Researchers have used flow cytometry to characterize distinct cell populations within heterogeneous samples since the technique’s invention. However, technological constraints held investigators back from exploring a robust suite of variables relevant to the biological phenomena they studied. The advent of multicolor flow cytometry has helped loosen these shackles, allowing today’s scientists to probe deeper and acquire more detailed information than ever before.
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The maximum number of variables researchers can examine in any given flow cytometry experiment hinges on the number of light sources and detectors available. An instrument possessing multiple lasers gives scientists the ability to simultaneously use fluorophores that do not share the same excitation spectrum, expanding the number of antibodies that can be employed. Furthermore, combining multiple lasers with multiple channels can even differentiate between two fluorophores with distinct excitation but similar emission spectra-as apparatuses with parallel laser arrangements can collect distinct emission signals from a single particle as it is excited sequentially by each laser.
Of course, the capacity to generate light at various wavelengths needs to be matched by the ability to detect light at an equal number of wavelengths. Detectors, whether photodiodes (PDs) or photomultiplier tubes (PMTs), convert all photons to electrical signals and are therefore not tuned to specific wavelengths. Rather, flow cytometers employ a system of filters and mirrors to ensure that each detector only receives light from a specific wavelength range corresponding to the emission spectra of a particular fluorophore. Filters do this by allowing all light either above (longpass), below (shortpass), or within (bandpass) a certain wavelength range, while mirrors are important not only for redirecting light to filters and detectors within the apparatus, but also splitting light by wavelength (dichroic mirrors). Together, filter-mirror-detector combinations enable instruments to capture emitted fluorophore signals and convert them into data.
The additional depth of information made possible by multicolor flow cytometry has not only made it possible to delineate increasingly comprehensive cellular profiles, but also to explore the rich world of cellular plasticity by examining single cells under different conditions or in response to different stimuli. For example, multicolor flow cytometry has paved the way for novel insights in the field of immunology. The ability to probe more cell surface markers simultaneously has revealed that many immune cells contain multiple subsets, identifiable by the presence of three or four common markers and one unique one, each with distinct and important functions and niches. This has tremendously benefitted oncology, where it is now possible to create in-depth profiles of individual cancer cells, identifying not only their phenotypes and behaviors, but also potential weak points and markers for targeted therapeutics.
Whether someone is just starting to use multicolor flow cytometry or an experienced veteran transitioning from one application to the next and looking for an upgrade, they ask for the same things.
Precision: Does the instrument have the sensitivity to identify the cells I’m looking for? Can it deliver accurate data even when using low sample volumes?
When choosing a model for your multicolor flow cytometry needs, it’s important to understand just exactly how the tool works. What is its signal detection mechanism? What elements—possibly in a reagent or appearing endogenously in cells—may cause interference? What settings can be adjusted to improve data accuracy and minimize noise, and how can I adjust these settings?
Flexibility: Are there sufficient filter channels to acquire the depth of data I require? Are customized antibody panels available to answer my research questions?
To properly set up a multicolor flow cytometry panel, researchers need to identify the markers they want to probe, obtain the necessary antibodies, ensure antibody specificity and selectivity, and also make sure that the fluorophores conjugated to the antibodies can be detected by the instrument but do not overlap in terms of emission spectra. This is not always easy, but many equipment manufacturers are helping to ease the burden by offering pre-assembled multicolor panels that align with the technical capabilities of their instruments. Many of these panels can be further customized in consultation with scientists to better meet their specific research needs.
Utility: Is the instrument user-friendly? Are there appropriate and compatible data acquisition, storage, and analysis software available?
Scientists today are not only looking for a wide array of features in the flow cytometry instruments they use, they’re examining whether accessing these features is simple, or whether they’ll have to spend more time inputting parameters and protocols than actually running the experiment. Multicolor flow cytometry has greatly increased the number of variables that a scientist can examine in any given experiment, and this has translated into a significant increase in the amount and complexity of acquired data. The modern researcher is looking for something that can deliver a fast and simple method of accessing, processing, parsing, and interpreting all of that information.
The ideal flow cytometer will vary for each individual depending on their own research needs and goals. Identifying that ideal mix of technical power, experimental adaptability, and user friendliness will go a long way towards finding an instrument that suits not only today’s projects, but also tomorrow’s.
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FACSMelodyTM Cell Sorter Platform
Manufactured By: BD BIOSCIENCES
The FACSMelodyTM Cell Sorter Platform combines user-intuitive sorting with excellent signal detection sensitivity. With its three spatially distinct lasers, the FACSMelody is capable of detecting up to nine colors simultaneously. Integrated automation streamlines your workflow, reducing time requirements and improving throughput, while FACSChorusTM software guides users through data acquisition and analysis step-by-step.
The above video was sponsored by and produced in conjunction with BD Biosciences.
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