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 How do we see it? • Dichroic and Filter System • Use specialised filters to split colours to see specific fluorescent probes Long Pass Filter • Typically permits transmission of all light above a set wavelength e.g. 500nm • Used for single labelled samples and for maximum light gain • Short Pass Filter Long Pass Short Band Pass Filter • Permits transmission of light between two defined wavelengths e.g. 530 + 15nm • Used for multiple labelled samples or to help reduce background fluorescence Band Pass Dichroic Filter • Reflects light up to one wavelength and transmits light beyond specified wavelength or vice versa • Used to excite sample with one wavelength, but also enables emission light to be directed to detector Dichroic No filter or dichroic is perfect! Always use controls FITC and PE 120 FITC PE 100 80 60 40 20 0 490 540 590 640 690 How does it work? Fluorescence Detection Forward Scatter Sideward Scatter Fluorescence Detection Reproduced from Terry Hoy Flow Detectors • Photomultiplier Tubes (PMT) and Photodiodes – PMTs are colour blind! They generate electron when photons are present, which in turn is converted into a digital signal. Therefore colours seen on the monitor is a pseudo colour. • Other Fluorescence Detectors – – – – Eyes Photographic Film Charge Couple Devices (CCD) Photodiodes 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 Limitations • Cost of Lasers • Restricted fluorochromes for lasers – Excitation wavelength – Broad emission – Spectral shifts • Number of particles to be studied Solutions • • • • More Lasers Conjugate FRET probes Novel probes Novel solutions Multi-line Systems • Multi-line systems • Many more probes available • Sequential analysis Tandem Dyes / FRET conjugates Energy transfer e.g. PE e.g. PE-Cy5 Tandem Dyes (FRET Probes) • e.g. – – – – PE-TxR PE-Cy5 PE-Cy7 APC-Cy7 Energy transfer transfer excitation A phycoerythrin-Texas Red phycoerythrin-cyanine5 emission B ECD PC5 Tandem Dyes 120 PE PE-Cy5 100 80 60 40 20 0 500 550 600 650 700 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 Thank you 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 Limitations of Fluorescence Imaging and Localisation Studies Poor Data MIT professor sacked for fabricating data! New Scientist - ‘A high-flying researcher has been fired from the prestigious Massachusetts Institute of Technology in Boston for fabricating data’ Immunity vol 8 pg 265 ………….one of at least 3 different papers But……. Not fraudulent, just plain wrong! Not fraudulent, just plain wrong! 25% positive 0% positive