非洲菊盘状花雄蕊发育与舌状花生长着色的对应关系

非洲菊(Gerbern hybrida)头状花序由外轮舌状花和内轮盘状花构成。通过观察内轮盘状花雄蕊花粉囊和花粉粒的形态结构与发育顺序,和测定外轮舌状花花瓣的长度、宽度、花色素苷含量等,对它们之间的对应关系进行了研究。花序外轮舌状花花瓣开始着色时为P3期,此时第1轮盘状花出现成熟花粉粒。研究明确了内轮盘状花花粉粒发育与外轮舌状花生长时期和花色素苷积累的对应关系。     

菊花不同花期及花序不同部位香气成分和挥发研究

以切花菊品种‘神马’为试材,采用顶空-固相微萃取和气相色谱-质谱联用(GC/MS)技术,分别测定菊花不同花期及花序不同部位的香气成分种类和含量,并利用生物显微镜观察花瓣的表皮细胞和横切面组织细胞的结构特征。结果表明:(1)菊花花蕾期共检测到香气成分24种,始花期31种,盛花期43种,终花期22种;随着花朵的开放和凋谢,酮类、萜烯类和醇类化合物的含量呈先上升后下降的趋势,在盛花期含量达到最高,而酯类、醛类和杂环类化合物则呈持续下降的趋势。(2)盛花期,在舌状花中共检测到香气成分种类31种,在管状花中共检测到50种;舌状花对菊花香气的贡献比管状花大;菊花舌状花由内轮向外轮香气成分种类变化不大,但是同类香气成分含量的变化出现由内轮向外轮逐渐减少的趋势。(3)异环柠檬醛、桉叶醇、α-蒎烯、β-金合欢烯和石竹烯等化合物可能为菊花的主要特征香气成分。(4)显微观察结果表明:舌状花的香气可能是通过表皮细胞间隙释放的,上表皮是菊花释放香气的主要部位。     

利用激光扫描共聚焦显微镜研究拟南芥气孔发生与发育

通过激光扫描共聚焦显微镜,利用不同种类(波长)的激光研究拟南芥叶片气孔发生与发育。结果表明,利用紫外激光(351nm)扫描可以清楚观察到拟南芥表皮各种细胞及其发生发育的形态变化,包括表皮毛细胞、副卫细胞、保卫细胞、铺垫表皮细胞等。气孔发生过程中,首先原表皮细胞不对称分裂产生拟分生组织和副卫细胞,接着分化出保卫细胞母细胞,进一步发育形成保卫细胞,最终形成气孔器。气孔分化完成后,保卫细胞在紫外激光下不产生荧光,但利用蓝光激发(488nm)辅助荧光素染色,可清晰地看到保卫细胞。结果表明,激光扫描共聚焦显微镜在拟南芥叶表皮细胞形态研究上有独特的功能。     

3D-SIM结构照明超分辨率显微镜实现蛋白质在植物亚细胞器内的定位

基因表达产物蛋白质的亚细胞定位是解析基因生物学功能的重要证据之一。近年来出现的超分辨率光学成像技术已成功应用于人类和动物细胞中,预示着显微成像技术继激光共聚焦技术后的又一重要进步。由于植物细胞的特殊性和成像技术的研发取向,超分辨率光学成像技术在植物细胞蛋白质亚细胞定位的应用尚未见报道。该研究利用Delta Vision OMX显微镜技术,克服了叶绿体基粒中叶绿素自发荧光与融合蛋白荧光不易区分的缺陷,解决了受分辨率局限无法将植物细胞中蛋白质在亚细胞器内可视化精确定位的技术难题,成功地将植物蔗糖合成酶Zm SUS-SH1定位在烟草表皮细胞叶绿体基粒周围。该研究同时建立了一套基于撕片制片法的简便OMX显微镜制片方法,并针对OMX显微成像技术在植物细胞中蛋白质亚细胞定位的应用进行了讨论。     

抗砷细胞荧光染色后的激光共聚焦显微镜检测

目的:应用激光共聚焦显微镜检测活细胞内荧光物质含量.方法:传代培养长期低剂量砷诱导的抗砷细胞,用荧光染料Rhodamine-123对细胞染色30min,实验组与维拉帕米(Verapamil)共同孵育,对照组为单加Rhodamine-123的抗砷细胞.应用激光共聚焦显微镜采集Rhodamine-123的荧光图像动态序列,并且记录不同时间段的细胞内荧光强度.结果:实验组细胞染色12h,24h,36h,48h,60h后,荧光强度依次为(51.567±0.7572)、(46.533±0.7095)、(39.557±0.601)、(38.6±0.6245)和(38.505±0.718),明显高于同时间段对照组的荧光强度,差异均有显著性(P＜0.01).结论:应用激光共聚焦显微成像技术能进行活细胞水平荧光物质实时定量检测.     

ABA诱导蚕豆气孔保卫细胞H2O2的产生

以蚕豆叶片下表皮为材料,将荧光探针HPTS导入蚕豆气孔保卫细胞内,利用荧光光谱和激光共聚焦显微技术,检测了ABA诱导蚕豆气孔关闭过程中H     

构建钙荧光探针细胞用于探究胞浆和线粒体钙对ATP刺激的响应

目的:构建表达基因编辑钙探针(GECIs)的细胞系HeLa-GECIs,探究细胞应答外界ATP刺激中钙离子在细胞内的响应和变化。方法:分别用能够直接通过荧光强度反映细胞胞浆内和线粒体内钙离子相对浓度的2种钙探针cyto-GCaMP6和4mt-GCaMP6感染HeLa细胞,获得2种表达钙离子探针的HeLa细胞系;在感染了2种腺病毒探针24 h后,用共聚焦荧光显微镜检测荧光探针在HeLa细胞内的表达情况;在表达2种钙探针的细胞的培养基中加入外源ATP,用Time-lapse成像动态观测技术观察HeLa细胞内钙离子对外环境中ATP的响应。结果:共聚焦荧光显微镜观察,确定95%以上的细胞表达了对应的钙离子指示荧光探针;Time-lapse成像动态观测技术观察发现,在细胞培养基中加入ATP后,细胞胞浆钙探针荧光强度瞬时(3～6 s)升至10倍,200 s后逐渐降低到基础水平;线粒体钙到达峰值(4倍)的时间稍滞后(5～8 s),并且回落更慢,300 s时至1.5倍。在ATP受体P2X7抑制剂A438079预处理的实验组,上述胞浆钙和线粒体钙浓度上升不明显。结论:构建了能在活体细胞内通过荧光探针实时监测钙离子响应胞外ATP刺激的细胞实验体系,为进一步深入探究ATP等危险信号导致细胞的炎性损伤机制奠定了基础。     

利用激光扫描共聚焦显微镜研究植物细胞发育形态学变化

通过激光扫描共聚焦显微镜,利用不同种类(波长)的激光研究植物细胞发育形态学变化。结果表明,利用紫外激光(351 nm)扫描可以清楚地观察到拟南芥叶片表皮细胞的形态及其变化,在已分化的叶片表皮上可观察到包括“铺垫”表皮细胞(epidermal pavement cells)、气孔保卫细胞(guard cell)、气孔伴胞(subsidiarycells)、表皮毛细胞(trichomes)和表皮毛的足细胞(socket cells)等多种形态不同的细胞种类;利用蓝光激光(488nm)辅助曙红浅染,可清晰地显示出拟南芥根生长区内部的各种原始细胞,包括静止区(quiescent center)细胞、皮层/内皮层原始细胞(cortex/endodermal initial cell)、表皮/根冠原始细胞(epidermal/root cap initial cell)和中柱/根冠原始细胞(columella/root cap initial cell)等。利用双光子激光(800 nm)连续扫描30 s可以诱发叶绿体产生自发荧光,并可观察到叶绿体在叶肉细胞中的运动轨迹。结果说明激光扫描共聚焦显微镜在植物细胞形态及发育研究上具有独特的功能。     

三角褐指藻光合与生长参数测量方法的研究

三角褐指藻是生产生物柴油的优势藻种,监测其光合和生长参数是利用它们生产生物柴油的关键环节。本研究通过细胞计数对细胞生长量进行监测,利用多激发波长调制叶绿素荧光仪(Multicolor PAM)、NanoDrop紫外分光光度计与荧光分光光度计分别检测叶绿素荧光参数、培养基中硝酸盐含量、细胞叶绿素荧光强度与中性脂相对含量。结果显示细胞密度与叶绿素荧光强度只有在生长期才具有线性关系,达到稳定期后,叶绿素荧光强度显著性下降,此时与细胞密度不成比例;PSII最大光化学量子效率F_v/F_m与叶绿素荧光值比生长速率呈正相关,在整个培养阶段F_v/F_m的变化趋势呈现两次上升与下降的过程;光化学淬灭系数qL在培养过程中呈上升趋势,而相对电子传递速率rETR与实际光合效率Y(II)均呈现先升后降的趋势;培养基中硝酸盐含量消耗到一定程度后保持恒定,不再降低;细胞中性脂相对含量在藻细胞接种1天后稍下降,随后持续上升,并在培养基硝酸盐含量不再下降后继续增加。本研究中光合与生长参数测量得到的相关数据,以及培养基硝酸盐含量和细胞中性脂相对含量测量方法的建立,可以为三角褐指藻实验室培养及其代谢产物积累的研究提供必要的研究基础与检测手段。     

超高灵敏度荧光显微成像技术对光敏剂细胞内分布的初步研究

目的：探讨应用基于ICCD的超高灵敏度荧光显微成像系统研究光敏剂细胞内分布的可行性。方法：传代培养内皮细胞、食管癌细胞和肺癌细胞,将不同浓度血卟啉单甲醚(HMME)与细胞共同孵育不同时间。采用荧光显微镜及ICCD组成的荧光显微成像系统采集不同浓度及不同孵育时间条件下HMME的荧光图像,并采用计算机图像处理技术进行图像增强、滤波后计算其细胞浆与细胞核的平均荧光强度比值。同时应用激光共聚焦显微镜图像采集进行对比。结果：HMME浓度为5μg／ml时,荧光显微镜采集到HMME的荧光图像;HMME浓度升高到160μg／ml,激光共聚焦显微镜获得HMME的荧光图像。两组图像的特点都为胞浆中荧光强度较高,细胞核区荧光较弱;细胞浆与细胞核的比值约为2～3：1。结论：荧光显微镜和ICCD采集细胞内光敏剂的荧光图像灵敏度高,方法可靠、实用。HMME较多分布在细胞质中,细胞核吸收较少。     

He-Ne激光对小麦ROP GTPase经增强UV-B辐射造成的损伤的修复研究

采用He-Ne激光辐照对增强UV-B辐射后小麦幼苗ROP GTPase损伤修复作用进行了研究。采用了SDS-PAGE电泳法检测各组ROP GTPase的含量和激光共聚焦显微镜对小麦微丝进行FITC荧光强度标记的测定。研究结果表明：经增强UV-B辐射后,小麦幼苗的Rop GTPase含量降低,LCSM扫描细胞原生质体形状发生改变,细胞骨架受到破坏,其被标记的荧光变暗,强度减弱,在整个UV-B处理期间均低于对照组（CK）,再以He-Ne激光处理后,其含量,形状和强度均有所提高,但仍低于对照组。由此说明,UV-B辐射能使小麦幼苗的Rop GTPase含量下降,微丝骨架受到破坏,一定剂量的激光对UV-B辐射后小麦的蛋白含量和细胞骨架有一定修复作用。Rop GTPase参与了微丝骨架重组的过程。     

Localization and quantification of epidermal growth factor receptors on single cells by confocal laser scanning microscopy.

We have established a method for quantifying binding of fluorescence-labeled growth factors to their receptors on single cells in situ with the confocal laser scanning microscope (CLSM). Biotinylated epidermal growth factor (EGF) coupled to phycoerythrin-labeled anti-biotin was used to compare the levels of fluorescence on three different cell types for which the number of EGF factors was known from Scatchard analysis of [125I]-EGF binding. The results showed that as few as 10,000 receptors/cell were detectable above back-ground. This method will provide a rapid and quantifiable alternative to autoradiography for ligand binding to single cells in situ.     

Visualization of plastids and lipophilic components in living colonies of a wild strain of the hydrocarbon-forming, green alga Botryococcus by laser scanning confocal microscopy

Colonies of a wild strain from Lake Burley-Griffin, Australia, of the hydrocarbon-producing green alga Botryococcus were examined by confocal laser scanning microscopy. The microscope was fitted with a dual wavelength krypton-argon laser, which permitted simultaneous detection of chlorophyll autofluorescence and lipophilic dye fluorescence. This quick and simple technique revealed the precise structural conformation of the autofluorescing plastids in living cells and their 3-dimensional spatial arrangement within the dense globular colonies. Cells stained with the lipophilic carbocyanine dye, DIOC6(3) contain an apical array of intensely staining granules as well as a more diffuse internal cisternal system thought to be endoplasmic reticulum. The cationic lipophilic dye rhodamine123 revealed a finer reticulate system in the outermost cytoplasm partially overlaying the plastid. Both dyes revealed the lipophilic nature of the extracellular matrix and enabled the secretion of lipid globules exuded from the colonies to be visualized. It is suggested that confocal laser scanning microcopy would make an ideal tool to screen isolates for their potential to form and secrete hydrocarbon, processes which are still far from clearly understood in this potentially commercially important alga. This revised version was published online in June 2006 with corrections to the Cover Date.     

Pigment composition and adaptation in free-living and symbiotic strains of Acaryochloris marina

Acaryochloris marina strains have been isolated from several varied locations and habitats worldwide demonstrating a diverse and dynamic ecology. In this study, the whole cell photophysiologies of strain MBIC11017, originally isolated from a colonial ascidian, and the free-living epilithic strain CCMEE5410 are analyzed by absorbance and fluorescence spectroscopy, laser scanning confocal microscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent protein analysis. We demonstrate pigment adaptation in MBIC11017 and CCMEE5410 under different light regimes. We show that the higher the incident growth light intensity for both strains, the greater the decrease in their chlorophyll d content. However, the strain MBIC11017 loses its phycobiliproteins relative to its chlorophyll d content when grown at light intensities of 40 microE m(-2) s(-1) without shaking and 100 microE m(-2) s(-1) with shaking. We also conclude that phycobiliproteins are absent in the free-living strain CCMEE5410.     

A corolla-and carpel-abundant,non-specific lipid transfer protein gene is expressed in the epidermis and parenchyma of Gerbera hybrida var. Regina (Compositae)

We are examining the floral organ differentiation in Compositae by isolating and characterizing corolla abundant genes. Differential screening of a cDNa library made from the ray floret corolla of Gerbera hybrida var. Regina revealed an abundant cDNA clone which is expressed in the corolla but not in leaves. This cDNA (gltp1) codes for a polypeptide similar to non-specific lipid transfer proteins of the plants. The gltp1 gene is expressed only in the corolla and carpels and is developmentally regulated during corolla development. The gltp1 mRNA accumulates both in epidermal cell layers and in the mesophyll of the corolla. In the stylar part of the carpel, the gltp1 mRNA can be detected in the epidermal and in parenchymal cells but not in the transmitting tissue. Analogous patterns of gltp1 expression in the corolla and carpel may indicate that similar genetic programmes operate during the development of these two tissues.     

Cell expansion and microtubule behavior in ray floret petals of Gerbera hybrida: responses to light and gibberellic acid

It is still unclear how light and gibberellins are integrated to regulate petal size. Here, we report that light improves both the length and the width of the ray floret petals in G. hybrid, but GA(3) promotes only the petal length. It is also revealed that the control of the petal size by light and GA(3) depends on modulating the cell size, which is governed by the behavior of cortical microtubule.Light and gibberellins are important regulators of plant organ growth. However, little is known about their roles in petal size determination. Here, we report how light and gibberellic acid (GA(3)) signals are integrated to regulate the ray floret (Rf) size in Gerbera hybrida. The inflorescences of G. hybrida at stages 1.5 were cultivated in vitro for 9 d followed by the determination of the Rf petal size. Results demonstrated that the light signal significantly enhanced both the length and the width of Rf petals, but GA(3) promoted only the petal length. Moreover, GA(3) displayed a synergistic positive effect on the length but an antagonistic effect on the width with the light signal. Measurements of the petal cells revealed that the cell size, not the cell number, exhibited a dominant contribution to the petal size in response to light and GA(3) signals. Furthermore, light and GA(3) signals not only induced an obvious reorientation of cortical microtubules (MTs) into transverse arrays but also promoted the recovery of the MT lengths in petal cells following oryzalin (an MT depolymerizing agent) treatment. Importantly, disruption of the MT lengths and arrays by oryzalin could inhibit the cell expansion and the petal enlargement induced by light or/and GA(3) signals. Taken together, it is concluded that the control of the petal size by light and GA(3) signals mainly depends on modulating the cell size and, moreover, the organization of the cortical MTs plays a crucial role in the control of the cell size and hence the Rf petal growth.     

Construction of a two-photon microscope for video-rate Ca imaging

We describe the construction of a video-rate two-photon laser scanning microscope, compare its performance to a similar confocal microscope, and illustrate its use for imaging local Ca(2+) transients from cortical neurons in brain slices. Key features include the use of a Ti-sapphire femtosecond laser allowing continuous tuning over a wide (700-1000 nm) wavelength range, a resonant scanning mirror to permit frame acquisition at 30 Hz, and efficient wide-field fluorescence detection. Two-photon imaging provides compelling advantages over confocal microscopy in terms of improved imaging depth and reduced phototoxicity and photobleaching, but the high cost of commercial instruments has limited their widespread adoption. By constructing one's own system the expense is greatly reduced without sacrifice of performance, and the microscope can be more readily tailored to specific applications.     

Application of the immunofluorescence technique and confocal laser scanning microscopy for studying the distribution of the luteinizing hormone/chorionic gonadotropin (LH/CG) receptor on rat luteal cells

We used confocal scanning microscopy to study the semi-quantitative distribution of luteinizing hormone/chorionic gonadotropin (LH/CG) receptors on rat luteal cells at both the two- and the three-dimensional level. The receptors were visualized in 6-microns sections of pseudopregnant rat ovaries using polyclonal rabbit antiserum to hCG-affinity-purified LH/CG receptor in conjunction with rhodamine-conjugated anti-rabbit immunoglobulins. Twenty to 30 optical sections were taken at different focal planes from representative luteal cells with a confocal laser scanning microscope and then processed digitally to two- and three-dimensional pseudocolored images. Distinct differences in fluorescence intensity could be demonstrated at both the two- and the three-dimensional level on the luteal cell surfaces, suggesting an uneven distribution of the LH/CG receptors on the cell membranes. This probably results in the compartmentalization and polarization of luteal cell function.