Transcript Document
Fluorescence and Fluorochromes Peter O’Toole [email protected] Tel: 01904 328722 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 Fluorescein – A Typical Fluorescent Probe 120 Intensity 100 80 60 40 20 0 380 480 nm 580 680 PE 120 120 100 100 80 80 Intensity Intensity FITC 60 60 40 40 20 20 0 0 380 480 nm 580 680 390 490 590 nm 690 790 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? PE-Cy7 Log Comp 10 4 10 3 10 2 10 1 10 0 100 101 102 103 PE Log Comp 104 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 Fluorophore Excited by Propidium Iodide (PI) blue DRAQ5 orange Chromomycin A3 violet Hoechst 33258 UV Hoechst 33342 UV DAPI UV Acridine Orange (AO)blue Emit used for red red blue blue blue blue green red DNA DNA (viable cells) DNA (chromosome analysis) DNA (chromosome analysis) DNA (viable cells) DNA DNA RNA Typical Fluorochromes (antibodies labels) 488 nm excitation 633 nm Fluorescein (FITC) 512 green Allophycyanin APC 660 Alexa 488 515 green Cy5 670 Phycoerythrin (PE) 565 yellow APC – Cy7 770 Cyanine 3 (Cy3) 570 yellow PE-Texas Red (ECD) 620 red 405 nm PE-Cy5 (PC5) Alexa 405 440 Pacific Blue (PB) 440 665 deep red Peridin-chlorophyll (PerCP)670 deep red PE-Cy5.5 (PC5.5) 695 deep red PE-Cy7 (PC7) 755 far red Cascade Blue (CB) 440 100 Intensity 80 60 40 20 0 380 480 nm 580 680 100 Intensity 80 60 40 20 0 380 480 nm 580 680 APC 120 120 100 100 80 80 Intensity Intensity FITC 60 60 40 40 20 20 0 0 380 480 nm 580 680 380 480 nm580 680 Energy transfer transfer excitation A phycoerythrin-Texas Red phycoerythrin-cyanine5 emission B ECD PC5 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 256 SSC Lin 192 128 64 0 0 64 128 FSC Lin 192 256 4372 Counts 3279 2186 1093 0 100 101 102 FL 3 Log 103 104