesis,but their expression rapidly declined at mature green and *** a sharp and even higher peak came at turning/pink *** six calmodulins,SlCaM1 had the highest expression during fruit enlargement,whereas SlCaM2 was the major calmodulin during fruit ***,SlCaMs showed different patterns in three ripening mutants rin,Nor and *** particular,at the stages corresponding to mature green and breaker,the expression levels of SlCaMs in those mutants were significantly higher than ***,SlCaMs,especially SlCaM2 were upregulated by *** overexpressing SlCaM2 in mature green fruit delayed ripening,while reducing SlCaM2 expression accelerated *** results suggest that SlCaMs play double roles to regulate fruit *** to the ethylene burst,the ethylene-independent repression of SlCaMs might be critical for fruit to initiate the ripening *** the ethylene burst,SlCaMs could participate in the ethylene coordinated rapid ripening.

关键词： development. calmodulin turning

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肿瘤坏死因子α对心肌细胞内受磷蛋白表达和细胞内钙的调节作用

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中国医学科学院学报 2005年第6期27卷 767-771,i0016页

作者：姚宇玫胡申江黄元伟杨春虎孙坚朱朝晖吴涛 Department of Cardiology Zhejiang Provincial Tongde Hospital 浙江大学医学院附属第一医院心内科暨心血管研究所杭州310003 confocal microscopy Lab Zhejiang Provincial Key Laboratory of BioelectromagneticsZhejiang University MedicalCollege

目的探讨肿瘤坏死因子α(TNFα)对培养的乳鼠心肌细胞内受磷蛋白(PLB)和肌浆网钙泵蛋白同功酶(SERCA2a)基因表达及细胞内游离钙离子浓度的影响。方法体外培养的乳鼠心肌细胞被随机分成对照组和不同浓度TNFα(1、10、30、50、70μg/L)干... 详细信息

目的探讨肿瘤坏死因子α(TNFα)对培养的乳鼠心肌细胞内受磷蛋白(PLB)和肌浆网钙泵蛋白同功酶(SERCA2a)基因表达及细胞内游离钙离子浓度的影响。方法体外培养的乳鼠心肌细胞被随机分成对照组和不同浓度TNFα(1、10、30、50、70μg/L)干预组,用单管一步法逆转录聚合酶链反应(RT-PCR)和Westernblotting技术,观察TNFα对乳鼠心肌细胞PLB和SERCA2a基因表达的影响。采用Fluo-3/Am荧光染色剂负载细胞,在激光共聚焦显微镜下测定TNFα对单个乳鼠心肌细胞内游离钙离子浓度的影响。结果TNFα浓度依赖性增高PLBmRNA和蛋白质的表达,10、30、50、70μg/LTNFα组PLBmRNA表达量分别较对照组增加66%、106%、141%、189%(P<0.05),蛋白质的表达分别较对照组提高30%、48%、73%、114%(P<0.001)。而1μg/LTNFα组与对照组PLBmRNA和蛋白质表达差异均无显著性。TNFα不影响SERCA2a的表达。50μg/LTNFα作用细胞24h,能降低异丙肾上腺素刺激的[Ca2+]i变化幅度的增高,[Ca2+]i变化幅度较对照组减少33%(P<0.01),但对静息状态下[Ca2+]i变化幅度无影响。结论TNFα可增强心肌细胞内PLB的表达,降低心肌细胞内[Ca2+]i的变化,这可能与TNFα对心肌细胞的负性肌力作用有关。

关键词：肿瘤坏死因子受磷蛋白肌浆网钙泵蛋白心肌细胞钙

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Localization and secretory pathways of a 58K-like protein in multi-vesicular bodies in callus of Arabidopsis thaliana

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Science China(Life Sciences) 2008年第9期51卷 827-832页

作者： XU Shan QIAN Jie SONG Xin ZHU Jian School of Life Science and Technology Tongji UniversityShanghai 200092China Laboratory of Electron microscopy Faculty of Basic Medicine ScienceSecond Military Medical UniversityShanghai 200433China

Multi-vesicular bodies in endocytosis and protoplasts are special cellular structures that are consid-ered to be originated from invagination of plasma membranes. However, the genesis and function of multi-vesicular bodies, the relationship with Golgi bodies and cell walls, and their secretory pathways remain controversial and ambiguous. Using a monoclonal antibody against an animal 58K protein, we have detected, by Western blotting and confocal microscopy, that a 58K-like protein is present in the calli of Arabidopsis thaliana and Hypericum perforatum. The results of immuno-electron microscopy showed that the 58K-like protein was located in the cisternae of Golgi bodies, secretory vesicles, multi-vesicular bodies, cell walls and vacuoles in callus of Arabidopsis thaliana, suggesting that the multi-vesicular bodies may be originated from Golgi bodies and function as a transporter carrying substances synthesized in Golgi bodies to cell walls and vacuoles. It seems that multi-vesicular bodies have a close relationship with the development of the cell wall and vacuole. The possible secretory pathways of multi-vesicular bodies might be in exocytosis, in which multi-vesicular bodies carry sub-stances to the cell wall for its construction, and in endocytosis, in which multi-vesicular bodies carry substances to the vacuole for its development, depending on what they carry and where the materials are transported. We hence propose that there is more than one pathway for the secretion of multi-vesicular bodies. In addition, our results provided a paradigm that a plant molecule, such as the 58k-like protein in callus of Arabidopsis thaliana, can be detected using a cross-reactive monoclonal antibody induced by an animal protein, and illustrate the existence of analog molecules in both animal and plant kingdoms.

关键词： 58K-like protein Arabidopsis thaliana localization multi-vesicular bodies secretory pathway

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DLST mutations in pheochromocytoma and paraganglioma cause proteome hyposuccinylation andmetabolic remodeling

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Cancer Communications 2023年第7期43卷 838-843页

作者： Sara Mellid Fernando García Luis Javier Leandro-García Alberto Díaz-Talavera Ángel Mario Martínez-Montes Eduardo Gil Bruna Calsina María Monteagudo Rocío Letón Juan María Roldán-Romero María Santos Javier Lanillos Carlos Valdivia Natalia Martínez-Puente Javier de Nicolás-Hernández Scherezade Jiménez Manuel Pérez-Martínez Emiliano Honrado Javier Coloma Ana Cerezo Clara María Santiveri Manel Esteller Ramón Campos-Olivas Eduardo Caleiras Cristina Montero-Conde Cristina Rodríguez-Antona Javier Muñoz Mercedes Robledo Alberto Cascón Hereditary Endocrine Cancer Group Spanish National Cancer Research Centre(CNIO)MadridSpain Proteomics unit Spanish National Cancer Research Centre(CNIO)MadridSpain Centro de Investigación Biomédica en Red de Enfermedades Raras(CIBERER) MadridSpain Monoclonal Antibodies Core unit Spanish National Cancer Research Centre(CNIO)MadridSpain confocal microscopy Core unit Spanish National Cancer Research Centre(CNIO)MadridSpain Anatomical Pathology Service Hospital of LeónLeónCastilla y LeónSpain Structural Biology Programme Spanish National Cancer Research Centre(CNIO)MadridSpain CNIO-Lilly Cell Signalling and Immunometabolism Section Spanish National Cancer Research Centre(CNIO)MadridSpain Spectroscopy and Nuclear Magnetic Resonance unit Spanish National Cancer Research Centre(CNIO)MadridSpain Josep Carreras Leukaemia Research Institute(IJC) BadalonaBarcelonaSpain Centro de Investigacion Biomedica en Red Cancer(CIBERONC) MadridSpain Institucio Catalana de Recerca i Estudis Avançats(ICREA) BarcelonaSpain Physiological Sciences Department School of Medicine and Health SciencesUniversity of Barcelona(UB)L’HospitaletBarcelonaSpain Histopathology Core unit Spanish National Cancer Research Centre(CNIO)MadridSpain Cell Signaling and Clinical Proteomics Group Biocruces Bizkaia Health Research InstituteBarakaldoSpain Ikerbasque Basque Foundation for ScienceBilbaoSpain

Dear Editor,Of all human tumors,pheochromocytomas and paragangliomas(PPGLs)have the highest heritability *** 15%of PPGLs harbor mutations in genes encoding tricarboxylic acid(TCA)cycle-related enzymes that cause oncometabolite accumulation and drive tumorigenesis via metabolic adaptation to hypoxia and global hypermethylation[1].The dihydrolipoamide S-succinyltransferase(DLST)gene was recently described as a new PPGL susceptibility gene[2].

关键词： glioma remodeling cytoma

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Expressions of chromogranin A and cathepsin D in human primary hepatocellular carcinoma

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World Journal of Gastroenterology 2000年第5期6卷 693-698页

作者： Xiao Feng Huang Chun Mei Wang Xiao Wen Dai Zhen Jiang Li Bo Rong Pan Li Bin Yu Bin Qian Li Fang Department of Electron microscopy Fourth Military Medical UniversityXi’an 710032Shaanxi ProvinceChina Department of Pathology Chinese PLA 117 HospitalHangzhou 310013Zhejiang ProvinceChina Room 12 Building 621Fourth Military Medical UniversityXi’an 710032Shaanxi ProvinceChina

AIM To determine the expression and clinicalsignificance of chromogranin A and cathepsin Din hepatocellular carcinoma(HCC).METHODS Double immunofluorescence stain-ing techniques combined with laser confocalscanning microscopy(LSCM)was used toinvestigate chromogranin A and cathepsin Dexpressions in 85 HCC *** Cathepsin D was expressed in :3normal liver tissues,while in HCC the stainingshowed regional variation and the fraction ofstrongly stained cells increased as the tumorsbecame less differentiated and usually clinicallymore *** which showed strongpositivity for cathepsin D were present in 71/85(83.5%)*** expression of cathepsinD in cancer cells was related tohistopathological *** were morecommon in grade 3-4(26/28,92.9%)and grade2(46/53,86.8%)tumors than in grade 1 tumors(1/4,25.0%)(P0.05)*** coexpression of CgA andcathepsin D was found by double labeledimmunofluorescence staining *** of cathepsin D was disturbed in HCCcells and accumulated in the *** Dhad proteolytic activity and autocrine mitogeniceffect,suggesting their functions in *** findings demonstrated that the expressionof cathepsin D in HCC had prognostic *** Chromogranin A and cathepsin Dare expressed in a high proportion of HCC andthe existence of cathepsin D in HCC might berelated to processing of *** is clearly asubject for further studies because of itspotential clinical applications.

关键词： carcinoma hepatocellular/pathology cathepsin D/metabolism chromogranins/metabolism microscopy confocal

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confocal-based fluorescence fluctuation spectroscopy with a SPAD array detector

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Light(Science & Applications) 2021年第2期10卷 289-300页

作者： Eli Slenders Marco Castello Mauro Buttafava Federica Villa Alberto Tosi Luca Lanzanó Sami Valtteri Koho Giuseppe Vicidomini Molecular microscopy and Spectroscopy Istituto Italiano di TecnologiaGenoaItaly Dipartimento di Eiettronica Informazione e BioingegneriaPolitecmco di MilanoMilanItaly Nanoscopy and NIC@IIT lstituto Italiano di TecnologiaGenoaItaly Dipartimento di Fisica e Astronomia Universita di CataniaCataniaItaly

The combination of confocal laser-scanning microscopy(CLSM)and fluorescence fluctuation spectroscopy(FFS)is a powerful tool in studying fast,sub-resolution biomolecular processes in living cells.A detector array can further enhance CLSM-based FFS techniques,as it allows the simultaneous acquisition of several samples-essentially images-of the CLSM detection ***,the detector arrays that have previously been proposed for this purpose require tedious data corrections and preclude the combination of FFS with single-photon techniques,such as fluorescence lifetime ***,we solve these limitations by integrating a novel singie-photon-avalanche-diode(SPAD)array detector in a CLSM *** validate this new implementation on a series of FFS analyses:spot-variation fluorescence correlation spectroscopy,pair-correlation function analysis,and image-derived mean squared displacement *** predict that the unique combination of spatial and temporal information provided by our detector will make the proposed architecture the method of choice for CLSM-based FFS.

关键词： spectroscopy correction integrating

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