Introduction
34 NovoCyte Quanteon Flow Cytometer Operator's Guide
• SSC - indicates the internal complexity or granularity of the cells or particles
Figure 6. Forward Scatter (FSC) and Side Scatter (SSC)
Fluorescence
When a cell or particle stained with fluorochrome-conjugated antibodies or other
fluorescent labels passes through the focused laser beam, the fluorochrome or
fluorescent labels (in general called dyes in this guide) can absorb photons
(energy) and be excited to a higher energy state. This higher energy state is not
stable so that the dyes will return to their ground state shortly, accompanied by the
release of energy. Most energy is released in the form of emitted light, whereas a
very small portion of the energy is released in the form of heat. The light emission
is known as fluorescence.
Fluorescence is always a longer wavelength (lower-energy photon) compared to
the excitation wavelength. The difference between the excitation maximum and
the emission maximum is known as the Stokes shift. Some fluorochromes such
as FITC have a smaller Stokes shift, absorbing blue light (488 nm) and emitting
green light (525 nm), while other fluorochromes such as PerCP exhibit a larger
Stokes shift, absorbing blue light (488 nm) and emitting red light (675 nm).
The emission spectra for some commonly used fluorochromes are shown in
Figure 7.
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