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We Tested 5 Major Flow Cytometry SPADE Programs for Speed – Here Are The Results

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Written By: Tim Bushnell, Ph.D.

As a follow-up to our post on tSNE where we compared the speed of calculation in leading software packages, let’s consider the case of SPADE (Spanning-tree Progression Analysis of Density-normalized Events). A favored algorithm in the flow cytometry community, SPADE is used when dealing with highly multidimensional or otherwise complex datasets. Like tSNE, SPADE extracts information across events in your data unsupervised and presents the result in a unique visual format.

Unlike tSNE, which is a dimensionality-reduction algorithm that presents a multidimensional dataset in 2 dimensions (tSNE-1 and tSNE-2), SPADE is a clustering and graph-layout algorithm. The result is quite different from the two-dimensional plot of tSNE, and rather resembles a phylogenetic tree in its branching structure (Fig. 1). As with a phylogenetic tree, similar clusters are grouped closer together, and dissimilar clusters are located more distally on the tree. If you’d like to read more on the theory and development of the SPADE algorithm, see the original literature [Qiu ...

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Mass Cytometry Revolves Around These 5 Things

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Written By: Tim Busnell, Ph.D.

1. How it works
2. Panel design
3. Sample preparation
4. Data analysis
5. Imaging mass cytometry

Today’s article will summarize the functionality of mass cytometry technology. This tech has been commercialized largely by Fluidigm in the CyTof systems. There are 5 key points to cover, or takeaways, that cytometrists should keep in mind as they perform their research.

How Does Mass Cytometry Work?

Traditional fluorescent flow cytometry has started to push the limit of the number of simultaneous parameters that can be measured. With the recent advent of spectral cytometry, as many as 40 simultaneous fluorescence parameters can be measured.

The first foray into high-dimensional cytometry didn’t use fluorescence. Rather, the antibodies were labeled with metal ions. To measure these labels, the cells had to be vaporized and the ion masses measured using a different detector. Thus cytometry time-of-flight, or CyTOF, more commonly known as Mass Cytometry, was born.

The mass cytometry process cycle

Figure 1: The CyTOF process, from Bendall et al. (2012).

The mass cytometry process is sh ...

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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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