How To Optimize Your Spectral Flow Experiment
by R.L. Becker
published 2/21/2022
In conventional flow cytometry, the signal from each fluorophore is measured by a single detector. Optical filters are set such that only a narrow range of the emission profile for each designated fluor is captured by the detector, while the rest of the signature is discarded. This is crucial for pairing each emission peak with the corresponding fluorescent label. However, even after only a narrow range of the spectral profile is captured, because fluors emit over a range of wavelengths, there is often overlap, or regions where multiple dyes are emitting at the same wavelength. This can cause the signal from one dye to be picked up by the primary detector of another, in a process called fluorescence spillover. When this occurs, the detector alone cannot differentiate from which fluor the signal is coming from, and compensation is necessary to ensure that the signal recorded by each detector is only that from the intended dye.
What is spectral flow cytometry?
In spectral flow, not only the peak emission range, but the entire emission signature is recorded for each dye using a set of detectors. The unique spectra for every fluor are differentiated through a process called spectral unmixing, which considers differences in emission across a broader range of wavelengths. Although mathematically distinct, basic compensation and spectral unmixing are conceptually similar and both rely on single stained controls.
Because the entire spectral profile of each dye is captured, this information can be used for downstream optical analysis. Additionally, similar dyes that could previously not be differentiated by conventional flow cytometry can be used side-by-side in spectral flow experiments. Furthermore, a greater number of fluorophores can be used simultaneously in spectral flow cytometry; the maximum is limited only by the total number of detectors in the instrument.
What are the advantages of spectral flow cytometry?
Due to many unique advantages of spectral flow cytometry, its popularity has increased significantly over the past decade, as scientists continue to rely on this technology for a widening breadth of applications. Still, because spectral flow relies on the sum of the differences in emission signatures to differentiate between dyes, there are a greater number of considerations in designing and compensating for a spectral flow experiment, and spectral compensation calculations are generally more intensive.
Spectral flow is cool and all, but what about compensation?
There are several barriers to successful spectral compensation that may present during an experiment. First, it is essential that the compensation control achieves a maximum fluorescence intensity without reaching saturation. This aids in the creation of a more accurate compensation matrix and can be especially vital in the detection of rare cell populations. Additionally, autofluorescence must be assessed, typically using an unstained control. Baseline autofluorescence can vary greatly depending on the sample type, therefore, to optimize autofluorescence extraction, a researcher should aim to use the same kind of sample for unstained controls, single-stained references, and experimental samples.
More often, this is not the case, and instead, solid-core plastic beads are used, as they are convenient, timesaving, and allow researchers to conserve valuable sample. However, these polystyrene compensation beads generally have a baseline autofluorescence much higher than that of cells. Additionally, the spectral signature generated by solid core beads is not a faithful match to that emitted by cells. Due to the importance of relatively small differences in emission profiles in separating fluorophores during spectral unmixing, even subtle mismatches in spectral signatures between compensation controls and samples can lead to over or under-correction, and in turn, compromise integrity of results.
There has to be a better way!
SpectraComp® by Slingshot Biosciences offers a convenient solution to single cell control staining during spectral compensation. These synthetic cells are engineered to replicate the scatter profile of lymphocytes, bind to the three most commonly used species of capture antibodies, and generate a spectral profile like that of single stained cellular controls.
SpectraComp compensation controls eliminate the discrepancies in scatter profiles seen between solid core beads and cells. These synthetic cell controls also allow for more accurate autofluorescence extraction, as their baseline autofluorescence matches that emitted by real cells. Additionally, they outmatch competitors in brightness, due to a greater surface area and a higher antibody-binding capacity than solid core compensation beads. SpectraComp controls also perform better in conjunction with tandem dyes, because they do not interfere with energy transfer as compensation beads often do. Altogether, SpectraComp allows the user to readily optimize compensation calculations and avoid spectral mismatches that can lead to distortion of data.
SpectraComp consistently outperforms competitor beads in matching the fluorescence spectra of single stained cell controls.
SpectraComp binds to all three most commonly used species of capture antibody (mouse, rat, and hamster) in addition to multiple IgG isotypes – all on one bead. These synthetic cell controls are stable at room temperature, and therefore far exceed competitors in terms of convenience and ease of use. In short, the use of these synthetic cell compensation controls provides the convenience of compensation beads, with the experimental rigor of using cellular controls.
What is spectral flow cytometry?
In spectral flow, not only the peak emission range, but the entire emission signature is recorded for each dye using a set of detectors. The unique spectra for every fluor are differentiated through a process called spectral unmixing, which considers differences in emission across a broader range of wavelengths. Although mathematically distinct, basic compensation and spectral unmixing are conceptually similar and both rely on single stained controls.
Because the entire spectral profile of each dye is captured, this information can be used for downstream optical analysis. Additionally, similar dyes that could previously not be differentiated by conventional flow cytometry can be used side-by-side in spectral flow experiments. Furthermore, a greater number of fluorophores can be used simultaneously in spectral flow cytometry; the maximum is limited only by the total number of detectors in the instrument.
What are the advantages of spectral flow cytometry?
Due to many unique advantages of spectral flow cytometry, its popularity has increased significantly over the past decade, as scientists continue to rely on this technology for a widening breadth of applications. Still, because spectral flow relies on the sum of the differences in emission signatures to differentiate between dyes, there are a greater number of considerations in designing and compensating for a spectral flow experiment, and spectral compensation calculations are generally more intensive.
Spectral flow is cool and all, but what about compensation?
There are several barriers to successful spectral compensation that may present during an experiment. First, it is essential that the compensation control achieves a maximum fluorescence intensity without reaching saturation. This aids in the creation of a more accurate compensation matrix and can be especially vital in the detection of rare cell populations. Additionally, autofluorescence must be assessed, typically using an unstained control. Baseline autofluorescence can vary greatly depending on the sample type, therefore, to optimize autofluorescence extraction, a researcher should aim to use the same kind of sample for unstained controls, single-stained references, and experimental samples.
More often, this is not the case, and instead, solid-core plastic beads are used, as they are convenient, timesaving, and allow researchers to conserve valuable sample. However, these polystyrene compensation beads generally have a baseline autofluorescence much higher than that of cells. Additionally, the spectral signature generated by solid core beads is not a faithful match to that emitted by cells. Due to the importance of relatively small differences in emission profiles in separating fluorophores during spectral unmixing, even subtle mismatches in spectral signatures between compensation controls and samples can lead to over or under-correction, and in turn, compromise integrity of results.
There has to be a better way!
SpectraComp® by Slingshot Biosciences offers a convenient solution to single cell control staining during spectral compensation. These synthetic cells are engineered to replicate the scatter profile of lymphocytes, bind to the three most commonly used species of capture antibodies, and generate a spectral profile like that of single stained cellular controls.
SpectraComp compensation controls eliminate the discrepancies in scatter profiles seen between solid core beads and cells. These synthetic cell controls also allow for more accurate autofluorescence extraction, as their baseline autofluorescence matches that emitted by real cells. Additionally, they outmatch competitors in brightness, due to a greater surface area and a higher antibody-binding capacity than solid core compensation beads. SpectraComp controls also perform better in conjunction with tandem dyes, because they do not interfere with energy transfer as compensation beads often do. Altogether, SpectraComp allows the user to readily optimize compensation calculations and avoid spectral mismatches that can lead to distortion of data.
SpectraComp consistently outperforms competitor beads in matching the fluorescence spectra of single stained cell controls.
SpectraComp binds to all three most commonly used species of capture antibody (mouse, rat, and hamster) in addition to multiple IgG isotypes – all on one bead. These synthetic cell controls are stable at room temperature, and therefore far exceed competitors in terms of convenience and ease of use. In short, the use of these synthetic cell compensation controls provides the convenience of compensation beads, with the experimental rigor of using cellular controls.
More Info:
- Get SpectraComp® for your next spectral flow cytometry experiment.
- Read A Workflow for Spectral Compensation with SpectraComp® Synthetic Cell Controls application note
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Cytek Biosciences, Aurora User’s Guide. Fremont, CA: 2019. https://www.umassmed.edu/globalassets/flow-cytometry-lab/documents/aurora-users-guide-103017.pdf. Accessed January 31, 2022
FlowJo LLC. Biologist's Guide to Spectral Compensation https://docs.flowjo.com/flowjo/experiment-based-platforms/plat-comp-overview/plat-comp-users/ Accessed February 1, 2022.
McKinnon KM. Flow Cytometry: An Overview. Curr Protoc Immunol. 2018;120:5.1.1-5.1.11. Published 2018 Feb 21. doi:10.1002/cpim.40
Nolan, J., & Condello, D. (2013). Spectral Flow Cytometry. Current Protocols In Cytometry, 63(1). doi: 10.1002/0471142956.cy0127s63
Roederer M. Compensation in Flow Cytometry. Current Protocols in Cytometry. 2002;22(1). doi:10.1002/0471142956.cy0114s22
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