激光共焦显微成像的最新进展及其在生命科学研究中的应用

激光扫描共聚焦显微镜近年来得到了迅速发展,是近代最先进的细胞生物医学分析仪器之一。通过它可以对观察样品进行无创断层扫描和成像,在生物学和医学研究诊断的各个方面都得到了广泛的应用。本文主要介绍了激光扫描共焦显微镜的基本原理和发展状况,并着重介绍了在共焦荧光显微镜中采用薄荧光层和切片成像特性图来表征成像状态的功能。这种方法一般用于表征共聚焦和多光子显微镜的成像特性,是比较显微镜切片成像条件、成像质量等相关性能的重要依据。     

激光共聚焦显微镜与光学显微镜之比较

激光扫描共聚焦显微镜在活细胞的动态检测、光学切片和三维结构重建等方面较光学显微镜有质的飞跃。本文对激光扫描共聚焦显微镜和光学显微镜进行了比较和讨论,并简单介绍多光子激光扫描显微镜。     

激光共聚焦显微技术在植物学中的应用

激光扫描共聚焦显微镜（Ｌａ－ｓｅｒ　Ｓｃａｎｎｉｎｇ　Ｃｏｎｆｏｃａｌ　Ｍｉｃｒｏｓｃｏｐｅ,ＬＳＣＭ）是近年来发展起来的一种新型高精度显微镜,它在荧光显微镜成像基础上加装了激光扫描装置,使用可激发的荧光探针对样品进行标记,利用计算机进行图像采集处理,从而可得到样品内部细微结构的荧光图像。目前此种显微技术不仅用于观察经固定的各种细胞和组织结构,而且还可对活细胞的形态、结构,离子实时动态等进行观察和定量荧光测定,以及定量图像分析。另外,该仪器还具备样品断层扫描,三维图像重建的独特功能。因此,共聚焦…     

激光共聚焦扫描技术与荧光组织化学染色

激光扫描共聚焦显微镜Confocal Laser Scanning Microscope)是20世纪80年代中期发展起来并得到广泛应用的新技术[1].在传统光学显微镜基础上,激光扫描共聚焦显微镜用激光作为光源,采用共轭聚焦原理和装置,并利用计算机对所观察的对象进行数字图像处理观察、分析和输出.其特点是可以对样品进行断层扫描和成像,进行无损伤观察和分析细胞的三维空间结构.同时,利用免疫荧光标记和离子荧光标记探针,该技术也可对活细胞内的生命活动进行动态观察和分析,是细胞生物学研究的重要手段和工具,极大地丰富了人们对细胞生命现象的认识.     

激光扫描共聚焦显微镜系统及其在细胞生物学中的应用

激光扫描共聚焦显微镜是近十年发展起来的医学图象分析仪器,现已广泛应用于荧光定量测量、共焦图象分析、三维图象重建、活细胞动力学参数监测和胞间通讯研究等方面。其性能为普通光学显微镜质的飞跃,是电子显微镜的一个补充。本文以美国Ｍｅｒｉｄｉａｎ公司的ＡＣＡＳＵＬＴＩＭＡ３１２为例简要介绍了激光扫描共聚焦显微镜系统的结构、功能和生物学应用前景。     

激光扫描共聚焦显微镜荧光探针的选择和应用

激光扫描共聚焦显微镜是检测生物荧光信号的最新技术手段。不仅广泛用于荧光定性、定量测量,还可用于活细胞动态荧光监测、组织细胞断层扫描、三维图象重建、共聚焦图象分析、荧光光漂白恢复、激光显微切割手术等。本文拟就激光扫描共聚焦显微镜常用的检测内容及其相关荧光探针的选择和应用做一简单的介绍。     

活细胞钙动态的共聚焦扫描显微镜检测技术

共聚焦激光扫描显微镜（Confocal Laser Scarming Microscope,CLSM）广泛应用于活细胞内钙敏感探针标记的钙水平的动态测量。较之传统的显微镜CLSM在钙成像分析上有着不可比拟的优越性,但也存在一些缺陷,近些年陆续出现了一些针对这些缺陷的改善措施,如比率法、葡聚糖探针及其他一些新技术与共聚焦显微镜的联合应用等,并且出现了诸如双光子显微镜等新型激光共聚焦显微镜。随着共聚焦钙成像技术的不断发展进步,其今后的应用前景将会越越广阔。     

共聚焦激光扫描显微镜技术

自1985年共聚焦激光扫描显微镜问世以来,该项技术一直在飞速发展和完善。近年来一系列新型的共聚焦显微镜相继研制成功和投入使用,如视频共聚焦激光扫描显微镜、双光子显微镜、4Pi显微镜、荧光寿命成像显微镜等,它们较传统共聚焦显微镜有着各自独特的优势,了解它们的基本性能和特点有助于其在生物学领域更为广泛深入的应用。     

共聚焦显微技术简介

共聚焦显微镜在生物学研究中得到广泛应用,共聚焦显微技术按照显微镜构造原理的不同分成激光扫描共聚焦和数字共聚焦显微技术两种,共聚焦技术具有成像清晰,获得三维图像,进行多标记观察,活细胞内动态生理反应的实时观察记录,定性定量分析等优势,与共聚焦显微技术相关的技术有荧光染料的选择,荧光指示剂装载以及图像数据处理等。     

激光扫描共聚焦显微镜在激光照射诱导细胞凋亡研究中的应用

激光扫描共聚焦显微镜结合荧光探针以及荧光共振能量转移技术,已成为近年来应用在活细胞中研究大分子行为的一种非常有效的研究工具。本文介绍这一技术在激光照射诱导细胞凋亡的研究中的应用。     

Multiple microscopic approaches demonstrate linkage between chromoplast architecture and carotenoid composition in diverse Capsicum annuum fruit

Increased accumulation of specific carotenoids in plastids through plant breeding or genetic engineering requires an understanding of the limitations that storage sites for these compounds may impose on that accumulation. Here, using Capsicum annuum L. fruit, we demonstrate directly the unique sub‐organellar accumulation sites of specific carotenoids using live cell hyperspectral confocal Raman microscopy. Further, we show that chromoplasts from specific cultivars vary in shape and size, and these structural variations are associated with carotenoid compositional differences. Live‐cell imaging utilizing laser scanning confocal (LSCM) and confocal Raman microscopy, as well as fixed tissue imaging by scanning and transmission electron microscopy (SEM and TEM), all demonstrated morphological differences with high concordance for the measurements across the multiple imaging modalities. These results reveal additional opportunities for genetic controls on fruit color and carotenoid‐based phenotypes.     

Principles and practices of laser scanning confocal microscopy

The laser scanning confocal microscope (LSCM) is an essential tool for many biomedical imaging applications at the level of the light microscope. The basic principles of confocal microscopy and the evolution of the LSCM into today's sophisticated instruments are outlined. The major imaging modes of the LSCM are introduced including single optical sections, multiple wavelength images, three-dimensional reconstructions, and living cell and tissue sequences. Practical aspects of specimen preparation, image collection, and image presentation are included along with a primer on troubleshooting the LSCM for the novice.     

用激光扫描共聚焦显微镜观察雪松花粉和花粉管

为更直观地观察和显示花粉和花粉管中细胞结构及其细胞核的状态与行为。雪松花粉和花粉管经卡诺液固定,分别以埃氏苏木精、曙红、Hoechst 33243单染和曙红-Hoechst 33342双染后,用冬青油整体透明,在激光扫描共聚焦显微镜下观察。4种染色法观察效果不同;以曙红-Hoechst 33342双染的样品观察效果最佳,在紫外光激发下清晰地显示出细胞核,在488 nm激光激发下不仅能清晰看到花粉和花粉管壁结构,且能分辨管细胞、柄细胞及体细胞的结构特点和空间位置关系。建立了一种快速简便的适于在激光扫描共聚焦显微镜下观察花粉和花粉管中成员细胞结构及其细胞核的状态、行为的制片技术;激光扫描共聚焦显微镜具有独特的共轭成像装置、连续光学扫描、图像三维重组和多通道检测等功能,极好地展示了雪松花粉和花粉管的结构特点,相比于传统的光学显微镜和荧光显微镜,其观察到的图像更清晰、更直观、更具立体感。     

利用激光扫描共聚焦显微镜的肿瘤血管在体成像研究

传统的观察血管的方法需将组织制成切片,然后通过光学显微镜进行观察。显示的只是血管的某一片段而无法观察到血管的全貌。应用激光扫描共聚焦显微镜,可对活体动物血管进行断层成像,从而再现血管的结构。本方法为对肿瘤等病变组织血管进行研究提供了一种新的检测手段。     

Confocal microscopy: principles and applications to the field of reproductive biology

Confocal microscopy allows analysis of fluorescent labeled thick specimens without physical sectioning. Optical sections are generated by eliminating out-of-focus fluorescence and displayed as digitalized images. It allows 3-dimensional reconstruction (XYZ) and time-analysis (XYT), thus providing unique chance to link morphology with cell function. Since images are obtained by scanning, excess illumination of the specimen and quick decrease of the fluorescent signal are avoided. Resolution obtained with a Laser Scanning Confocal Microscopy (LSCM) is theoretically better than that of a conventional microscope. The preparation of the specimen may be based on standard techniques, such as immunocytochemistry applied to fixed cells, or on staining of living cells, following the use of different fluorescent probes at the same time (colocalization). In our laboratory, we use the LSCM system Fluoview version 2.1 (Olympus) to study reproductive biology of animals and humans. We work on stainings of oocytes and blastocysts (mouse, bovine, human), and human ovarian tissues. We study mitochondrial distribution, cortical granule migration, calcium oscillations and spindle quality to link culture conditions and oocyte quality. Staining of F-actin is used to check transzonal projections (in zona pellucida) or to detect abnormalities following experimental treatment. Blastocyst quality is analyzed in sequential optical sections for microfilament organization and counting of total cell number (staining with phalloidin (actin) and picogreen (DNA). Trophectoderm and inner cell mass distribution (differential staining), apoptotic cells (TUNEL method) and viable cells (live/dead test) are also evaluated. Confocal imaging can be helpful for rapid determination of follicle density (staining with AM Calcein) and follicle morphology (picogreen) in ovarian cortical biopsies. The current review describes the principles of confocal microscopy and illustrates its applications to the field of reproductive biology by a large collection of pictures.     

激光扫描共聚焦显微镜在花粉研究中的应用

激光扫描共聚焦显微镜（Laser scanning confocal microscope,LSCM）是普通光学显微镜、激光、计算机及其相应的软件技术结合的产物,能实现连续光学切片和生物三维结构重组及动态分析.作为一种先进的技术手段,LSCM技术已经为花粉生物学的研究提供了有价值的新资料,大大地推动了植物生殖生物学的发展.本文综述了LSCM技术在花粉粒形态（形状、大小及三维重建）、花粉的内部结构（如细胞骨架）、花粉的遗传学、花粉的发育、花粉的萌发、花粉自发荧光特性、孢粉研究等方面中的应用,并指出了LSCM的不足之处,最后提出了LSCM技术在花粉研究中的应用展望.未来LSCM技术应该与多光子技术、活细胞工作站技术相结合使用,才能更准确地进行花粉动态研究的实时监测.     

Immunolabeled microtubules and microfilaments are visible in multiple cell layers of rye root tip sections

Summary A protocol was developed to observe plant microtubules and actin microfilaments in large tissue samples without physical sectioning. Rye (Secale cereale L. cv. Rymin) root tip pieces from two-day-old seedlings were fixed and processed for immunolabeling. Incubation times of 24–48 h were required to insure adequate penetration of fixatives, antibodies, and washing buffers. Clearing of the tissue with methyl salicylate reduced background auto-fluorescence that would otherwise interfere with the resolution of cytoskeletal structures. Microtubules or microfilaments in 5–7 cell layers were visualized using the optical-sectioning capability of laser scanning confocal microscopy (LSCM) and projected as three-dimensional images. The three-dimensional character of the cytoskeletal elements is retained when viewing stained cells of intact tissue. The net-like character of a microfilament array radiating out from a single point into the cytoplasm is maintained when the cells are stained in intact root tip pieces and imaging is accomplished in situ.Abbreviations Cy3 cyanine 3.18-conjugated goat anti-mouse IgG - FITC-M fluorescein isothiocyanate conjugated anti-mouse IgG - IFB immunofluorescence buffer - LSCM laser scanning confoeal microscopy - TPBS phosphate-buffered saline with 0.1% Triton X-100     

四种显微技术观测大鼠颌下腺细胞与丝素-壳聚糖体外共培养的形态学特点

目的采用倒置显微镜、扫描电镜（scanning electron microscopy,SEM）、荧光显微镜和激光共聚焦显微镜（（laser scanning confocal microscopy,LSCM））技术对大鼠颌下腺细胞（rat submandibular gland cells,RSMGs）与丝素-壳聚糖（silk fibroin-chitosan,SFCs）的体外复合培养进行形态学观察。为观测、评估种子细胞在三维支架的内部生长情况提供技术支持。方法取0～8 d龄SD大鼠的颌下腺,对大鼠颌下腺细胞进行原代培养、分离纯化并传代;用抗细胞角蛋白单克隆抗体（CK8）及淀粉酶抗体的免疫细胞化学染色鉴定细胞来源。选取传至第二代的对数生长期的RSMGs作为种子细胞,选取SFCs共混膜（5×5×2）mm作为支架材料构建组织工程化涎腺样结构。将种子细胞与支架材料复合培养并分别于倒置显微镜、SEM、荧光显微镜和LSCM下观察二者复合生长情况。结果倒置显微镜可以直接观察活细胞与支架复合生长情况,方法简单易行。SEM可以较精确的展示细胞支架复合生长的表面超微结构。经过荧光染料的着色,荧光显微镜和LSCM都可以观察到支架上锚定的种子细胞。荧光显微镜可见细胞核的荧光信号均匀的分布在支架孔隙内。LSCM通过层扫描及三维重建技术对较厚的标本获取图像;并可以通过旋转图像,从不同角度观察细胞支架复合物的三维剖面或整体结构,得到更为准确的定位信息。结论四种显微技术均可应用于RSMGs与SFCs体外共培养的形态学观测。LSCM的三维重建技术结合荧光染料标记可以较好地获得RSMGs与SFCs复合生长的情况,有着较广泛的应用价值。     

激光扫描共聚焦显微镜在植物学中的应用

激光扫描共聚焦显微镜(LSCM)是普通光学显微镜与激光和计算机及其相应的软件技术组合的产物,实现了连续光学切片,能在亚细胞水平观察细胞骨架的动态变化、细胞内特异蛋白、钙等离子的变化,并结合电生理等技术观察细胞生理活动与细胞形态及运动变化的相互关系。并广泛应用于生物三维结构重组及动态分析。本文综述了应用激光扫描共聚焦显微镜技术在植物细胞学、植物发育、组织化学以及基因表达、检测等领域取得的进展。     

CRISPR/dCas9系统在活细胞成像领域的研究进展

研究不同基因、染色体以及基因与染色体之间的时空关系在遗传学、发育生物学和生物医学等领域具有重要意义。CRISPR/Cas9基因编辑技术具有优异的靶向性,已经成为应用最广泛的基因编辑工具。近年来,研究人员基于Cas9的核酸酶失活突变体dCas9发展了一系列先进的活细胞成像技术,为染色质、基因组特定位点的高分辨成像提供了快速、方便的研究工具。文中从细胞递送方式、荧光信号优化以及正交多色成像3个方面对CRISPR/dCas9系统在活细胞成像中的研究进展进行了综述,并对该领域的发展趋势进行了展望。