Why spectral flow cytometry is advantageous over conventional flow cytometry in multi-color experiments?

Conventional flow cytometry employs the one color, one detector paradigm, which disregards much of the valuable information of the emission spectrum and can be limited by the broad emission peaks of available fluorophores. Filters and mirrors need to be broad enough to capture enough light for detection, yet narrow enough to minimize the fluorescence spillover from the nearby fluorophores. Fluorescence compensation may be performed to reduce the influence of spillover, allowing for multi-color analysis. However, fluorescent molecules with very minor differences in their spectra, such as allophycocyanin (APC) and Alexa Fluor 647 dye, still cannot be distinguished by conventional flow cytometry.

In contrast, spectral flow cytometers capture the full emission spectrum of the fluorophore, making more fluorescence data available for further analysis. Dyes with very similar spectral features, again such as APC and Alexa Fluor 647 dye, can be easily differentiated by comparing their overall spectra rather than the intensity in an individual channel at a specific wavelength. Therefore, spectral flow cytometry enables the compatibility and distinction of many fluorescent combinations that were previously difficult or impossible to separate by conventional flow cytometry, which makes it more advantageous in multi-color experiments.