Fluorescence and Fluorochromes Peter O’Toole Tel:

Main Principles Fluorescence Fluorophores, native or man made Excite with one colour (wavelength A) Emits with a different colour (wavelength B) Different fluorophores have different colour properties Use specialised filters to split colours to see specific fluorescent probes Use of new Fluorescent Proteins (XFPs e.g. GFP)

Fluorescence - Photon Release Electron excited form ground state by absorption of light Fluorescence observed as electron decays - photon release Energy lost so light emitted at a longer wavelength

Fluorescence excitation emission non-radiative(quenching) Ground State Excited State

Fluorescein – A Typical Fluorescent Probe

FITCPE

Fluorescent Properties Absorption Efficiency –Chose to suit lasers Emission Properties –Peak and broadness Quantum Efficiency – they do not always fluoresce! Environment Dependence –pH –Binding properties Bleaching

Why do we need fluorescence in flow cytometry? Many cells appear the same Fluorescence enables us to mark specific components/particles –Identify and characterise sub-populations Fluorescence enables quantification Enables specific discrimination –e.g. live/dead, cell cycle

Identify sub populations

Fluorescence Quantification e.g. DNA

Morphological Information …..but statistics?

Fluorochromes Used to label covalently other probes –e.g. fluorescein attached to an antibody Used to label cell components directly –e.g. propidium iodide which binds to DNA Used to explore their environment –e.g. pH sensitive dyes

Fluorochromes used to label nucleic acids Propidium Iodide (PI)blueredDNA DRAQ5orangeredDNA (viable cells) Chromomycin A3violetblueDNA (chromosome analysis) Hoechst 33258UVblueDNA (chromosome analysis) Hoechst 33342UVblueDNA (viable cells) DAPIUVblueDNA Acridine Orange (AO)bluegreenDNA redRNA FluorophoreExcited byEmitused for

Typical Fluorochromes (antibodies labels) Fluorescein (FITC) 512 green Alexa green Phycoerythrin (PE) 565 yellow Cyanine 3(Cy3)570 yellow PE-Texas Red (ECD) 620 red PE-Cy5 (PC5)665 deep red Peridin-chlorophyll (PerCP)670 deep red PE-Cy5.5 (PC5.5)695 deep red PE-Cy7 (PC7)755 far red 488 nm excitation 633 nm Allophycyanin APC 660 Cy5 670 APC – Cy nm Alexa Pacific Blue (PB) 440 Cascade Blue (CB) 440

FITCAPC

non-radiative(quenching) excited states ground state AB Energy transfer Molecule A absorbs light and is excited excitation transfer A passes the energy onto molecule B emission Molecule B emits light

Energy transfer excitation emission transfer A B phycoerythrin-Texas RedECD phycoerythrin-cyanine5PC5

Fluorescence Many colours Many probes –Antibody stains, DNA stains, ion dyes… Many uses in flow cytometry –Immunophenotyping, cell cycle, calcium flux, apoptosis, transfection, receptor quantification, protein interaction, cell proliferation… Many uses in microscopy and spectroscopy

Lipid Droplets