登录查看更多内容

What are the best ways to optimize sample preparation for microscopy?

由人工智能和领英社区提供技术支持

Microscopy is a powerful tool for bioengineering, but it requires careful sample preparation to obtain high-quality images and data. In this article, you will learn some of the best ways to optimize your sample preparation for different types of microscopy, such as fluorescence, electron, and atomic force microscopy. You will also discover some common pitfalls and tips to avoid them.

此文章中的业界达人

由社区从 2 条内容中精选。了解更多

Saxon Christopher Whittle, PhD

Scientific Team Lead at Parexel

1 Choose the right fixation method

Fixation is the process of preserving the structure and function of your biological samples before microscopy. Depending on your research question and the type of microscopy you use, you may need to fix your samples with chemical agents, heat, or freezing. Chemical fixation can cross-link proteins and lipids, but it may also alter their fluorescence or antigenicity. Heat fixation can denature proteins and damage membranes, but it is fast and simple. Freezing fixation can preserve the native state of your samples, but it requires special equipment and protocols. You should choose the fixation method that best suits your sample type, microscopy technique, and analysis goals.

添加您的观点

2 Optimize the staining and labeling process

Staining and labeling are the methods of enhancing the contrast and specificity of your samples for microscopy. You can use dyes, antibodies, or fluorescent probes to stain or label different components of your samples, such as nuclei, membranes, organelles, or molecules. However, staining and labeling can also introduce artifacts, background noise, or quenching effects. You should optimize the concentration, incubation time, and washing steps of your staining and labeling process to achieve the best signal-to-noise ratio and minimize non-specific binding. You should also use appropriate controls and validation methods to ensure the accuracy and reproducibility of your staining and labeling results.

添加您的观点

Saxon Christopher Whittle, PhD

Scientific Team Lead at Parexel
举报内容
Often it can be useful to produce more than one sample to demonstrate that replications of what you are staining for can be observed, and it isn't just a one-off or artifact/non-specfic binding. Normally three or more samples can show that results are reproducible.

已翻译

赞

3 Select the suitable mounting medium and coverslip

Mounting medium and coverslip are the materials that you use to mount your samples on slides for microscopy. They can affect the optical quality, stability, and preservation of your samples. You should select the mounting medium and coverslip that match the refractive index, fluorescence properties, and compatibility of your samples and microscopy objectives. For example, if you use fluorescence microscopy, you should avoid mounting media that contain autofluorescent compounds or that quench your fluorophores. You should also use coverslips that have the appropriate thickness and flatness for your microscopy technique and objective magnification.

添加您的观点

Saxon Christopher Whittle, PhD

Scientific Team Lead at Parexel
(已编辑)
举报内容
Also need to make sure that your slides are labelled with the sample and the type of fixation/staining - someone else may be using the slides in the future so you may need to ensure that your notes can be read. If you use abbreviations then consider a key to decipher them in the box where they are stored

已翻译

赞

4 Adapt to the specific requirements of your microscopy technique

Different microscopy techniques have different advantages and limitations for bioengineering applications. You should adapt your sample preparation to the specific requirements of your microscopy technique to optimize your image quality and data analysis. For example, if you use electron microscopy, you may need to dehydrate, embed, and section your samples before imaging. If you use atomic force microscopy, you may need to immobilize your samples on a suitable substrate and adjust the scanning parameters and force feedback. If you use confocal or multiphoton microscopy, you may need to optimize the laser power, scanning speed, and pinhole size.

添加您的观点

5 Avoid common pitfalls and follow best practices

Sample preparation for microscopy can be challenging and prone to errors. You should avoid some common pitfalls and follow some best practices to ensure the reliability and validity of your microscopy results. Some of the common pitfalls are: contamination, degradation, evaporation, shrinkage, swelling, distortion, or photobleaching of your samples. Some of the best practices are: using fresh and sterile reagents, storing and handling your samples properly, performing quality checks, documenting and standardizing your protocols, and comparing and calibrating your microscopy settings.

添加您的观点

6 Here’s what else to consider

This is a space to share examples, stories, or insights that don’t fit into any of the previous sections. What else would you like to add?

添加您的观点

Bioengineering

+ 关注

给文章评分

我们借助人工智能创建了此文章。您认为这篇文章怎么样？

很棒不太好

举报此文章

查看全部

What are the best ways to optimize sample preparation for microscopy?

1

2

3

4

5

6

1 Choose the right fixation method

2 Optimize the staining and labeling process

3 Select the suitable mounting medium and coverslip

4 Adapt to the specific requirements of your microscopy technique

5 Avoid common pitfalls and follow best practices

6 Here’s what else to consider

Bioengineering

给文章评分

感谢您的反馈

更多Bioengineering相关文章

更多相关阅读内容