Flow Cytometry Fluorescence

Immunophenotyping of cells via flow cytometry relies on release of fluorescent signals from fluorochromes bound to monoclonal antibodies. When these fluorochromes are excited by specific wavelengths of light (the excitation or absorption wavelength of a fluorochrome), they absorb the light, gaining energy and releasing it as photons of light. The emitted wavelength can be recorded and analysed to determine the phenotype of the cell.

Flow Cytometry: Excitation and Emission Wavelengths
Chart showing how excitation and emission wavelengths differ. Fluorochromes are excited by specific wavelengths of light (red curve). This energy is stored and released as an emission wavelength (blue curve). The difference between the maximal absorption / excitation and emission spectra is known as Stoke’s shift. Different colours can be used to trigger a varying number of fluorochromes, increasing the number of variables that can be characterised simultaneously.

Common Excitation and Emission Wavelengths

FluorochromeExcitation (nm)Emission (nm)
Allophycocyanin (APC)650660
APC-cyanine 7 (APC-Cy7)650785
Fluorescein isothiocyanate (FITC)494519
Pacific blue (PB)410455
Peridinin-chlorophyll (PerCP)482678
PerCP-cyanine 5.5 (PerCP-Cy5.5)482678
Phycoerythrin (PE)496, 546578
PE-cyanine 5 (PE-Cy5)496, 546667
PE-cyanine 5.5 (PE-Cy5.5)495, 564696
PE-cyanine 5 (PE-Cy7)495, 564767

Spectral Overlap

As demonstrated in the figure and table above, light emissions occur over a spectrum. Different fluorochromes can have overlapping emission spectra, which can result in emissions from one fluorochrome spilling over into a detector designed for another fluorochrome. This is known as spectral overlap, and must be corrected by compensation to reduce the analytical interference from this phenomenon.

Figure illustrating how emission wavelengths from two different fluorochromes can overlap, resulting in interference and analytical issues if not compensated for.
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