Semiquantitative Confocal Laser Scanning Microscopy Applied to Marine Invertebrate Ecotoxicology

Confocal laser scanning microscopy (CLSM) represents a powerful, but largely unexplored ecotoxicologic tool for rapidly assessing in vivo effects of toxicants on marine invertebrate embryo quality and development. We describe here a new semiquantitative CLSM approach for assessing relative yolk quantity in marine invertebrate embryos (harpacticoid copepods) produced by parents reared from hatching to adult in the polycylic aromatic hydrocarbon chrysene. This method is based on fluorogenic labeling of embryo yolk and subsequent statistical analysis of areal pixel intensities over multiple Z-series using a general linear model (GLM)–nested analysis of variance. The fluorescent yolk-labeling method described here was able to detect statistically significant differences in yolk concentrations in marine copepod (Amphiascus tenuiremis) eggs or embryos from females exposed to ultraviolet light and chrysene-contaminated sediments. Yolk intensities in embryos from females cultured throughout their life cycles in clean sediments were statistically identical with or without UV exposure. In constrast, yolk intensities in embryos of females cultured throughout their life cycle in chrysene-contaminated sediments were significantly higher in the non-UV-exposed treatment with chrysene at 2500 ng/g sediment (65.7% higher) and the UV-exposed treatment with chrysene at 500 ng/g sediment (76.6% higher).     

双光子及共聚焦激光扫描显微术研究天花粉蛋白对人绒癌细胞的作用机制

天花粉蛋白(trichosanthin, TCS)是从中草药栝楼根中提取的一种核糖体失活蛋白,具有抗肿瘤和抗HIV功能.应用双光子及共聚焦激光扫描显微术结合特异性荧光探针Hoechst 33342、2′,7′-二氯荧光黄双乙酸酯 (DCFH-DA)、Indo-1和Fluo 3-AM,首次同时观察了TCS诱导人绒癌细胞（JAR细胞）凋亡过程中活性氧自由基（ROS）和细胞内钙离子浓度（［Ca²⁺］_i）的变化,实验结果表明TCS引起的［Ca²⁺］_i升高和ROS形成参与了TCS诱导的JAR细胞凋亡,并且ROS形成和［Ca²⁺］_i升高有关.共聚焦激光扫描显微术的研究结果表明,［Ca²⁺］_i升高不是导致ROS形成的主要原因,TCS诱导产生的ROS可能是通过TCS与JAR细胞膜表面受体作用介导的.     

中国对虾卵子活化及卵裂的激光扫描共聚焦显微镜研究(英文)

有报导,与其它动物不同,对虾的卵子一接触海水、不经过受精,就可以被活化。但是,只有受精卵才能卵裂发育。而且,各种对虾卵子内皮质棒及皮层颗粒的形态结构、活化过程各有不同。本文就中国对虾卵子的活化及卵裂过程进行了激光扫描共聚焦显微镜（ＬＳＣＭ）研究。该技术可以清晰地显示细胞的立体图像。采集中国对虾,人工诱导产卵,未受精卵取自产卵虾体内,受精卵分期分批取样,固定。供试样品经ＰＢＳ漂洗,ＡＯ、ＦＩＴＣ和ＰＩ染色,ＬＳＣＭ观察、图像处理。刚产出的、未活化卵子形态不规则,皮层内富含皮质棒和皮层颗粒（Ｆｉｇ１）。不论受精与否,产入海水的卵子都能被激活并恢复减数分裂。活化后的卵子释放出皮层囊泡内的皮质棒（Ｆｉｇｓ２＆３）。释放出的皮质棒转变为均质的胶质层（Ｆｉｇｓ４＆５）。随后,皮层颗粒释放并在卵子表面转化为均质化的孵化膜。卵子开始举起,与孵化膜之间形成卵周隙。卵周隙内具有絮状物质（Ｆｉｇｓ６,７＆８）。卵子产出后笫８～１０ｍｉｎ,经过减数分裂,第一极体排放,并随着孵化膜的举起而被推向卵外（Ｆｉｇ５）。笫２０ｍｉｎ,第二极体排放,并由卵子表面到达孵化膜的内缘（Ｆｉｇ６）。在２０℃水温条件下,受精卵产入海水中６０     

共聚焦显微技术研究SA对蚕豆气孔保卫细胞的影响

水杨酸(salicylic acid,SA)作为植物体内一种内源性的信号分子,具有多种生理功能.实验表明水杨酸以浓度依赖的方式诱导气孔关闭,抑制气孔张开.20U/mL的CAT与SA共同处理时可逆转SA诱导气孔关闭作用的83%~90%.以H2O2荧光探针H2DCFDA结合激光扫描共聚焦显微术直接检测到SA处理可引起保卫细胞内H2O2的产生;在保卫细胞内显微注射CAT可完全阻止SA导致的DCF荧光增强.表明SA诱导的气孔关闭可能与H2O2的产生有关.     

利用激光共聚焦扫描显微镜对溃疡病菌侵染柑橘叶片的实时观察

柑橘溃疡病对柑橘产业造成了巨大损失,而研究柑橘与溃疡病菌的互作关系以及柑橘的感病和抗病性均需要观察溃疡病菌在柑橘寄主中的侵染和定殖过程。激光共聚焦扫描显微镜不仅可以观察活细胞,活组织的动态代谢过程,而且可以获得三维图像,对于病原菌在柑橘植物组织内的繁殖和致病机制研究具有重要意义。但是,选择适宜的植物材料和制片方法对激光共聚焦扫描显微镜的观察效果影响很大。本文对激光共聚焦扫描显微镜所观察的材料在其处理和观察方法上加以改进,获得了质量更好的图片和实验结果,也使得实验更为方便快捷。激光共聚焦扫描显微观察还在瞬时表达分析中得到应用,提高了柑橘瞬时表达分析的效果。通过将切片和压片相结合观察到溃疡病菌在不同时间点对柑橘叶片的侵染情况,而通过3D建模能观察到柑橘叶片不同组织层面中的病菌数量和病菌位置,为研究溃疡病菌在叶片中的定殖方式和入侵数量提供了前期基础。     

利用激光扫描共聚焦显微术对同源四倍体水稻胚囊形成与发育的进一步观察

应用改进的整体染色透明激光扫描共聚焦显微术(WCLSM),对同源四倍体水稻PDER-2B-4x胚囊的形成与发育过程进行观察。发现其胚囊的形成发育过程与二倍体的一致,可以清楚地划分为8个发育时期,即孢原细胞形成期、大孢子母细胞形成期、大孢子母细胞减数分裂期、功能大孢子形成期、单核胚囊形成期、胚囊有丝分裂期、八核胚囊发育期和成熟胚囊期。除正常发育的过程外,大孢子发育的各个过程均出现一些异常现象,包括:细胞退化、核位置异常、核数目异常和细胞分化异常等。这些异常可能最终导致多种结构异常成熟胚囊的形成。     

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).^1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.^3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.^4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.^18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),^8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),^9,10 and Bacteroidetes (Bac303).¹¹ In addition, specific probes binding to Streptococcus mutans (MUT590)^12,13 and Porphyromonas gingivalis (POGI)^13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.^5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations^3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.