Documentation of floral secretory glands in Pleurothallidinae (Orchidaceae) using Scanning Electron Microscopy (SEM)

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A clear, step by step description of the treatment of orchid flowers, subtribe Pleurothallidinae, with Critical Point Drying for SEM is presented. It shows that a simple, short fixation and dehydration method prior to Critical Point Drying is sufficient to obtain good results.


Pleurothallidinae (Orchidaceae) have relatively small flowers that exhibit a wide variety of ornaments on the perianth parts, especially on the sepals and lip. It is clear that most of those play an important role in attracting and arresting pollinators, which in most cases are known to be small flies. However, it is difficult to judge just from looking at photographs from the stereoscopic microscope if the observable thickenings, papillae or hairs are actually secretory glands. Scanning Electron Microscopy (SEM) can be a powerful tool to explore these flowers in greater detail, aiding to locate, compare and identify those glands (Karremans et al., 2015).

Materials and Reagents

  1. Snap cap vials (10 ml) (VWR, catalog number: 548-0621 )
  2. Gloves (nitrile, powder free)
  3. Petri dish
  4. Double sided ultra-smooth carbon adhesive tape, 12 mm diameter (Electron Microscopy Sciences, catalog number: 77827-12 )
  5. Plastic box or dessicator with dry silicagel
  6. Ethanol
    Note: For steps using diluted ethanol use a 96% non denatured ethanol with distilled water, for steps using pure ethanol use a min. of 99.9%, water free ethanol.
  7. Acetone, HPLC Plus, for HPLC, ≥ 99.9% (Sigma-Aldrich, catalog number: 650501-1L )
  8. Carbon dioxide, technical grade (in gas cylinder with feed pipe)
  9. Argon gas 4.6, technical quality


  1. Aluminium specimen mount, JEOL(Ted Pella, catalog number: 16231 )
  2. Soft pincet (e.g., light forceps, [VWR, catalog number: 232-2119 ])
  3. SEM specimen mount tweezers (Electron Microscopy Sciences, catalog mumber: 76800 )
  4. Platina/palladium (Pt/Pd) target, 57 x 0.1 mm (Pt/Pd ratio, 80:20)
  5. Automated critical point dryer (Leica Microsystems, model: EM CPD300 )
  6. Rotary mixer (Ted Pella, Pelco®, model: R1 )
  7. Holders and baskets for Leica critical point dryer (e.g., Fine mesh specimen holder with mesh specimen baskets or filter disc holder with 4 wells)
  8. Sputter coater (Quorum Technologies, model: Q150T S ) with rotating holder for 6 SEM stubs (Q150T S/E/ES Sample Preparation System Instruction Manual), film thickness monitor and RV3 Rotary Vane pump (Edwards, model: A65201903 )
  9. Field emission scanning electron microscope (JEOL, model: JSM-7600F ) (Figure 1)
    Note: This product is discontinued but any standard scanning electron microscope will do (for example JEOL, model: JSM-IT100 InTouchScopeTM)

    Figure 1. Field emission scanning electron microscope in place and ready for use


  1. Jeol PC-SEM version


  1. Preparing the samples for critical point drying
    1. Carefully harvest fresh flowers using a conventional pincet. Take care of collecting only young, undamaged flowers. It is important to collect material from plants that are in good, healthy condition (for example the same day of anthesis; Figure 2). Place the flower in a plastic tube containing ethanol (70%), covering it completely. Leave it to soak for at least one week (this is highly dependent on the size of the material, and it is likely that a shorter period of a few days will also be enough for Pleurothallidinae flowers). The condition of the cell structures observed in SEM is mostly dependent on the quality of the collected material.

      Figure 2. Flowers of Specklinia remotiflora. A. Shortly after anthesis, flower in healthy condition. B. Damaged flower, a few days after anthesis, not recommended for visualization with SEM.

    2. Place flowers directly from ethanol 70% into snap cap vials (10 ml) with acetone pure grade. Covering the flower completely. Try to leave as little time as possible between cutting the flowers from the plant and placing them in ethanol 70%.
    3. Turn slowly in a rotary mixer for 30 min at room temperature.
    4. Replace the used for fresh acetone, once again covering the flowers immediately. Flowers should be exposed to air as short as possible because this damages the cells.
    5. Turn slowly for 30 min once again.
    6. Submerge the holders/baskets for critical point drying in a Petri dish with acetone. Put flowers in the holders. Remember not to let them dry up! Add a small (5 mm) piece of paper with (pencil written) sample number to each sample well.
    7. Proceed to critical point drying following the Leica EM CPD300 protocol (Leica EM CPD300 application booklet) (Figure 3).

      Figure 3. Settings used for critical point drying using the Leica EM CPD300

  2. Placing and displaying the samples for coating
    1. Take samples out of the dryer. They should be papery, light, brittle and practically colorless. Be careful handling them, they can easily get damaged.
    2. Place the aluminum specimen mounts in acetone for 60 sec to clean them. Take them out and place on a tissue, allowing the acetone to evaporate completely by exposing to air for a few minutes. Handle stubs with gloves from now on. Paste carbon adhesive double sided tape on one (top) end. Label stubs on the bottom according to samples
      Note: You might need more than one stub per sample depending on size of the flowers.
    3. Handle flowers carefully using a soft pincet. Consider that every part touched with the pincet will be damaged, therefore hold them from a point which is not interesting to visualize later. In most pleurothallids the rachis, pedicel or ovary will do as they may not be of interest for the visualization
      Note: You can have multiple flowers dried so that you can have the different organs represented once.
    4. Place flower on tape. You will need to open up the flowers as much as possible to allow visualisation. Flat flowers like Platystele and Stelis can just be placed entirely (Figure 4), flat on the tape, more tubular flowers like Acianthera and Specklinia have to be dismembered (Figure 5). In those cases, after the synsepal is fixed you can remove the dorsal sepal and petals. It is important to touch the segments as little as possible, to place them on the tape in final position (you will not be able to change position after it was placed), and to consider all possible viewing points you would like.

      Figure 4. Small and flat flowers like that of Stelis can be displayed complete. Scale bar = 500 μm.

      Figure 5. Stubs showing diverse Pleurothallidinae flowers, dismembered and placed according to the visualization needs. A. Lankesteriana sp. B, E. Specklinia spp. C, D. Echinosepala spp.

    5. Store stubs in a Petri dish, and in a closed plastic box or desiccator with some dry silica gel until they will be sputter-coated. Samples can be stored for years at room temperature in a relatively dry place. In places where the humidity is high samples can be stored together with silica gel. If necessary sputter-coating can be repeated before the re-visualization.

  3. Sputter-coating the samples
    1. Open Argon cylinder allowing gas to reach the Q150T S sputter coater (Q150T S/E/ES Sample Preparation System Instruction Manual). Turn the machine on.
    2. Carefully remove the glass chamber and place the stubs in the holder distributing them evenly so that it is balanced when rotating. Place the glass chamber back on. Lower the cover taking care that it does not touch the samples. Select a ‘Profile’ on the operating screen. It is recommended that you use a coat thickness of PT/Pd 20 nm because Pleurothallidinae flowers are quite irregular. (A PT/Pd 10 nm would probably be enough otherwise.) Run the profile (Figure 6). Anthers and pollinaria often become charged with electrons during observation in the SEM, which disrupts the image acquisition. A thicker coating may help prevent this problem.

      Figure 6. Micrographs of the glandular areas of the sepals of Specklinia species taken without coating using a stereoscopic microscope (1) and with coating using the scanning electron microscope (2). A. S. barbae; B. S. pfavii; C. S. tribuloides. Scale bars = 1 mm (A1, B1 and C1); Scale bars = 100 μm (A2, B2 and C2).

    3. After the run has completely finished the vacuum should allow for the opening of the cover. The samples can be removed using a pincet and returned to the Petri dish.

  4. Visualization and photographs
    1. Turn the JEOL JSM-7600F field emission scanning electron microscope on, following the microscope manual.
    2. Use the multiple 12 mm stub holder and place up to six stubs tightly. Always use powder free gloves when handling the holder and the stubs.
    3. Follow the microscope manual to place the samples inside using the specimen exchange rod.
    4. Follow the microscope manual to visualize the material. Set the parameters for visualization: Set the work distance (Z) to 15 mm, and set the acceleration voltage to 10 KV. Probe current is 6 and detector mode is SB.
      Note: The work distance may be varied, a shorter distance gives higher resolution but lower focus depth, a higher work distance will give lower resolution but higher focus depth. Accelerating voltage can be varied, at higher kV the resolution becomes better but there is more chance of charging. It is advised not to use more than 15 kV for biological samples. Lower kV can be useful if you need to avoid charging (e.g., 5 kV) or if you want to work without coating your material (e.g., 2 kV). However, a lower kV gives less resolution and noisier images. For our work with this specific SEM we always use Probe current 6 and a detector mode SB for our biological samples. This setting combines collection of side scattered and backscattered electrons, which gives good image results. 
    5. When working with magnifications higher than 500x you can switch from ‘Low Mag’ to ‘High Mag’ mode.
    6. Save pictures as bitmap (Figure 7).

      Figure 7. Scanning electron micrographs of diverse glands found in the flowers of Pleurothallidinae. A. Possible osmophore on the abaxial surface of the sepals of Echinosepala. B. Sunken trichome on abaxial surface of Condylago (= Stelis). C. Nectaries on the lip of Platystele. D. Nectar secreting stomata on adaxial surface of the sepals of Specklinia. The meassurements represent 100, 100, 100 and 50 μm.


  1. If no fresh flowers are available it is possible to use specimens kept in the liquid collections at herbaria (Procedure A, step 1). Flowers preserved in FAA and its variations will still be suitable for visualization through the method described here.
  2. When Critical Point Drying (Procedure A, steps 2-6), instead of acetone, you can also use 100% ethanol (absolute). In our experience this also gives good results.


The Alberta Mennega Foundation funded the first author at several stages of the study that resulted in the protocol here described. This protocol has been modified from the original application booklet by Leica Microsystems.


  1. Karremans, A. P., Pupulin, F., Grimaldi, D., Beentjes, K. K., Butot, R., Fazzi, G. E., Kaspers, K., Kruizinga, J., Roessingh, P., Smets, E. F. and Gravendeel, B. (2015). Pollination of Specklinia by nectar-feeding Drosophila: the first reported case of a deceptive syndrome employing aggregation pheromones in Orchidaceae. Ann Bot 116(3): 437-455.


提出了一个清晰,一步一步描述的兰花花,分类Pleurothallidinae,关键点干燥SEM。 它表明在临界点干燥之前的简单,短暂的固定和脱水方法足以获得良好的结果。

[背景] Pleurothallidinae(兰科)具有相对小的花 在花被部分,特别是萼片和唇部展示各种各样的装饰品。 显然,其中大多数在吸引和逮捕传粉媒介方面发挥重要作用,在大多数情况下,这些传粉媒介被认为是小苍蝇。 然而,如果可观察到的增厚,乳突或毛发实际上是分泌腺,则很难从观察立体显微镜的照片来判断。 扫描电子显微镜(SEM)可以是更详细地探索这些花的有力工具,帮助定位,比较和鉴定这些腺(Karremans等人,2015)。


  1. 将盖帽小瓶(10ml)(VWR,目录号:548-0621)
  2. 手套(腈,无粉)
  3. 培养皿
  4. 双面超光滑碳胶带,直径12 mm(Electron Microscopy Sciences,目录号:77827-12)
  5. 带干燥硅胶的塑料盒或干燥器
  6. 乙醇
    注意:对于使用稀释的乙醇的步骤,使用96%非变性乙醇和蒸馏水,对于使用纯乙醇的步骤使用最小。 的99.9%,无水乙醇。
  7. Acetone,HPLC Plus,用于HPLC,≥99.9%(Sigma-Aldrich,目录号:650501-1L)
  8. 二氧化碳,工业级(在带有进料管的气瓶)
  9. 氩气4.6,技术质量


  1. 铝试样台,JEOL(Ted Pella,目录号:16231)
  2. 软针(,例如,轻型镊子,[VWR,目录号:232-2119])
  3. SEM试样安装镊子(Electron Microscopy Sciences,目录号:76800)
  4. 铂/钯(Pt/Pd)靶,57×0.1mm(Pt/Pd比,80:20)
  5. 自动临界点干燥器(Leica Microsystems,型号:EM CPD300)
  6. 旋转混合器(Ted Pella,Pelco ,型号:R1)
  7. 徕卡关键点干燥器(例如,带有网孔样品篮的细网样品架或带有4个孔的滤膜支架)的支架和篮筐
  8. (Q150T S/E/ES样品制备系统使用手册),膜厚度监测器和RV3旋转叶片泵(Edwards,型号:A65201903)的旋转夹具的溅射涂布机(Quorum Technologies,型号:Q150T S) >
  9. 场发射扫描电子显微镜(JEOL,型号:JSM-7600F)(图1)
    注意:本产品已停产,但任何标准扫描电子显微镜均可(例如JEOL,型号:JSM-IT100 InTouchScopeTM)



  1. Jeol PC-SEM版本2.1.0.3


  1. 准备样品进行临界点干燥
    1. 使用传统的针头小心地收获鲜花。照顾收集只有年轻,未受损的花。重要的是从处于良好健康状态的植物(例如开花的同一天)收集材料;图2。将花放在含有乙醇(70%)的塑料管中,完全覆盖。让它浸泡至少一个星期(这高度依赖于材料的大小,并且可能更短的几天也将足够Pleurothallidinae花)。在SEM中观察到的细胞结构的状况主要取决于所收集材料的质量

      图2. 的花朵。 B.损坏的花,开花后几天,不建议用SEM进行可视化
    2. 将花直接从乙醇70%放入具有丙酮纯级的卡通小瓶(10ml)中。完全覆盖花。尝试离开尽可能少的时间,从植物切花之间,并将其放置在乙醇70%
    3. 在室温下,在旋转混合器中缓慢转动30分钟
    4. 更换用于新鲜丙酮,再次覆盖鲜花立即。花应该暴露在尽可能短的空气中,因为这会损坏细胞
    5. 再次慢慢转动30分钟。
    6. 将支架/篮子浸入带有丙酮的培养皿中进行临界点干燥。把花放在持有人。记住不要让他们干涸!向每个样品孔中加入一个小(5毫米)的纸(样品编号为铅笔)。
    7. 按照Leica EM CPD300协议进行临界点干燥( Leica EM CPD300应用手册)(图3)。

      图3.使用Leica EM CPD300进行临界点干燥的设置

  2. 放置和显示涂层样品
    1. 将样品从干燥器中取出。它们应该是纸质的,轻的,脆的和几乎无色的。小心处理它们,它们很容易损坏。
    2. 将铝样品安装在丙酮中60秒,以清洁它们。取出它们并放置在组织上,使丙酮通过暴露于空气几分钟完全蒸发。从现在起用手套处理残余物。在一(顶部)端粘贴碳胶双面胶带。根据样品
      在底部标记存根 注意:根据花的大小,您可能需要为每个示例设置多个存根。
    3. 使用软针织小心地处理花。考虑到与针脚接触的每个部分都将被损坏,因此保持它们从一个不感兴趣的点以后可视化。在大多数pleurothallids,轴,pedicel或卵巢会做,因为他们可能不感兴趣的可视化
    4. 放置在磁带上的花。你将需要尽可能多地打开花,以允许可视化。扁平花如Platystele和Stelis可以完全放置(图4),平放在磁带上,更多的管状花如Acianthera和Specklinia必须被解散(图5)。在这些情况下,synsepal固定后,你可以删除背萼片和花瓣。重要的是尽可能少地触摸段,将它们放置在磁带上的最终位置(在放置之后将不能改变位置),并考虑所有可能的观察点。 >

      图4.像Stelis这样的小花朵和扁平花朵可以完整显示。比例尺= 500μm。

      图5.根据可视化需求显示多种Pleurothallidinae花,分解和放置的根。 A. Lankesteriana Specklinia spp。 C,D。 Echinosepala spp。

    5. 将桩存储在培养皿中,并在封闭的塑料盒或干燥器中用一些干燥的硅胶直到它们被溅射涂覆。样品可以在室温下在相对干燥的地方储存数年。在湿度高的地方,样品可以与硅胶一起储存。如果需要,可以在重新可视化之前重复溅射镀膜
  3. 溅射样品
    1. 打开氩气筒,使气体到达Q150T S溅射镀膜机( Q150T S/E/ES样品制备系统使用手册)。打开机器。
    2. 小心地取出玻璃室,将短棒放在支架上,使其均匀分布,以便在旋转时保持平衡。将玻璃室重新放回。放下盖子,小心不要接触样品。在操作屏幕上选择"配置文件"。建议使用PT/Pd 20 nm的涂层厚度,因为Pleurothallidinae花相当不规则。 (否则PT/Pd 10nm可能是足够的。)运行轮廓(图6)。花药和花粉在SEM中观察期间经常变得带电荷,这破坏了图像采集。较厚的涂层可能有助于防止此问题。

      图6.使用立体显微镜(1)和使用扫描电子显微镜(2)进行涂覆而未涂覆的Specklinia物种的萼片的腺体区域的显微照片。 A. B. pfavii ; C. S。 tribuloides 。比例尺= 1mm(A1,B1和C1);比例尺=100μm(A2,B2和C2)
    3. 在运行完成后,真空应允许盖的打开。样品可以使用针头取出并返回培养皿。

  4. 可视化和照片
    1. 按照显微镜手册打开JEOL JSM-7600F场发射扫描电子显微镜
    2. 使用多个12 mm短轴支架,并紧紧地放置六个短截线。处理支架和支柱时,请始终使用无粉手套。
    3. 按照显微镜手册使用样品交换棒将样品置于内部
    4. 按照显微镜手册可视化材料。设置可视化参数:将工作距离(Z)设置为15 mm,并将加速电压设置为10 KV。探头电流为6,探测器模式为SB 注意:工作距离可以变化,较短的距离给出较高的分辨率但较低的聚焦深度,较高的工作距离将给出较低的分辨率,但较高的聚焦深度。加速电压可以变化,在较高的kV下分辨率变得更好,但是有更多的充电机会。建议不要使用超过15 kV的生物样品。如果需要避免充电(例如,5kV)或者如果希望在不涂覆材料(例如,2kV)的情况下工作,则较低的kV可能是有用的。然而,较低的kV给出较少的分辨率和噪声图像。对于我们使用这个特定的SEM,我们总是使用探针电流6和探测器模式SB为我们的生物样品。此设置结合侧向散射电子和背散射电子的收集,从而获得良好的图像效果。 
    5. 当使用放大率高于500x时,可以从"低磁场"切换到"高磁场"模式
    6. 将图片保存为位图(图7)。

      图7.在Pleurothallidinae的花中发现的不同腺体的扫描电子显微照片。 B.在Condylago的后轴表面上的凹陷毛刺(= Stelis )。 C. D.在仙客来萼片的近轴表面上分泌气孔的花蜜。测量值为100,100,100和50μm。


  1. 如果没有鲜花,可以使用保存在液体收集品中的标本(步骤A,步骤1)。保存在FAA及其变体中的花将仍适于通过这里描述的方法可视化
  2. 当临界点干燥(步骤A,步骤2-6),而不是丙酮,你也可以使用100%乙醇(绝对)。根据我们的经验,这也有很好的效果。


艾伯塔省Mennega基金会资助第一作者在研究的几个阶段,导致这里描述的协议。此协议已从Leica Microsystems的原始应用手册修改。


  1. Karremans,AP,Pupulin,F.,Grimaldi,D.,Beentjes,KK,Butot,R.,Fazzi,GE,Kaspers,K.,Kruizinga,J.,Roessingh,P.,Smets,EF和Gravendeel, (2015)。  授粉 Specklinia 通过花蜜喂养果蝇:第一个报道的使用聚集信息素在兰科中的欺骗性综合征的病例。 Ann Bot 116(3):437-455 。
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引用:Karremans, A. P., van Heuven, B., Langelaan, R. and Gravendeel, B. (2016). Documentation of floral secretory glands in Pleurothallidinae (Orchidaceae) using Scanning Electron Microscopy (SEM). Bio-protocol 6(22): e2021. DOI: 10.21769/BioProtoc.2021.

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