The Power Of Spectral Viewers And Their Use In Full Spectrum Flow Cytometry
By: Tim Bushnell, PhD
What photon from yonder fluorochrome breaks? It is … umm… hmmm. Let me see. Excitation off a 561 nm laser, with an emission maximum of 692 nm. I’m sure if Shakespeare was a flow cytometrist, he might have written that very scene. But the play is lost in time. However, since the protagonist had difficulty determining what fluorochrome was emitting photons, let’s consider how this could be figured out. In my opinion, one of the handiest flow cytometry tools is the spectral viewer. This tool helps visualize the excitation and emission profile of different fluorochromes, as well as allowing you…
3 Must-Have High-Dimensional Flow Cytometry Controls
By: Tim Bushnell, PhD
Developments such as the recent upgrade to the Cytobank analysis platform and the creation of new packages such as Immunocluster are reducing the computational expertise needed to work with high-dimensional flow cytometry datasets. Whether you are a researcher in academia, industry, or government, you may want to take advantage of the reduced barrier to entry to apply high-dimensional flow cytometry in your work. However, you’ll need the right experimental design to access the new transformative insights available through these approaches and avoid wasting the considerable time and money required for performing them. As with all experiments, a good design begins…
The Fluorochrome Less Excited: How To Build A Flow Cytometry Antibody Panel
By: Tim Bushnell, PhD
Fluorochrome, antibodies and detectors are important. The journey of a thousand cells starts with a good fluorescent panel. The polychromatic panel is the combination of antibodies and fluorochromes. These will be used during the experiment to answer the biological question of interest. When you only need a few targets, the creation of the panel is relatively straightforward. It’s only when you start to get into more complex panels with multiple fluorochromes that overlap in excitation and emission gets more interesting. FLUOROCHROMES Both full spectrum and traditional fluorescent flow cytometry rely on measuring the emission of the fluorochromes that are attached…
Flow Cytometry Year in Review: Key Changes To Know
By: Meerambika Mishra
Here we are, at the end of an eventful year 2021. But with the promise of a new year 2022 to come. It has been a long year, filled with ups and downs. It is always good to reflect on the past year as we move to the future. In Memoriam Sir Isaac Newton wrote “If I have seen further, it is by standing upon the shoulders of giants.” In the past year, we have lost some giants of our field including Zbigniew Darzynkiwicz, who contributed much in the areas of cell cycle analysis and apoptosis. Howard Shapiro, known for…
What Star Trek Taught Me About Flow Cytometry
By: Tim Bushnell, PhD
It is no secret that I am a very big fan of the Star Trek franchise. There are many good episodes and lessons explored in the 813+ episodes, 12 movies (and counting). Don’t worry, this blog is not going to review all 813, or even 5 of them. Instead, some of the lessons I have taken away from the show that have applicability to science and flow cytometry. “Darmok and Jalad at Tanagra.” (ST:TNG season 5, episode 2) This is probably one of my favorite episodes, which involves Picard and an alien trying to establish a common ground and learn…
5 Flow Cytometry Strategies That Sun Tzu Taught Me
By: Tim Bushnell, PhD
Sun Tzu was a Chinese general and philosopher. His most famous writing is ‘The Art of War’, and has been studied by generals and CEOs, to glean ideas and strategies to help their missions. I was recently rereading this work and thought to myself if any of Sun Tzu’s lessons could apply to flow cytometry. So I have identified 5 points that I think lend themselves to thinking about flow cytometry. “Quickness is the essence of the war.” In flow cytometry, speed is of the essence. The longer the cells are out of their natural environment, the less happy they…
A Basic Guide To Flow Cytometry (3 Foundational Concepts)
By: Meerambika Mishra
Mastering foundational concepts are imperative for successfully using any technique or system. Robert Heinlein introduced the term ‘Grok’ in his novel Stranger in a Strange Land. Ever since then it has made its way into popular culture. To Grok something is to understand it intuitively, fully. As a cytometrist, there are several key concepts that you must grok to be successful in your career. These foundational concepts are the key tools that we use day in and day out to identify and characterize our cells of interest. Cells Flow cytometry measures biological processes at the whole cell level. To do…
Which Fluorophores To Use For Your Microscopy Experiment
By: Heather Brown-Harding, PhD
Fluorophore selection is important. I have often been asked by my facility users which fluorophore is best suited for their experiments. The answer to this is mostly dependent on whether they are using a widefield microscope with set excitation/emission cubes or a laser based system that lets you select the laser and the emission window. Once you have narrowed down which fluorophores you can excite and collect the correct emission, you can further refine the specific fluorophore that is best for your experiment. In this blog we will discuss how to determine what can work with your microscope, and how…
4 No Cost Ways To Improve Your Microscopy Image Quality
By: Heather Brown-Harding, PhD
Image quality is critical for accurate and reproducible data. Many people get stuck on the magnification of the objective or on using a confocal instead of a widefield microscope. There are several other factors that affect the image quality such as the numerical aperture of the objective, the signal-to-noise ratio of the system, or the brightness of the sample. Numerical aperture is the ability of an objective to collect light from a sample, but it contributes to two key formulas that will affect your image quality. The first is the theoretical resolution of the objective. It is expressed with the…
What Is Total Internal Reflection Fluorescence (TIRF) Microscopy & Is It Right For You?
By: Heather Brown-Harding, PhD
TIRF is not as common as other microscopy based techniques due to certain restrictions. We will discuss these restrictions, then analyze why it might be perfect for your experiment. TIRF relies on an evanescent wave, created through a critical angle of coherent light (i.e. laser) that reaches a refractive index mismatch. What does it mean in practice? A high angle laser reflects off the interface of the coverslip and the sample. Although the depth that this wave penetrates is dependent on the wavelength of the light, in practice it is approximately 50-300nm from the coverslip. Therefore, the cell membrane is…
5 Drool Worthy Imaging Advances Of 2020
By: Heather Brown-Harding, PhD
2020 was a difficult year for many, with their own research being interrupted- either by lab shutdowns or recruitment into the race against COVID-19. Despite the challenges, scientists have continued to be creative and have pushed the boundaries of what is possible. These are the techniques and technologies that every microscopist was envious of in 2020. Spatially Resolved Transcriptomics Nature Methods declared that spatially resolved transcriptomics was the 2020 method of the year. These are a group of methods that combine gene expression with their physical location. Single-cell RNA sequencing (scRNAseq) was originally developed for cells that had been dissociated…
Getting A New Flow Cytometer? Try Before You Buy (And 2 Other Tips)
By: Tim Bushnell, PhD
One question that I get asked on a regular basis is what flow cytometer should I purchase? It’s not as simple as you might imagine. In fact, you need to treat this process as carefully as you would a valuable experiment. There are a lot of variables, and if you’re not asking yourself a huge list of questions, you may miss something critical that will result in the instrument being less than desired. This is a shortlist of questions to ask as you go about the process – your 3-part pocket guide to acquiring the flow cytometer that’s right for…