Mass Cytometry Revolves Around These 5 Things

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.

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

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 shown in figure 1. Cells are labeled following standard procedures, and are introduced into the system by the formation of mist droplets (nebulizer), these droplets pass through the ICP – an induced coupled plasma that vaporizes the cells, leaving the ion cloud. This cloud passes through a quadrupole that is designed to filter out the common/abundant ions leaving the heavy metal ions that were coupled to the antibodies. These are passed into the Time-of-Flight chamber where the mass and quantity of these masses is captured. Thee values are converted into typical FCS data for analysis.

Designing the Panel

A lot has been made about the fact that in mass cytometry there is no ‘compensation’ required. This is because there is no equivalent to autofluorescence of cells in the mass cytometry world. On the other hand, there are several factors including the purity of the metal ion, the oxidation of some metals and the sensitivity of the detector that need to be considered during panel design Figure 2 shows the differential sensitivity of the detector based on mass and figure 3 shows the predicted issues with oxidation and purity.

CyTOF differential sensitivity based on mass

Figure 2: Differential sensitivity of the CyTOF based on mass.

Oxidation and purity issues of different metals in mass cytometry operation

Figure 3: The potential oxidation and purity issues of different metals contributing and receiving error from other metals.

It should be noted that if a patient that a sample is derived from has been exposed to barium (used in some imaging tests), this can impact the instrument sensitivity. Likewise cisplatin, a common chemotherapeutic can also reduce instrument sensitivity. In fact, cisplatin has become a useful viability indicator for mass cytometry experiments.

There is a useful panel design tool that helps take these factors into account. After adding the target populations, you can run an optimized metals and a table, such as shown in figure 4 is generated, making the best recommendations for the proposed panel, taking into account the factors mentioned above.

Output from the CyTOF mass cytometry panel design tool

Figure 4: Output from the CyTOF panel design tool.

Preparing the Sample

Sample preparation, although similar to traditional fluorescent flow, requires that the sample be extremely clean. Unlike fluorescence, where the emitted photons are measured by the detector, in mass spectrometry, all the ions that make it into the TOF will interact with the detector, leading to signal, this is shown in Figure 5. Additionally, with the cells being vaporized the size and complexity parameters that are typically used in fluorescent flow are lost.

Poorly washed CyTOF sample and the negative impact on mass cytometry experiments

Figure 5: The impact of a poorly washed CyTOF sample, showing that the excess contaminates impact the signal. Data from Fluidigm (formerly DVS sciences)

There are some very specific considerations that need to be taken into account since the samples also are finally resuspended in water, rather than a protein containing buffer. Care must be taken when pelleting cells, so make sure to mark where the pellet should be located to reduce the chance of losing the pellet. Second, before running on the instrument, cells are stained with DNA intercalator IR191/IR193 which is used to help identify the target cells

Cell concentration is also important, so that there is clean separation between the ion clouds from each cell, the system should be run at no more than approximately 1,000 events per second. Practically, less than this maximum is better, to take into account uneven delivery of the sample through the nebulizer. If the cells are run to fast, this will lead to cloud fusion, and the resulting data will be suspect. Figure 6 shows this effect.

Here is what happens when cells are running too fast during mass cytometry operation

Figure 6: impact on the data of running cells to fast. The bottom left panel shows the output of the ‘raindrop plot’ when cells are run close together. For comparison, on the bottom right panel, is an example of good cell concentration, with a single cell highlighted in the red box.

Barcoding is a powerful tool for mass cytometry assays. In this process, each individual sample is labeled with a combination of one or more barcoding metal tags, giving each sample a unique signature. The samples are mixed together and antibody staining continues. This ensures all the samples are stained equivalently, reducing the effect of staining individual tubes can have on signal variation, as well as reducing the total amount of antibody needed. This was discussed in this article by Zunder et al (2015), and shown in Figure 7 from that paper.

CyTOF barcoding

Figure 7: Barcoding for CyTOF samples.

Finally, CyTOF data lends itself to normalization between runs. This is accomplished using a collection of beads with known intensity of five metals. From those signals, using software the cell data can be corrected. This is described in this paper by Finck et al (2013), and an example of data from figure 2 of that paper is shown below (Fig. 8)

CyTOF data normalization with beads

Figure 8: Normalization of CyTOF data using beads. From Finck et al (2013).

Data Analysis

Data analysis of high-dimensional data continues to evolve. While traditional bivariate gating can be performed, automated tools for visualization, clustering and comparing are being developed. These packages can be found as freeware as R scripts, and are getting implemented into commercially available software. The power of each of these tools is such that they require separate articles, so stay tuned to the Expert Cytometry blog for more information. Data analysis from a clustering program (SPADE) and data reduction (tSNE) analysis are shown in figure 9.

CyTOF data clustering

Figure 9: Data analysis of CyTOF data via clustering (left panel) and data reduction (right panel)

Researchers are developing pipelines such that data will effectively go in one end and undergo a normalization, a data reduction, a dimensionality reduction, and a gating partition – all of this automatically. This technology has the potential to profoundly facilitate the analysis process.

Imaging Mass Cytometry

CyTOF analysis has been expanded into imaging mass cytometry. In this process, shown in figure 10, a tissue section is labeled with metal-tagged antibodies, the tissue is interrogated with a laser to ionize a part of the sample. The vaporized tissue is carried away and enters the mass cytometer. This process is shown in figure 10, from the paper by Chang and colleagues (2017).

Mass cytometry imaging process

Figure 10: Imaging Mass Cytometry process.

This methodology provides expression level data in a location specific manner, allowing for better understanding and interrogation of complex microenvironments. While an emerging technology, it is rapidly becoming more popular. There are over 20 publications using this technology, including this one from Carvajal-Hausdorf and coworkers (2019), where the authors used an 18-plex analysis of breast cancer patient samples to examine cytotoxic T-cell infiltration after treatment (example data in figure 11).

Tissue mass cytometry results

Figure 11: Results of tissue mass cytometry: From Carvajal-Hausdorf and coworkers (2019)

In conclusion, mass cytometry is a powerful and expanding tool for the complex analysis of both suspension cells and tissues. The ability to measure over 30 targets at the same time allows researchers to probe biological processes in a deep and meaningful way, making cells understandable from an exciting whole-cell angle. The 5 core features of cytometry research include panel design, sample preparation, data analysis, and imaging mass cytometry. It’s always a good idea–for veterans and neophytes alike–to review the core principles of cytometry technology’s functionality.

To learn more about how Mass Cytometry Revolves Around These 5 Things, and to get access to all of our advanced materials including 20 training videos, presentations, workbooks, and private group membership, get on the Flow Cytometry Mastery Class wait list.

Join Expert Cytometry's Mastery Class
Tim Bushnell, PhD
Tim Bushnell, PhD

Tim Bushnell holds a PhD in Biology from the Rensselaer Polytechnic Institute. He is a co-founder of—and didactic mind behind—ExCyte, the world’s leading flow cytometry training company, which organization boasts a veritable library of in-the-lab resources on sequencing, microscopy, and related topics in the life sciences.

Similar Articles

5 Flow Cytometry Strategies That Sun Tzu Taught Me

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)

A Basic Guide To Flow Cytometry (3 Foundational Concepts)

By: Tim Bushnell, PhD

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…

4 Critical Rules For Spectral Unmixing

4 Critical Rules For Spectral Unmixing

By: Tim Bushnell, PhD

Spectral unmixing is the mathematical process by which a spectrum is broken down into the abundances of the different fluorochromes that make up the observed spectrum. This was described in the paper by Novo et al., (2013), which presented a generalized model for spectral unmixing of flow cytometry data. Of course, like compensation in traditional fluorescent flow cytometry, there are important rules to observe regarding the controls that are used to unmix the sample. If you need a refresher on the rules for TFF compensation, you can read about them here.    This blog will discuss the generalized process of spectral unmixing…

How To Buy A Flow Cytometer - What You Need To Evaluate From A To Z

How To Buy A Flow Cytometer - What You Need To Evaluate From A To Z

By: Tim Bushnell, PhD

So you have the money to buy a flow cytometer. Is it a sorter? Or perhaps a spectral analyzer? No wait, maybe an imaging mass cytometer?  Big or small?  What to choose?  How to choose?  More importantly, once you sign the contract to purchase the instrument, you don’t want to be struck with buyers remorse.  It is indeed a big decision and we have the best advice for you to consider before making the purchase. Let’s discuss some of the steps you should take to prevent buyers remorse and ensure you are getting the best instrument for your needs.  Do…

How small can you go? Flow cytometry of bacteria and viruses

How small can you go? Flow cytometry of bacteria and viruses

By: Tim Bushnell, PhD

Flow cytometers are traditionally designed for measuring particles, like beads and cells. These tend to fall in the small micron size range. Looking at the relative size of different targets of biological interest, it is clear the most common targets for flow cytometry (cells) are comparatively large (figure 1). Figure 1:  Relative size of different biological targets of interest. Image modified from Bioninja.    In the visible spectrum, where most of the excitation light sources reside, it is clear the cells are larger than the light. This is important as one of the characteristics that we typically measure is the amount…

What Is Spectral Unmixing And Why It's Important In Flow Cytometry

What Is Spectral Unmixing And Why It's Important In Flow Cytometry

By: Tim Bushnell, PhD

As the labeled cell passes through the interrogation point, it is illuminated by the excitation lasers. The fluorochromes, fluoresce; emitting photons of a higher wavelength than the excitation source. This is typically modeled using spectral viewers such as in the figure below, which shows the excitation (dashed lines) and emission (filled curves) for Brilliant Violet 421TM (purple) and Alexa Fluor 488Ⓡ (green).  Figure 1: Excitation and emission profiles of BV421TM and AF488Ⓡ  In traditional fluorescent flow cytometry (TFF), the instrument measures each fluorochrome off an individual detector. Since the detectors we use — photomultiplier tubes (PMT) and avalanche photodiodes (APD)…

How To Extract Cells From Tissues Using Laser Capture Microscopy

How To Extract Cells From Tissues Using Laser Capture Microscopy

By: Tim Bushnell, PhD

Extracting specific cells still remains an important aspect of several emerging genomic techniques. Prior knowledge about the input cells helps to put the downstream results in context. The most common isolation technique is cell sorting, but it requires a single cell suspension and eliminates any spatial information about the microenvironment. Spatial transcriptomics is an emerging technique that can address some of these issues, but that is a topic for another blog.  So what does a researcher who needs to isolate a specific type of cell do? The answer lies in the technique of laser capture microdissection (LCM). Developed at the National…

The Importance Of Quality Control And Quality Assurance In Flow Cytometry (Part 4 Of 6)

The Importance Of Quality Control And Quality Assurance In Flow Cytometry (Part 4 Of 6)

By: Tim Bushnell, PhD

Incorporating quality control as a part of the optimization process in  your flow cytometry protocol is important. Take a step back and consider how to build quality control tracking into the experimental protocol.  When researchers hear about quality control, they immediately shift their attention to those operating and maintaining the instrument, as if the whole weight of QC should fall on their shoulders.   It is true that core facilities work hard to provide high-quality instruments and monitor performance over time so that the researchers can enjoy uniformity in their experiments. That, however, is just one level of QC.  As the experimental…

How To Optimize Instrument Voltage For Flow Cytometry Experiments  (Part 3 Of 6)

How To Optimize Instrument Voltage For Flow Cytometry Experiments (Part 3 Of 6)

By: Tim Bushnell, PhD

As we continue to explore the steps involved in optimizing a flow cytometry experiment, we turn our attention to the detectors and optimizing sensitivity: instrument voltage optimization.  This is important as we want to ensure that we can make as sensitive a measurement as possible.  This requires us to know the optimal sensitivity of our instrument, and how our stained cells are resolved based on that voltage.  Let’s start by asking the question what makes a good voltage?  Joe Trotter, from the BD Biosciences Advanced Technology Group, once suggested the following:  Electronic noise effects resolution sensitivity   A good minimal PMT…

Top Technical Training eBooks

Get the Advanced Microscopy eBook

Get the Advanced Microscopy eBook

Heather Brown-Harding, PhD

Learn the best practices and advanced techniques across the diverse fields of microscopy, including instrumentation, experimental setup, image analysis, figure preparation, and more.

Get The Free Modern Flow Cytometry eBook

Get The Free Modern Flow Cytometry eBook

Tim Bushnell, PhD

Learn the best practices of flow cytometry experimentation, data analysis, figure preparation, antibody panel design, instrumentation and more.

Get The Free 4-10 Compensation eBook

Get The Free 4-10 Compensation eBook

Tim Bushnell, PhD

Advanced 4-10 Color Compensation, Learn strategies for designing advanced antibody compensation panels and how to use your compensation matrix to analyze your experimental data.