The Spectral Viewers You Should Be Using For Your Flow Cytometry. (2024)

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

The Spectral Viewers You Should Be Using For Your Flow Cytometry. (1)

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.

The Spectral Viewers You Should Be Using For Your Flow Cytometry. (2)

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?

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?

The Spectral Viewers You Should Be Using For Your Flow Cytometry. (3)

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.

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.

The Spectral Viewers You Should Be Using For Your Flow Cytometry. (4)

Here is the spectrum of BV605™…

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

The Spectral Viewers You Should Be Using For Your Flow Cytometry. (5)

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.

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.

The Spectral Viewers You Should Be Using For Your Flow Cytometry. (6)

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.

The Spectral Viewers You Should Be Using For Your Flow Cytometry. (7)

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?

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

How To Optimize Flow Cytometry Filters

The Spectral Viewers You Should Be Using For Your Flow Cytometry. (8)

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:

The Spectral Viewers You Should Be Using For Your Flow Cytometry. (9)

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:

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?

The Spectral Viewers You Should Be Using For Your Flow Cytometry. (10)

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.

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.

The Spectral Viewers You Should Be Using For Your Flow Cytometry. (11)

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.

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 viewersand 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.

The Spectral Viewers You Should Be Using For Your Flow Cytometry. (2024)

FAQs

The Spectral Viewers You Should Be Using For Your Flow Cytometry.? ›

Spectral flow cytometry provides more information for each fluorophore which allows for increased resolution and sensitivity, and fluorophores having similar emission maxima but differing off-peak emissions can be differentiated using unmixing. This provides greater flexibility and capability in panel design.

What is the use of spectral flow cytometry? ›

Spectral flow cytometry provides more information for each fluorophore which allows for increased resolution and sensitivity, and fluorophores having similar emission maxima but differing off-peak emissions can be differentiated using unmixing. This provides greater flexibility and capability in panel design.

How do you choose fluorophores for flow cytometry? ›

Tips for Selecting Appropriate Fluorescent Dyes

Choose the brightest set of fluorophores for your flow cytometer instrument configuration. Choose fluorophores to help minimize the spectral overlap. Reserve the brightest fluorophores for the weaker antibodies and vice versa.

What are the most commonly used detectors for flow cytometry? ›

There are several detector types that can be used on a flow cytometer, the most common types being the photodiode (PD) and the photomultiplier tube (PMT).

What is the difference between spectral analyzer and flow cytometry? ›

While conventional flow cytometry optics block, reflect, or transmit a photon based on its wavelength, spectral flow cytometers disperse photons according to wavelength. The two most common types of dispersive optics are prisms and gratings.

What is spectral analysis used for? ›

Spectral analysis is the process of estimating the power spectrum (PS) of a signal from its time-domain representation. Spectral density characterizes the frequency content of a signal or a stochastic process.

What is the purpose of spectral lines? ›

A spectral line is like a fingerprint that can be used to identify the atoms, elements or molecules present in a star, galaxy or cloud of interstellar gas. If we separate the incoming light from a celestial source using a prism, we will often see a spectrum of colours crossed with discrete lines.

What are the 3 main components of flow cytometry? ›

Flow Cytometry Instrumentation and Methodology

The three main components of a flow cytometer are the fluidics, optics, and electronics. The fluidics system of a flow cytometer is responsible for transporting samples from the sample tube to the flow cell, past the laser, sorted and/or discarded.

What is the most common use of flow cytometry? ›

The most common application performed on the cytometer is immunophenotyping. This technique identifies and quantifies populations of cells in a heterogeneous sample - usually blood, bone marrow or lymph.

What is needed for flow cytometry? ›

Cell populations can be analyzed and/or purified based on their fluorescent or light scattering characteristics. A variety of fluorescent reagents are utilized in flow cytometry. These include, fluorescently conjugated antibodies, DNA binding dyes, viability dyes, ion indicator dyes and fluorescent expression proteins.

What is a spectrum analyser? ›

A spectrum analyzer is a device used to measure the strength of an RF signal over a defined band of frequencies. The signal passes through a filter that allows only a specific range of frequencies, and the resulting signal is then passed through an amplifier and displayed on a screen.

What is full spectrum flow cytometry? ›

Spectral flow cytometry is based on many of the fundamental aspects of conventional flow cytometry but has unique optical collection and analytical capabilities. With spectral flow cytometry, the emission spectrum of every fluorescence molecule is captured by a set of detectors across a defined wavelength range.

What is the main purpose of flow cytometry? ›

Healthcare providers commonly use it to evaluate bone marrow, peripheral blood and other fluids in your body. Flow cytometry can help diagnose several conditions, including infectious diseases and cancer.

What is the use of spectral evidence? ›

Spectral evidence is testimony entered into the court record by witnesses who testified that a specter or spirit came to them in a dream and caused harm to them. A specter is a spirit, ghost, or apparition of a witch that is projected to do the deeds of the witch.

What is the use of spectral data? ›

Spectral information can for example be used to determine spectral power distribution, presence of undesired wavelengths, color rendering properties of light sources or for transmissive or reflective properties of materials.

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