木本植物的赛莱默水分布,用低温扫描电子显微镜可视化
Summary
观察木本植物中的水分布,可提供有关木本植物水流动力学的重要信息。在这项研究中,我们演示了使用低温和低温-SEM来观察原位赛莱默水分布的实用方法,消除了样品制备过程中水状态的伪性变化。
Abstract
安装低温单元(Cryo-SEM)的扫描电子显微镜允许在零度以下温度下观察标本,并用于利用液氮(LN)结合冷冻固定技术探索植物组织中的水分布2).然而,对于木本物种,由于木纤维的定向,观测木质横切表面的准备工作遇到了一些困难。此外,赛莱默导管中水柱的张力较高偶尔会导致水分布的伪性变化,尤其是在样品固定和收集过程中。在这项研究中,我们演示了一种使用低温和低温SEM来观察木本植物木质植物木质植物在原位中的水分布的有效程序。首先,在样品采集过程中,测量赛莱默水位应确定赛莱默导管中是否存在高张力。当赛莱默水位低时(<ca. ±0.5 MPa),需要一种张力松弛程序,以利于在样品冷冻固定期间更好地保持赛莱默导管中的水状态。接下来,在树茎周围安装一个防水衣领,并填充LN2,用于冻结固定木莱姆的水状态。收获后,应注意确保样品保持冷冻,同时完成样品制备的观察程序。使用低温器来清楚地暴露木赛默横切表面。在 Cryo-SEM 观测中,需要对冷冻蚀刻进行时间调整,以消除霜尘并突出观察表面的细胞壁边缘。我们的研究结果证明了低温SEM技术在细胞和亚细胞水平上观察木莱默体内水分布的适用性。低温SEM与无损原位观测技术的结合,将大大提高对木本植物水流动力学的探索。
Introduction
水资源的可得性(即降水、土壤含水量)严格决定植物物种的死亡率和地理分布,因为它们需要从土壤中吸收水分并将其输送到叶子进行光合生产。工厂必须在水供应波动的情况下维护其水输送系统。特别是,木本植物在蒸腾流的管道中产生高度张力,因为在某些情况下,它们需要保持冠冠距地面100米以上。为了在如此高的负压下保持水柱,赛莱默导管由具有刚性和疏水性-玻璃化细胞壁1的管状细胞连续体组成。每个物种的赛莱默导管对赛莱默导管的脆弱程度是物种在波动的供水条件下生存的一个很好的决定因素。此外,研究赛莱默导管的水状况对于评估受非生物或生物胁迫的个别树木的健康状况非常重要。由于赛莱默导管的集成液压功能,测量树液流量或水位可以提供木本植物水状态的估计值。此外,可视化木马单元中的水分布可以阐明赛莱默液压系统各个部件的状况。
有几个技术来可视化赛莱默导管的水状态3。观察木质组织中的水通通的经典和有用的方法包括将切割树枝的末端浸入染料中或将染料注入立立的树茎4,从而染色水柱。软X射线摄影还允许可视化水分布的切片木盘,由于差异X射线吸收强度的水分在赛莱默5,6。然而,这些方法只提供水运动的轨迹或演示水的宏观分布。最近,无损观测技术,如微聚焦X射线计算机断层扫描(μCT)7,8,9,10和磁共振成像(MRI)11,12,已显著改善,允许观察水在木苗内的赛莱默管道内。这些无损方法具有很大的优点,即无需人工切割效果即可观察赛莱默的水状态,并通过顺序成像或引入对比剂10来跟踪水流动态。然而,我们需要使用定制的MRI进行植物成像或基于同步加速器的专门设施,以获得能够识别细胞水平含水量的图像。此外,虽然基于同步加速器的μCT系统能够获得具有高空间分辨率的精细图像,这与光显微镜7、8、9相媲美,但活细胞可能因此受伤。高能X射线辐射13,14。采用扫描电子显微镜,其中安装低温单元(cryo-SEM)是一种非常有用的方法,用于在细胞水平上精确定位木莱莱中的水,尽管这需要破坏性地采集样品进行观察。为了固定赛莱默导管中的水,部分茎(即树枝、树枝或茎)被液氮(LN2)原位冷冻。Cryo-SEM 对修剪的冷冻标本表面的观察提供了高度放大的赛莱默结构图像,从中我们可以将赛莱默管道中的水识别为冰。这种方法的一个重要限制是不可能对同一样品中的水可移动性进行连续观察。然而,应用_CT或MRI对生活在田地中的树木进行顺序观察是极具挑战性的,因为这些仪器是不可携带的。相比之下,Cryo-SEM有潜力在大树上使用这种技术进行实地实验,以清楚地显示水含量不仅在细胞水平,而且在更精细的结构水平,例如,水在血管间坑15,水细胞间空间16,或水柱17中的气泡。
许多研究观察木质水的低温SEM已报告5,12,18,19,20,21,23。 Utsumi等人(1996年)最初建立了观察木耳原地的规程,通过将LN2填充到固定在茎21上的容器中,将活的树干冷冻固定。样品在样品采集期间和低温SEM制备期间保持温度保持在-20°C以下,以避免在赛莱默导管内熔化冰。这种方法已用于观察赛莱默的水,以澄清在不断变化的水制度11,12,24,25,26下的水分布, 27、28、水分布的季节变化21、29、30,冻融周期的影响17、31 32、湿木中的水分布5、从树木到心木20的过渡过程中水分布的变化、季节的季节过程和容器的分化33,和由某些生物应力引起的气穴23,34。使用低温SEM35,36,也验证了液压导电性和导管容易气穴的影响。Cryo-SEM配备了能量分散X射线光谱仪(EDX或EDS),用于研究含有水37的试样表面的元件分布。
在高液压张力下,含有导管的活躯干的冷冻固定有时会导致人工气穴,而Cryo-SEM在导管38、39的流明中观察到这种气穴是断裂的冰晶。特别是,具有更长和更宽的导管的阔叶物种容易受到张力引起的伪影,例如样品切割引起的气穴,即使在水中进行3、40。在取样一棵透射树(即白天取样)或严重干旱条件下,气穴伪影变得显眼,它们可能会误导对气穴发生3、38的高估。 39.因此,必须释放在管道中工作的张力,以避免伪气穴3、12、39。
使用安装在试样室中的刀的冻裂技术通常用于暴露试样表面,以便进行低温-SEM 观察。然而,木质植物组织的冻裂平面,特别是次生木莱默的横截面,过于粗糙,无法清楚地观察组织6中的解剖特征和水。应用低温处理修剪试样,可快速、高质量地制备样品表面20、23。该方法的总体目标是为各种赛莱默细胞的原位水分布提供电子显微镜分辨率的证据,而没有出现采样伪影。我们介绍了我们更新的程序,自我们首次采用它以来,在样品表面的取样、修剪和清洁方面,为了获得木莱的低温固定样品的高质量电子显微图,程序得到了稳步改进。
Protocol
Representative Results
Discussion
本文引入的低温-SEM观测方法对于在细胞尺度上清晰地可视化水分布具有实用意义。通过这种方法,探索木莱默内水分布的变化,可能有助于阐明树种对非生物应激(缺水或冷冻)或生物应激(树病)的耐受性机制。
该方法最关键的步骤是在样品采集和随后的样品制备过程中保持原生水状态的水分布特性。具有长导管的物种的赛莱默组织(尤其是环状孔状树木的早期木容器)在收获和冷?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了JSPS KAKENHI(No. 20120009, 20120010, 19780129, 25292110, 23780190, 23248022, 15H02450, 16H04936, 16H04948, 17H03825, 18H02258)
Materials
| coating material | JOEL Ltd., Japan | Gold wire, 0.50 × 1000 mm, 99.99 %, Parts No. 125000499 | |
| cryo scanning electron microscope | JOEL Ltd., Japan | JSM-6510 installed with MP-Z09085T / MP-51020ALS | |
| cryostat | Thermo Scientific | CryoStar NX70 | |
| microtome blade | Thermo Scientific | HP35 ULTRA Disposable Microtome Blades, 3153735 | |
| tissue freezing embedding medium | Thermo Scientific | Shandon Cryomatrix embedding resin, 6769006 |
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