What is the Stokes shift in flow cytometry?

Stokes Shift in Flow Cytometry

The Stokes shift refers to the difference in wavelength between the peak excitation and peak emission of a fluorophore used in flow cytometry. A fluorophore is a molecule that can re-emit light upon light excitation. Excitation occurs when a photon of a certain wavelength is absorbed by the fluorophore, which elevates the fluorophore to a higher energy state. As the fluorophore returns to its ground state, it emits a photon of light, which is the emission process.

The significance of Stokes shift is multifold in flow cytometry:

• It enables the separation of the excitation light from the emitted fluorescence, allowing for more accurate and sensitive measurement of the fluorescence signal.
• It permits multiplexing, where multiple fluorophores, each with different Stokes shifts, can be used simultaneously to evaluate several parameters of a single cell.

Stokes shift is quantified by the wavelength difference (usually measured in nanometers) between the absorption peak and the emission peak. A larger Stokes shift is generally beneficial as it minimizes the overlap between the emission spectrum of one fluorophore and the excitation spectrum of another, reducing spectral spillover.

In flow cytometry, lasers are used as the excitation source, and detectors are equipped with filters to isolate the emission wavelengths. The Stokes shift can be easily visualized on a fluorescence spectrum graph, where the separation between the excitation peak and the emission peak is clearly depicted.

The flow cytometer can distinguish each fluorophore's emission due to the unique spectral properties conferred by the Stokes shift. Optimizing fluorophore panels involves taking into consideration the Stokes shift to minimize overlap and maximize detection capabilities.

Example Stokes Shifts for Common Fluorophores Used in Flow Cytometry