大视场高分辨HiLo光切片显微成像系统
doi: 10.37188/CO.2022-0087
Wide-field-of-view and high-resolution HiLo optical sectioning microscopy system
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摘要:
现代生物学和生物医学领域迫切需要研制兼顾大视场、高分辨率的显微成像技术和仪器以对生物样品实现跨尺度观测,满足重大科学问题的研究需求。受限于系统的空间带宽积,传统商业显微镜无法满足这一需求,且现有高空间带宽积显微成像系统存在体积庞大、实施成本高昂等问题。本文基于HiLo光切片技术和自主设计的大视场高分辨显微物镜,研发了具有高空间带宽积特点的大视场高分辨HiLo光切片显微成像系统,测试了系统的成像视场和分辨率。应用该系统对小鼠脑切片开展了白光照明明场成像实验,并与OLYMPUS商业显微镜成像结果做了对比;对小麦种子荧光切片开展了光切片成像和宽场荧光成像对比实验。实验结果表明, 大视场高分辨HiLo光切片显微成像系统的成像视场达到4.8 mm×3.6 mm (对角视场为6.0 mm),横向分辨率达到0.74 μm,轴向分辨率达到4.16 μm。大视场高分辨HiLo光切片显微成像系统兼有大视场和高分辨率成像的优势和快速光切片成像的能力,能够对大体积生物样本开展快速三维成像,将为胚胎发育、脑成像、数字病理诊断等研究提供有力的技术支撑。
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关键词:
- 大视场 /
- 高分辨率 /
- 高空间带宽积 /
- 光切片显微技术
Abstract:The fields of modern biology and biomedicine urgently need wide-field-of-view (FOV), high-resolution microscopic technology and instruments for trans-scale observation of biological samples to meet the requirement of major scientific for research. Limited by the spatial bandwidth product, traditional commercial microscopes cannot meet this demand. Besides, the existing high spatial bandwidth product microscopy systems have problems such as bulky volume and high implementation costs. In this paper, based on the HiLo optical sectioning technology and the self-designed wide-field-of-view and high-resolution objective, a wide-field-of-view and high-resolution HiLo optical sectioning microscopy system was developed. The FOV and imaging resolution of this system were tested. Brightfield imaging experiments were carried out on mouse brain slices by this system and the results were compared with that of OLYMPUS commercial microscope. At the same time, wide-field fluorescence imaging comparison experiments were carried out on wheat seed fluorescent slices. The experiment results show that the FOV of this system reaches 4.8 mm×3.6 mm (the diagonal FOV is 6.0 mm), the lateral resolution reaches 0.74 μm, and the axial resolution reaches 4.16 μm. The comparative experiment proved that this system has the advantages of wide FOV, high resolution and the ability of fast optical sectioning imaging simultaneously. This system can carry out rapid 3D imaging of large-volume biological samples, which will provide strong technical support for researches such as embryonic development, brain imaging, and digital pathology diagnosis.
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Key words:
- wide field of view /
- high resolution /
- high space bandwidth product /
- optical sectioning microscopy
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图 1 大视场高分辨显微物镜光学设计评价图
Figure 1. Design evaluation graphs of the wide-field-of-view and high-resolution objective
图 2 大视场高分辨HiLo光切片显微成像系统光学原理图
Figure 2. Schematic diagram of wide-field-of-view and high-resolution HiLo optical sectioning microscopy system
图 3 系统实物照片
Figure 3. Photo of the system
图 4 系统成像视场测试结果图
Figure 4. The test results of FOV of the system
图 5 系统横向分辨率测试结果图
Figure 5. The test results of lateral resolution of the system
图 6 系统轴向分辨率测试结果图
Figure 6. The test results of the axial resolution of the system
图 7 HE染色的小鼠脑切片白光照明明场成像结果对比图
Figure 7. Comparison of brightfield imaging results of a HE-stained mouse brain slice illuminated by white light
图 8 FITC-WGA染色的小麦种子荧光切片三维光切片成像和三维宽场荧光成像结果对比图(50层图像切片,单层图像切片厚2 μm)
Figure 8. Comparison of 3D optical sectioning and wide-field fluorescence imaging results of a FITC-WGA-stained wheat seed fluorescence slice (50 slices, 2 μm thick of every slice)
表 1 大视场高分辨显微物镜设计参数
Table 1. Design parameters of the wide-field-of-view and high-resolution objective
参数 指标 物方视场(Field of View, FOV) Φ6.0 mm 数值孔径(Numerical Aperture, NA) 0.5 工作距(Working Distance, WD) 2 mm 焦距(Focus, F) 40 mm 工作波段(Working Waveband) 436 ~ 800 nm 浸没介质(Immersion Medium) 空气 表 2 系统参数
Table 2. Parameters of the system
参数 指标 照明光源 白光、488 nm/561 nm激光 成像视场 4.8 mm×3.6 mm(对角6.0 mm) 分辨率 横向0.74 μm,轴向4.16 μm 放大倍率 11.1 × 系统空间带宽积 151 M 工作模式 明场成像、宽场荧光成像、HiLo光切片成像 外形尺寸 565 mm(宽)×890 mm(长)×830 mm(高) 深圳坪山网站建设公司邢台网站优化制作公司seo的网站内部优化技巧网站优化文章怎么秒收怎么样优化网站排首页武昌企业网站优化公司沁阳百度网站优化价格网站整站优化思惶.大将军25网站排名优化哪家效果好大连专业的网站seo优化价格长葛品牌网站优化哪家好网站优化中404页面怎么处理网站关键词优化方案松岗教育网站优化网站内页关键词优化梅州首页网站关键词优化排名深圳企业网站优化网站权重优化多少钱做网站优化公司行业门户优化网站免费网站排名优化内江网站seo优化选哪家辽阳网站优化网站优化 图片医疗行业单页网站怎么做优化网站优化简历下载安化网站优化沙头角免费网站优化自适应网站排名优化网站地址优化怎么优化网站网速香港通过《维护国家安全条例》两大学生合买彩票中奖一人不认账让美丽中国“从细节出发”19岁小伙救下5人后溺亡 多方发声卫健委通报少年有偿捐血浆16次猝死汪小菲曝离婚始末何赛飞追着代拍打雅江山火三名扑火人员牺牲系谣言男子被猫抓伤后确诊“猫抓病”周杰伦一审败诉网易中国拥有亿元资产的家庭达13.3万户315晚会后胖东来又人满为患了高校汽车撞人致3死16伤 司机系学生张家界的山上“长”满了韩国人?张立群任西安交通大学校长手机成瘾是影响睡眠质量重要因素网友洛杉矶偶遇贾玲“重生之我在北大当嫡校长”单亲妈妈陷入热恋 14岁儿子报警倪萍分享减重40斤方法杨倩无缘巴黎奥运考生莫言也上北大硕士复试名单了许家印被限制高消费奥巴马现身唐宁街 黑色着装引猜测专访95后高颜值猪保姆男孩8年未见母亲被告知被遗忘七年后宇文玥被薅头发捞上岸郑州一火锅店爆改成麻辣烫店西双版纳热带植物园回应蜉蝣大爆发沉迷短剧的人就像掉进了杀猪盘当地回应沈阳致3死车祸车主疑毒驾开除党籍5年后 原水城县长再被查凯特王妃现身!外出购物视频曝光初中生遭15人围殴自卫刺伤3人判无罪事业单位女子向同事水杯投不明物质男子被流浪猫绊倒 投喂者赔24万外国人感慨凌晨的中国很安全路边卖淀粉肠阿姨主动出示声明书胖东来员工每周单休无小长假王树国卸任西安交大校长 师生送别小米汽车超级工厂正式揭幕黑马情侣提车了妈妈回应孩子在校撞护栏坠楼校方回应护栏损坏小学生课间坠楼房客欠租失踪 房东直发愁专家建议不必谈骨泥色变老人退休金被冒领16年 金额超20万西藏招商引资投资者子女可当地高考特朗普无法缴纳4.54亿美元罚金浙江一高校内汽车冲撞行人 多人受伤
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