Guide to Spectral Flow Cytometry

Flow cytometry interrogates biological features by measuring fluorescence of (typically) fluorochrome-tagged antibodies with specificity to those features. It transforms a cell’s invisible molecular information into colored light. Multiple molecules can be simultaneously interrogated using different fluors to yield information about not only their abundance but also their co-expression, on tens of thousands of cells per second.

A new paradigm for parsing out the contributions of each individual fluor has been gaining favor over the last decade, allowing researchers to interrogate upward of 60 biological features at one time. Here we discuss how spectral flow cytometry is able to capture that information and translate it into meaningful data, in the process pointing out some benefits compared to conventional flow cytometry.

Conventional flow cytometry

In conventional flow cytometry, cells stained with different fluors are passed by a series of lasers that excite the fluors. Depending on the properties of the fluor, multiple fluors can be excited by the same laser. Detectors then pick up the light emitted as the cells pass, reporting the brightness of each color to the instrument. Each distinct color requires its own detector. 

Yet while a fluorescent dye can be excited by a specific wavelength laser—say, 488 nm in the case of a blue laser used to excite FITC, PE, and many others—its emission spectrum is typically broad. “So that means that the signal you get from a given fluorochrome will spread across many detectors,” says Peter Mage, Associate Principal Engineer in BD Biosciences’ Advanced Technology Group. “You get what used to be called ‘fluorescence spillover.’ So the signal from FITC will be mostly in the FITC detector, but the emission tail bleeds over into the PE detector.”

This is not an issue when running a single-color experiment, but becomes one when concurrently using dyes—like FITC and PE—that cause spillover into other detectors and artificially increase their signal. Determining the signal from a cell dyed with only a single fluor (single-positive) in all detectors allows that fluor’s relative contribution to other fluors’ signals to be mathematically eliminated—a process known as ‘compensation.’

Yet as the number of dyes used to interrogate a given cell increases, overlaps in their emission spectra become more pronounced, with the spread between combinations of fluors making compensation ever more challenging. At some point there is no way to differentiate spectra, given the instrument’s bandpass filter configurations through which the light must pass on its way to the detectors, from each other.

Mind the gap: Enter spectral flow cytometry

“Spectral flow cytometers are made up of the same major components as conventional flow cytometers: fluidics, optics, and electronics,” points out Laura Johnston, Technical Application Specialist, Cytek^® Biosciences. The overall workflow, including sample preparation, reagents, and controls, are essentially the same as well.

The key difference is that “spectral flow cytometers utilize many more detectors in order to capture as much of the full spectrum as possible, Johnston notes. “While each detector, or channel, captures a small range of wavelengths, the combined arrays of detectors capture the full spectrum of light with little to no gaps.”

Fluorochromes can then be defined by a unique signature based on the signal intensity across all detector arrays. The spectrum collected is then deconvoluted using a mathematical process originally developed for satellite imaging, and later taken up by fluorescence microscopy. ‘Spectral unmixing,’ akin to compensation in conventional flow cytometry, teases out the individual fluor’s contributions so that they can be independently evaluated. “We’re not limited anymore to the narrow range of a filter,” Mirko Corselli, Associate Director of Market Development at BD Biosciences, explained in a webinar. “The only thing we need to worry about is whether we have the right laser to excite a given fluorochrome.”

Three benefits of spectral flow cytometry

At least three incremental benefits or new capabilities of spectral flow cytometry are commonly pointed out.

Overlapping profiles With spectral flow cytometry it is often possible to measure and utilize fluorescent molecules that have similar, overlapping emissions—including those that would in conventional flow cytometry have been assigned the same detector.

“There’s a huge caveat to that, which is that while you can technically do that, … any place where you have overlapping fluorescence from one dye into another, you can end up with increased measurement noise,” notes Mage. Corselli adds that such experiments are challenging, and “should be limited to high value panels or when there is no other option available.”

Flexibility As a consequence of being able to utilize highly overlapping fluors, “designing a multicolor flow experiment or designing a panel—that set of dyes that you’re looking at simultaneously—is a little bit more flexible with a spectral cytometer,” Mage explains. Rather than choosing fluors based on what detectors are available, “if the dye I’m looking at is excited sufficiently by one of the lasers on my system, and the emission peak is somewhere in the measurement range of the system (usually between about 350 and 850 nm), then I should be able to use it.”

Autofluorescence (AF) Some cells—such as tissue-derived samples and cell lines—tend to fluoresce even in the absence of exogenous staining. This can impact the ability of conventional flow cytometers to separate the autofluorescent signal from that of dyes (such as BD Horizon™ BV510) captured by the same detector. “The resulting data will appear as a high background for the negative population in the BD Horizon™ BV510 parameter, resulting in reduced resolution between the negative and positive populations,” explains Johnston. Spectral flow cytometers are able to define the autofluorescent signature and extract its contribution from the total measured signal, improving resolution between background and the real fluorescent signal of interest.

“The other thing is that AF can itself be an interesting biologically informative measurement parameter,” adds Mage. “It gives you a sort of label-free parameter that can be related to cell cycle, or related to the cell’s stimulation or activity.”

Mage sees the flow cytometry market “shifting toward making spectral more standard.” Corselli agrees, noting that “spectral flow is already changing the landscape of dyes and changing the way we develop dyes.”