1. Fluorescence and Fluorochromes Peter O’Toole pot1@york.ac.uk Tel: 01904 328722

2. 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)

3. 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

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

5. Fluorescein – A Typical Fluorescent Probe

6. FITCPE

7. 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

8. How do we see it? Dichroic and Filter System Use specialised filters to split colours to see specific fluorescent probes

9. 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 Pass

10. 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

11. 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

12. No filter or dichroic is perfect! Always use controls

13. Emission Filter (Long Pass or Band Pass) Excitation Filter (Short Pass) Dichroic Mirror Light Source Emission Detector Dichroic and Filter system Sample

14. FITC and PE

15. How does it work? Reproduced from Terry Hoy Forward Scatter Sideward Scatter Fluorescence Detection

16. 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

17. 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

18. Identify sub populations

19. Fluorescence Quantification e.g. DNA

20. Morphological Information …..but statistics?

21. 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

22. 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

23. Typical Fluorochromes (antibodies labels) Fluorescein (FITC) 512 green Alexa 488515 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 – Cy7 770 405 nm Alexa 405 440 Pacific Blue (PB) 440 Cascade Blue (CB) 440

27. FITCAPC

28. Limitations Cost of Lasers Restricted fluorochromes for lasers –Excitation wavelength –Broad emission –Spectral shifts Number of particles to be studied

29. Solutions More Lasers Conjugate FRET probes Novel probes Novel solutions

30. Multi-line Systems Multi-line systems Many more probes available Sequential analysis

31. Tandem Dyes / FRET conjugates

32. Energy transfer e.g. PE e.g. PE-Cy5

33. Tandem Dyes (FRET Probes) e.g. –PE-TxR –PE-Cy5 –PE-Cy7 –APC-Cy7

34. 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

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

37. Tandem Dyes

38. 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

39. Thank you

40. Lipid Droplets

41. Limitations of Fluorescence Imaging and Localisation Studies

42. Poor Data

43. MIT professor sacked for fabricating data! Immunity vol 8 pg 265 New Scientist - ‘A high-flying researcher has been fired from the prestigious Massachusetts Institute of Technology in Boston for fabricating data’ ………….one of at least 3 different papers

44. But…….

45. Not fraudulent, just plain wrong!

46. 25% positive 0% positive