What is the difference between conventional and spectral flow cytometry?

Conventional and spectral flow cytometry are based on the same fundamental principles and even use some common hardware. However, spectral flow cytometry is a more advanced technique and offers several advantages over the conventional technique.

Here’s a look at the differences between conventional and spectral flow cytometry.

Conventional flow cytometry:

• Employs mirrors and optical filters to reflect, block, or transmit a photon based on its wavelength.
• Uses a one detector – one fluorophore setup where each detector is assigned to a specific fluorophore.
• Detectors used are individual photodiode (PD) or photomultiplier tube (PMT).
• Only a fraction of the fluorophore emission spectra is captured and recorded.
• Is capable of detecting signals from specific fluorophore over defined wavelengths.
• Fluorophores with similar emission spectra are different to distinguish.
• Autofluorescence can hamper data analysis, particularly in larger and more granular cells. 

Spectral flow cytometry:

• Employs dispersive optics such as spectrographs or prisms to disperse photons according to wavelength across an array of detectors.
• All detectors in the cytometer capture a signature of the full emission spectra of each fluorophore.
• Uses a linear array of detectors to measure the continuous spectrum generated by dispersive elements. Common detectors used are arrays of charge coupled devices (CCDs) and multianode PMTs
• Spectral flow cytometers are capable of detecting multi-parametric panels with more than 30 different fluorophores.
• Is capable of collecting the entire spectral profile of fluorophores from multiple lasers. 
• Fluorophores with similar emission spectra can be easily distinguished.
• Autofluorescence is treated as another color and can be removed through spectral unmixing and analysis, allowing for clearer identification of low-expressing targets and dim fluorescence signals.