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Use These 5 Techniques for Super Resolution

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Written By: Heather Brown-Harding, Ph.D.

When you need better resolution than what can be achieved using a traditional microscope, it can be very intimidating to figure out which microscopes will work best for your experiment. Super-resolution imaging methods require software reconstruction after image acquisition. This is because multiple images are acquired, and they need to be combined. Additionally, the points of light need to be reassigned to their true location.

Today, we’re going to discuss 5 different super resolution methods their pros and cons. Although Rayleigh Criterion is not broken, these techniques each feature creative ways to get around it.

1. Structured illumination microscopy (or SIM).

SIM uses lasers through diffraction grating

SIM uses polarized light sources, like lasers, filtered through diffraction grating. The orientation of the grating is changed several times, and the resulting multiple images are used to reconstruct an image with better resolution than any of the originals.

The points of lights, which would normally interfere with their neighbors, are excited and captured at different times. ...

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Understanding Reproducibility in Flow Cytometry – It’s the Antibodies!

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Reproducibility in flow cytometry depends on antibody quality

Written By: Tim Busnell, Ph.D.

Reproducibility is key to the scientific method. After the results of a study are published, the community validates the findings and extends them. If the findings are not reproducible, the second step is impossible. With performable experiments increasing in complexity, and the concurrent increase in the cost of equipment and reagents to perform these experiments, it is important to find the best way to maximize the money spent on advancing research.

In flow cytometry, there are many places where improvements can be made to increase the consistency and reproducibility of an experiment. The most obvious place is in the instrument, which was the focus of a previous blog post. The focus of this blog entry will be on the reagents we use to identify the cells of interest: Antibodies and fluorochromes.

Why the focus on antibodies? The commentary on this topic (with 110 co-signers) by Bradbury and Plückthun (2015) is informative. The authors cite a review from 2008, in which the authors stated that “fewer than half of around 6,000 routinely-used commercia ...

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3 Components Of Every Flow Cytometer You Don’t Know Enough About

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Written By: Tim Bushnell, Ph.D.

All flow cytometer instruments have a certain 3 components you likely don’t know enough about. The way they are put together will dictate the performance of the system. As a user, you’ll be interacting heavily with these components, so you need to know both what they are and how they work.

The 3 components are the fluidics, optics, and electronics. Fluidics will be managed in order get interactions at the proper flow rate, ensuring that your data keep a tight CV. After cells have passed the laser intercept, you need a way of measuring the signal. This is where the second component will be used: optics. There are a few different types of optics you can use to measure your signal, like PMTs or APDs. Lastly, there are electronics, which process the photon into an electronic signal that is ultimately digitized and stored in a file known as the “FCS file.” This is key for interacting with your data once you have your results.

Let’s look at these 3 components in more detail…

1. Fluidics.

Fluidics will take the cells from your tube and bring ...

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4 Ways To Analyze Tissues By Flow Cytometry

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Written By: Tim Bushnell, Ph.D.

Why are we interested in cytometry? What are its practical applications? Let’s start by defining cytometry as the measurement of cellular processes at the whole-cell level. This definition is useful because it includes not only flow cytometry, but any technique that measures at the level of the whole cell. Microscopy, for instance, is a great example of cytometry.

But, why measure in tissues? When flow cytometry is practiced, the cells are broken up. Therefore, any cellular interactions within the sample are also broken up. This includes cell-to-cell contact and virtually any information about the microenvironment. As we continue to discover, the microenvironment can play a dramatic role in cell development, influencing how cells grow and change.

Here are 4 ways to analyze tissues by flow cytometry…

1. Multi-photon cytometry

Multi-photon microscopy doesn’t rely on traditional fluorescent excitation. In the traditional process, a single photon will excite the molecule. Following this, there is a fall back to the ground state along with the de ...

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Ask These 7 Questions Before Purchasing A Flow Cytometer

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Written By: Tim Bushnell, Ph.D.

Using funds to make a capital purchase can be an exciting time in a facility. If you don’t have the funds in hand, planning for future purchases requires due diligence to make sure the investment is worth it, and that it will satisfy the needs of the community. That $200,000 or more (sometimes much more) is an investment in the future research capabilities of the facility, so invest wisely.

Over the course of writing instrumentation grants, developing business plans, and acquiring instruments, the following questions should be your go-to checklist — what I look at and what you need answered before spending that hard-won funding.

1. What role does the instrument need to fill?

At the top of the list is identifying the instrument’s role. What does the research community need at the moment, and in the near future? Develop good tracking metrics on current trends in usage, and ask major users about their 1-to-3-year plans. And, don’t forget to investigate funding status.

Your data may suggest you need another cell sorter, but as you investigate gran ...

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