发光排放及其在透射电子显微镜（TEM）及以后的使用

In this interview, AZoM talks to Anna Walkiewicz, Application Specialist at Quorum Technologies, about the sample preparation technique glow discharge, how it can be used in Transmission Electron Microscopy and even beyond that.

Can you introduce yourself, your role and Quorum Technologies’ work?

My name is Anna Walkiewicz. I am an application specialist at Quorum Technologies, a company that specialises in manufacturing sample preparation systems and cryo solutions for Electron Microscopy Imaging.

We produce equipment like critical point driers, coaters and glow discharge systems that is necessary to make samples suitable for imaging techniques that involve electron beam scanning.

用户对Glow放电治疗的期望有什么好处？

我们主要使用发光放电进行传输的电子显微镜进行准备，但是该技术也可用于其他显微镜，其中分散在液体中的样品在成像之前将其应用于表面上。
With glow discharge we can change the substrate surface properties and this way direct how our sample will behave on that substrate.
对于TEM，我们使用具有带有金属网眼的磁盘形式的TEM网格。这些网格可能具有不同的构建，它们可以有其他聚合物，蕾丝或孔碳的层。直接从框中取出的TEM网格的表面特性并不均匀。这可能会导致不必要的效果，例如支撑上的聚集，聚集或很少的样本。
发光排放用于改变表面的润湿特性，并使表面均匀。这有助于样品在表面上的均匀分散，也可以将分子定向在支撑上。

使用Glow放电时要考虑的因素有哪些？

当使用发光排放时，我们需要考虑如何改变表面的性质或换句话说，如果在特定样品的情况下，如果亲水或疏水表面是最好的。
For bio molecules, especially proteins and nucleic acids, the type of the charge is an important factor. Positively charged molecules will bind better to the negatively charged surface.

Hydrophilic, negatively charged surfaces tend to be in the highest demand.

图片来源：法定技术

Glow放电治疗背后的科欧洲杯线上买球学是什么？

The name ‘glow discharge’ refers to a characteristic glow of plasma - an ionised gas that is overall neutral but consists of negatively and positively charged regions.
When a gas is ionised, ions and electrons are produced. When there are enough electrons, they form more ions when colliding with a neutral atom of gas.
此过程通过无弹性碰撞发生。弹性碰撞对我们的目的无济于事，但是无弹性碰撞会产生离子或自由基 - 两种感兴趣的物种，它们与表面反应并改变其特性。
当自由基被中和时，它会产生特征波长的光子。该光子负责我们在发光放电中看到的非常特征的发光颜色。

在甲醇（A）和淀粉胺（B）发光放电处理特征发光中。图片来源：法定技术

发光排放是清洁和修饰表面的流行技术，尤其是在TEM样品制备中。最常见的治疗方法是在空光排放中导致离开表面亲水性和负电荷。

Could you tell our readers more about the sort of treatments possible with glow discharge?

对于大多数样品，我们需要带负电的亲水表面，因为通常分散在水基溶液中的分子或颗粒会积极地充电。欧洲杯猜球平台这样的表面可以通过空气发光排放处理产生。

但是，在某些情况下，我们想要具有亲水性和积极充电的表面。这可以通过使排放表面沉浸在乙酸镁的低百分比水溶液中来实现。

如果我们使用化学蒸气进行发光排放，我们可以将处理升至更高的水平。我们可以使表面疏水，并具有积极或负电荷的 - 对生物分子，蛋白质，抗体和核酸非常有用的处理。

Could you elaborate on the use of glow discharge to support biomolecular research?

In-chemical vapor glow discharge treatment is very useful for working with proteins, especially if there is a need to model their active centres. A directed orientation of the protein on the TEM grid could be a game changer in the modelling part of the research, as it was in the case of proteasome. The issue with proteasome is that its active centres are located on the sides of the molecule, it was impossible to model them when having a top view of the proteasome molecule. When the surface properties that the proteasome sample was set on were changed, it was possible to achieve majority of side views thus a lot of molecules that could be used for modelling. These special surface properties were achieved by in amylamine glow discharge treatment and render the surface slightly hydrophobic, positively charged and attracted negatively charged sides of proteasome molecule.

Figure 7: (Left, no glow discharge, middle, in-air glow discharge, right, in-amylamine glow.) TEM (Tencai F20) images of 20s human proteasome complex showing the effect of altering the surface charge of the carbon support film on the orientation of the protein molecules. Carbon film of 2.5nm thickness on Quantifoil 1.2/1.3 400 mesh was used as a support for the sample, courtesy of Edward Morris, ICR, London

This in-amylamine treatment has been in use since the 1970s. The first person to use this technique was Jaque Dubouchet, who worked in Paris, France, at the Institute of Cancer Research.
他的设备是完全手动的，因此，使用特氟龙阀将一滴氨基胺引入了发光排放系统中。该设备今天仍在使用。

这种方法是否有任何问题，法定技术如何帮助克服这些问题？

Dubouchet设备的主要问题是无法确定治疗将取得成功。由于其手动性质，淀粉胺的压力或过程时间可能存在变化。

我们开发了Quorum GloQube system解决此问题，确保治疗是完全自动的，并且无需手动控制该过程。

Developing an instrument that can automatically control this treatment was challenging for a number of reasons.

Amylamine is toxic and should never be inhaled. The apparatus, therefore, had to be robustly sealed, and it is important to ensure that all amylamine vapor was removed after the treatment before the door could be opened. Furthermore, when exposed to air, amylamine forms enol, which crystallises, clogs instrumentation and prevents the treatment from working.
Quorum Technologies’ GlowQube - glow discharge system has been specifically designed to overcome these issues. Procedures are in place to ensure there is no air in the system and only amylamine is present in the chamber when you run the treatment, and the system is automatically cleaned after using amylamine.

The procedure is safe and can be used to modify surfaces in consecutive runs with no issues.
还有其他可能的治疗方法。例如，将甲醇作为大气进行发光排放提供了负电荷和疏水性的表面。这种治疗方法对于使用铁蛋白很有用，尤其是在准备样品以研究天然样品负荷时。

您还会给开始使用Glow放电的用户提供其他建议吗？

If in-air glow discharge does not provide suitable results, it is always possible to try something else, such as an in-chemical vapor method.

Figure 8: TEM images (Tencai F20 G2) of ferritin protein complex from horse spleen (Sigma Aldrich) applied to in-air and in methanol vapor glow discharged carbon support TEM grids, courtesy of Imperial College London

许多发光排放系统仅包含一个腔室，这意味着在空气发光放电后立即运行实体蒸气发光放电，而不必先进行彻底的手动清洁过程。Gloqube的清洁程序是内置的，使得在短时间内尝试不同的方法变得更加容易。

While most of my answers have focused on glow discharge as a treatment for TEM samples, I should point out that a lot of people do use glow discharge for many different purposes; for example, to modify surfaces and to anchor samples for AFM, especially when imaging DNA.

该方法的最大挑战之一来自其手动和可变性质。重要的是使用无需调整阀门或不断手动处理设备的自动化系统。

Time is money for many processes, and the GloQube is an ideal option for automating this process.

关于Walkiewicz博士

Anna E. Walkiewicz博士是Quorum Technologies的应用专家。她拥有伯明翰大学的博士学位，在那里她研究了纳米级的手性认可。在Keysight Technologies中担任SEM和AFM产品科学家的申请中，她承认了对高分辨率成像的适当样品准备的极端重要性。她感兴趣的主要领域在于发现在表面最高部分发生的过程的结果。

该信息已从Quorum Technologies Ltd提供的材料中采购，审查和改编。欧洲杯足球竞彩

有关此消息来源的更多信息，请访问Quorum Technologies Ltd.

该信息已从Quorum Technologies Ltd提供的材料中采购，审查和改编。欧洲杯足球竞彩

有关此消息来源的更多信息，请访问Quorum Technologies Ltd.