BD FACSCanto - Fluorescence

112 BD FACSCanto Flow Cytometer Reference Manual
Fluorescence
When cells or particles stained with fluorochrome-conjugated antibodies or other
dyes pass through a laser beam, the dyes can absorb photons (energy) and be
promoted to an excited electronic state. In returning to their ground state, the
dyes release energy, most of which is emitted as light. This light emission is
known as fluorescence.
Fluorescence is always a longer wavelength (lower-energy photon) than the
excitation wavelength. Some fluorescent compounds emit at a much longer
wavelength than their excitation wavelength. PerCP absorbs blue light (488 nm)
and emits red light (675 nm); other fluorochromes, such as FITC, absorb blue
light (488 nm) and emit green light (530 nm). These differences between
excitation and emission allow one laser to excite many fluorochromes.
The emission spectra for some commonly used fluorochromes are shown in
Figure A-5.
Figure A-5 Emission spectra of commonly used fluorochromes
0%
100%
400
500
700600 800
FITC APC PerCP-Cy5.5
PE-Cy7
APC-Cy7
PE-Cy7
APC-Cy7
PE
wavelength (nm)
normalized intensity