ANALYTICAL AND LABORATORY EQUIPMENT 25
Spectral analyser technology F
Marsha Griffin and Mike Zordan introduce a new spectral flow cytometry solution.
low cytometry is one of the most commonly used methods to perform cellular analysis. Te 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. Tis 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. Te increase in parameters has historically been accomplished by adding more excitation lasers and detectors to the instruments.
Fig. 1. An animated comparison between the optical design strategy of conventional flow cytometry instruments and the SP6800 spectral cytometer.
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.
Te 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. Tese 66 channels fully maximise the information obtained from each cell. Te 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. Te 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. Tis 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. Te new system is capable of performing analysis of multiple fluorescent proteins with over 10 colour immunofluorescence.
Te 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. Tese algorithms are able to discriminate fluorescence from different probes based on both spectral location and spectral shape. Tis
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