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5 Essential Controls For Reproducible Fluorescent Microscopy Imaging

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Written By: Heather Brown Harding, Ph.D.

Fluorescent imaging has the potential to bring great insights into your research project.

But, there is a lot of room for error.

And one of the most common and completely preventable errors that many researchers make is not having the necessary controls.

Having all the necessary controls may seem tedious.

I know in graduate school I was not a huge fan of controls, and would sometimes put off doing all the controls I needed until the end.

However, doing your controls after the fact is a terrible idea.

You could realize that all the data you recorded was just an artifact and that you wasted your time and samples.

It’s incredibly important to plan out your controls and perform them ahead of time so that you know that you have specific staining and you’re not picking up random noise, auto-fluorescence, and non-specific staining, etc.

Here are 5 fluorescent microscopy controls that you should be including in all your experiments…

1. Unlabeled sample.

The first and easiest control to an unlabeled sample.

In an unlabeled sample ...

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3 Action Steps You Can Take Right Now To Improve Your Flow Cytometry Reproducibility

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Written By: Tim Bushnell, Ph.D.

Reproducibility is a key issue in science.

Massive amounts of time and money are wasted when the results of experiments are not reproducible.

For example, I was called into a lab to look at their data because they had spent thousands of dollars sorting precious human samples and were now doing genomics analysis with the isolated cells.

Unfortunately, the results of the genomics analysis made no sense based on the sorted populations. The lab was working backward through every step of the process to try to identify what might have happened and if the experiments were salvageable.

As I reviewed the sorting process, one of the striking factors was that the quality control of the cell sorting experiments was very, very poor. In fact, it was non-existent.

So whoever was running their sorter was not performing quality control on the instrument, so the sorting results were all over the place. Voltages were changed dramatically for each experiment, and the separation of the target cells ranged from barley differentiated to well separated. The compensation cont ...

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Microscopy – 5 Reasons Coverslips Are Important For High-Quality Imaging

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Written By: Heather Brown Harding, Ph.D.

Most people are familiar with coverslips being placed on slides to protect the sample, but that’s not the only reasons that coverslips are important.

They also affect the image quality.

Coverslips function by working with your microscope to focus light to a single point and avoiding unnecessary noise in your image.

Having the wrong type of coverslip will damage the quality of your images and the quality of the data you extract from those images.

So today, we will discuss five reasons why coverslips, and utilizing the right ones, will improve your imaging…

1. Objectives expect there to be a coverslip in the light path.

Objectives are designed to compensate for a coverslip in the light path, and specifically for a 0.17mm thick glass to be in the light path.

This correlates with a number 1.5 coverslip.

You will also find number 1 (0.15mm) and number 2 (0.22mm) coverslips available for sale, but those will not be as effective.

If you check out the Nikon website MicroscopyU, you will see the objective with a numerical aperture of 0.95 ...

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3 Considerations To Ensure Your Cell Sorting Flow Cytometry Experiments Run Smoothly

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Written By: Tim Bushnell, Ph.D.

Cell sorting is such an important technique because it’s a gateway tool for many other downstream applications.

Assays such as culturing cells, genomic analysis, proteomics, injections into mice and the like are enabled by cell sorting, allowing researchers to use a homogeneously defined population of cells in their experiments.

With single-cell genomics we can readily characterize a population in great detail, but we have to have a single purified population for it to work.

That’s where the cell sorter comes in.

There are all sorts of applications we can do with a purified population and with the advances in sorting and fluorescent technologies, it is possible to isolate complex phenotypes from rare populations.

Fluorescent proteins are a great example of being able to look at the expression of the inter se or the marker coupled to a fluorescent protein so we can sort.

We can do two or three or four of these GFP, YFP and some of the fruit fluorochrome so you can look at the expression patterns of two or three different proteins at the sam ...

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3 Questions You Should Be Asking About Flow Cytometry Controls For Your Experiments

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Written By: Tim Bushnell, Ph.D.

What are the controls that you should be considering when planning and designing your flow cytometry experiments?

Are you using isotype controls? Should you be?

Isotype controls are one of the controls used in flow cytometry today and is very controversial. In fact, I encourage people to not use it.

Are you following the rules of compensation and including the appropriate controls?

Compensation is such an important thing to get correct when running your experiments, especially if you’re doing polychromatic flow cytometry, which many of us are doing nowadays.

What types of quality controls should you have in place? Quality control is an important part of the whole experimental process any yet many people don’t worry about it or even think about it.

But if left unchecked, poor quality control can cause major problems.

For example, a colleague of mine was searching for a biomarker for a disease that they were studying. They had done several years worth of analysis and over time acquired many samples. When they went to analyze the data they fo ...

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