Marsha Griffin and Mike Zordan introduce a new spectral flow cytometry solution.
Flow cytometry is one of the most commonly used methods to perform cellular analysis. The power of this technology is that it is able to perform single-cell fluorescence measurements at a very high speed. Since its rise to prominence in the 1970s, every commercial flow cytometer has used independent detectors for each fluorescence signal that is measured. This use of independent detectors requires that the fluorescent probes used for multicolour analysis have limited overlap in their emission spectra.
Each successive generation of flow cytometers has been able to measure more fluorescence parameters. The increase in parameters has historically been accomplished by adding more excitation lasers and detectors to the instruments. Such an approach has resulted in reduced improvements over the past decade because the potential number of channels (across the full emission spectrum) that can be separated by optical filters and dichroic mirrors is approaching capacity. Sony has developed and launched a new flow cytometer that breaks this paradigm, the SP6800 spectral analyser.
The SP6800 is unlike conventional cytometers because it utilises a 32-channel photomultiplier tube (PMT) and a custom prism array to perform spectral detection of fluorescence (Fig. 1). It features spatially separated 488nm and 638nm diode lasers, and can be equipped with an additional 405nm diode laser. Where a conventional flow cytometer will use dichroic mirrors and interference filters to direct fluorescence signals to an individual PMT, the prism array in the system spreads light from 500nm to 800nm across a linearly arrayed 32-channel PMT (Fig. 2).
One of the major advantages of this detection scheme is that it enables the measurement of up to 66 channels of fluorescence for each cell. These 66 channels fully maximise the information obtained from each cell. The system records separate measurements on the 32 channel PMT for both the 488nm laser and the 405/638nm lasers. When a system is equipped with a 405nm laser, two additional PMTs are added to measure fluorescence between 420 and 470nm. The ability to record fluorescence at this resolution allows the SP6800 to record complete fluorescence spectra from each cell (Fig. 3). One direct result of this increase in sampling power is that spectral flow cytometers are able to analyse more colours of fluorescence using fewer lasers than conventional flow cytometers. Sony’s system will return a minimum of 66 parameters for every cell analysed. This increased parameter power allows it to analyse over 20 colours using just three lasers, where a conventional flow cytometer must use at least five lasers to measure 20 colours. Additionally, fluorescent proteins have broad emission spectra that make it extremely difficult to combine multiple fluorescent proteins and multi-colour immunofluorescence in the same experiment on a conventional flow cytometer. The new system is capable of performing analysis of multiple fluorescent proteins with over 10 colour immunofluorescence.
The ability to record complete spectra allows the spectral analyser to perform applications that a conventional flow cytometer cannot. Signals from each individual fluorescent probe are resolved using the process of spectral un-mixing (Fig. 4). Spectral un-mixing algorithms process the entire measured spectra to determine the abundance of each fluorescent probe present. These algorithms are able to discriminate fluorescence from different probes based on both spectral location and spectral shape. This allows for the discrimination of fluorescent probes that overlap spectrally, as long as the spectra have different profiles. This is a great advantage because there are many fluorescent probes that cannot be used together in conventional flow cytometry, such as FITC and GFP, because they overlap spectrally and are measured by the same detector. In spectral flow cytometry, fluorescent probes such as these can be resolved since their spectral profiles are different. Another advantage to spectral flow cytometry is that it also allows for the analysis and measurement of cellular autofluorescence. In conventional flow cytometers, autofluorescence is a noise source that limits the sensitivity of the instrument. By looking at the entire fluorescence spectrum, the SP6800 is able to treat autofluorescence as an additional parameter. This enables sub-populations of cells to be identified by their autofluorescence signals. Additionally, being able to un-mix autofluorescence from the other fluorescent parameters allows for much higher sensitivity in assays. This is particularly useful when measuring the effect of pharmaceutical treatments on cells during in vitro assays.
Looking toward the future, the arrival of spectral flow cytometry with this new system will enable new methods of data analysis for the highly-dimensional datasets that are produced. Flow cytometry data analysis has traditionally been performed by sequential gating of cell populations based on two parameter histograms in a manual or semi-automated fashion. The high-resolution collection in the SP6800 produces single cell fluorescence spectrographs. New data analysis techniques will be able to perform spectral fingerprinting, where subpopulations of cells will be identified based on similarities of their total fluorescence spectra (Fig. 5). This allows for automated classification of cellular subpopulations that does not rely on sequential gating of populations.
Spectral flow cytometry is a powerful new technology that makes it easier to perform large multicolour studies, as well as perform new applications that were not possible on previous flow cytometers. These features make spectral flow cytometry an important tool for cellular analysis.
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Marsha Griffin and Mike Zordan are with Sony Biotechnology Inc in California, USA.
