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4 Spectral Viewers You Should Be Using For Your Flow Cytometry Experiments

Written by Tim Bushnell, PhD

At the heart of flow cytometry is the ability to make meaningful measurements of fluorescently tagged cells.

These fluorochromes can be bound to antibodies, a fluorescent protein, a reporter fluorochrome, and the like. Free online spectral viewers are useful in a variety of ways, all of which help improve experimental design and troubleshooting.

These spectral viewers have a special place on every scientist’s browser toolbar. I refer to them regularly, at least weekly.

During the process of panel design, it is useful to have these open to check and compare different fluorochromes. In fact, with recent upgrades to FluoroFinder, there is an integrated spectral viewer based on the filter configuration.

There are a host of different spectral viewers available online. Each one has its strengths and provides specific information. This often necessitates having to use two or three of them to get the information you want. These are the spectral links I use (in alphabetical order):

  1. AffymetrixFluorPlan Spectra Viewer
  2. BD Bioscience Spectrum Viewer
  3. Biolegend Spectra Analyzer
  4. ThermoFisher Fluorescence SpectraViewer

All the spectral viewers listed have several common and important features. These all allow the investigator to specify the laser excitation lines, the filter configurations and the fluorochromes. The one caveat with fluorochrome choice revolves around proprietary spectra and some will only be available with specific vendors.

The spectral viewers also output similar information, as illustrated below. This information can include how much of a given excitation curve is found in a given filter, the percentage of maximal emission, and more.

What Is A Spectral Viewer And Why You Should Use It

One of the primary benefits of spectral viewers is that they are useful in learning more about fluorochromes. For example, the popular tandem dye PerCP-CY5.5.  fluorescence spectrum analyzer | Expert Cytometry | fluorescence spectra viewer

This shows the excitation at 488 nm, and the nice emission. The BD Bioscience Spectrum Viewer gives you a nice additional feature–the %Max excitation, in this case it’s 98.4%. The excitation curve (shown in dashed blue) ends at about 450 nm. So, how can the spectral viewer help us explain the following data?fluorescence spectrum analyzer | Expert Cytometry | fluorescence spectra viewer

In this experiment, the beads were stained with either PerCP-Cy5.5 or Qdot705 and measured with either blue laser excitation (Blue B 710/50) or violet laser excitation (Violet B 710/50). As you can see from the graph, it’s clear that there is significant PerCP-Cy5.5 signal in the Violet B channel (Blue line).

Where is this signal coming from?

Seeing that the PerCP-Cy5.5 excitation profile ends, the following figure shows the excitation profile from PerCP.  Our answer is revealed in the full excitation spectrum.fluorescence spectrum analyzer | Expert Cytometry | fluorescence spectra viewer

PerCP, it turns out, can be excited by a 405 nm laser, at about 27% efficiency. Coupled to an efficient transfer to the Cy5.5 acceptor on the tandem, it explains why we see this signal in the violet laser.

This is significant if you were going to be using those two dyes in a polychromatic panel.

How To Evaluate New Fluorochromes On The Market

The Brilliant Violet™ dyes, produced by Sirigen, have been a boon to users of the violet laser. These dyes are extremely bright and have become very popular. The Brilliant Violet™ series of dyes include both polymer dyes (BV421™ and BV510™), and tandem dyes with the polymer core (BV570, BV605™, BV650™, BV711™, BV785™). While they may not be named as tandems, they may have some issues that the spectral viewer can reveal.

Here is the spectrum of BV605™…fluorescence spectrum analyzer | Expert Cytometry | fluorescence spectra viewer

Notice that there is a second excitation (here shown in the green 561 nm line). This excitation means that this tandem may have issues affecting the spillover of this dye into a PE and PE-Texas Red®-like channel.

Anytime a new fluorochrome comes out, it’s good to learn about it using these spectral viewers.  The Biolegend Spectra Analyzer is quick and easy to use, and they even have an app for the program, so you can go mobile with it. This is useful when you’re reviewing data with someone and don’t want to access your traditional computer browser.

How To Identify Areas Of Spectral Spillover

Another powerful use of the spectral viewers is to understand what channels on an instrument a given fluorochrome will spill into. The following example is using Alexa Fluor® 488, with three instrument filters placed on the graph.fluorescence spectrum analyzer | Expert Cytometry | fluorescence spectra viewer

With the ThermoFisher Fluorescence SpectraViewer, if you hover over the filter, it will report the percentage of the curve that is contained within that filter. In this case, the 530/30 bandpass filter captures about 49% of the curve.

fluorescence spectrum analyzer | Expert Cytometry | fluorescence spectra viewer

About 12% of the Alexa Fluor® 488 fluorescence is captured with the 585/42 filter, and about 1% with the 630/20 filter.

It is important to note that this is not the amount of compensation that needs to be applied, just the amount of the curve that is present in the filter.

Here is an example of another use for these spectral viewers, courtesy of the AffymetrixFluorPlan Spectra Viewer. In the results tab, it shows in table form the percentage of a given fluorochrome’s emission curve found in the filter in question.  Looking at this figure, with the PE and PE-Cy7 curves plotted, what can be said about the PE-Cy7 fluorochrome?fluorescence spectrum analyzer | Expert Cytometry | fluorescence spectra viewer

It turns out that the FRET between the PE emission and the Cy7 excitation is not optimal.

How To Optimize Flow Cytometry Filters

Another great use of the spectral viewers is to optimize the filters for a given fluorochrome on a specific instrument. Take for example, the following data. A researcher noticed some sensitivity issues off of two detectors when an instrument was installed. Beads were stained with FITC (488 nm excitation) and QDot545 (405 nm excitation) and run on the instrument. The data looked like this:fluorescence spectrum analyzer | Expert Cytometry | fluorescence spectra viewer

When the 532 nm laser was on, it was clear that the dim signals were shifted to the right. The instrument came with the vendor supplied filters. In modeling this issue, putting these two filters in, along with the laser lines, we see the following:fluorescence spectrum analyzer | Expert Cytometry | fluorescence spectra viewer

Notice that the 532 nm laser line was directly in the middle of these two filters. The cause of the loss of resolution was a result of the scatter from cells as they pass through the 532 nm laser. A fraction of this scatter wound up in the fibers leading to the blue and violet detectors.

The lesson learned from this was to always model your filters before ordering an instrument.  A tweak of the filter solved this problem, and the experiments continued.

What Is The FluoroFinder Spectral Viewer?

Recently, FluoroFinder released a new version of their panel design package.  As part of that package, when the cursor hovers over a given fluorochrome, the system will provide the researcher information based on the instrument configuration and filters on the machine. This is shown below.fluorescence spectrum analyzer | Expert Cytometry | fluorescence spectra viewer

This is an excellent resource when designing polychromatic panels. It is nice to get a view of the spectra of the fluorochrome choices on the instrument being used and quickly get a feel for how well a given filter/detector combination will capture the photons emitted from the fluorochrome. It also helps to see what other channels might be affected by the fluorochrome choice.

For example, on this instrument, there are several possible filters that can be used for QDOT565. Based on the filter configuration and emission profile of the fluorochrome, this emission may impact three other detectors. For that reason, it may be better to choose a different fluorochrome. This addition to FluoroFinder is a great feature to help make those critical fluorochrome choices during the design process.fluorescence spectrum analyzer | Expert Cytometry | fluorescence spectra viewer

Fluorochrome emission is the lifeblood of flow cytometry. The use of in silico tools can save a lot of effort and missed opportunity by allowing for the modeling of excitation and emission profiles in the context of what filters a given instrument is equipped with. Using these tools, it is easy to identify where a new fluorochrome will be measured on an instrument, where a fluorochrome may cause issues with other fluorochromes, and what filters are best for detection. These tools can save a lot of troubleshooting at the beginning of an experiment, and also help understand when issues do pop up. Bookmark them and use them at every opportunity.

To learn more about spectral viewers 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.

Tim Bushnell, PhD

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