1. Slide 1 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Lecture 3 Fluorescence and Fluorescence Probes BMS 524 - “Introduction to Confocal Microscopy and Image Analysis” 1 Credit course offered by Purdue University Department of Basic Medical Sciences, School of Veterinary Medicine UPDATED January 2000 J.Paul Robinson, Ph.D. Professor of Immunopharmacology Director, Purdue University Cytometry Laboratories These slides are intended for use in a lecture series. Copies of the graphics are distributed and students encouraged to take their notes on these graphics. The intent is to have the student NOT try to reproduce the figures, but to LISTEN and UNDERSTAND the material. All material copyright J.Paul Robinson unless otherwise stated, however, the material may be freely used for lectures, tutorials and workshops. It may not be used for any commercial purpose. The text for this course is Pawley “Introduction to Confocal Microscopy”, Plenum Press, 2nd Ed. A number of the ideas and figures in these lecture notes are taken from this text.

2. Slide 2 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Overview Fluorescence The fluorescent microscope Types of fluorescent probes Problems with fluorochromes General applications

3. Slide 3 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Excitation Sources Lamps Xenon Xenon/Mercury Lasers Argon Ion (Ar) Krypton (Kr) Helium Neon (He-Ne) Helium Cadmium (He-Cd) Krypton-Argon (Kr-Ar)

4. Slide 4 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Fluorescence Chromophores are components of molecules which absorb light They are generally aromatic rings

5. Slide 5 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Fluorescence What is it? Where does it come from? Advantages Disadvantages

6. Slide 6 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Fluorescence ENERGY S0S0 S1S1 S2S2 T2T2 T1T1 ABS FL I.C. ABS - AbsorbanceS 0.1.2 - Singlet Electronic Energy Levels FL - FluorescenceT 1,2 - Corresponding Triplet States I.C.- Nonradiative Internal ConversionIsC - Intersystem CrossingPH - Phosphorescence IsC PH [Vibrational sublevels] Jablonski Diagram Vibrational energy levels Rotational energy levels Electronic energy levels Singlet StatesTriplet States

7. Slide 7 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Simplified Jablonski Diagram S0S0 S’ 1 Energy S1S1 hv ex hv em

8. Slide 8 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Fluorescence The longer the wavelength the lower the energy The shorter the wavelength the higher the energy eg. UV light from sun causes the sunburn not the red visible light

9. Slide 9 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Fluorescence Excitation Spectra Intensity related to the probability of the event Wavelength the energy of the light absorbed or emitted

10. Slide 10 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Allophycocyanin (APC) Protein 632.5 nm (HeNe ) Excitation Emisson 300 nm 400 nm 500 nm 600 nm 700 nm

11. Slide 11 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Arc Lamp Excitation Spectra Irradiance at 0.5 m (mW m -2 nm -1 )         Xe Lamp Hg Lamp

12. Slide 12 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Ethidium PE cis-Parinaric acid Texas Red PE-TR Conj. PI FITC 600 nm300 nm500 nm700 nm400 nm 457350514610632488 Common Laser Lines

13. Slide 13 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Fluorescence Stokes Shift –is the energy difference between the lowest energy peak of absorbence and the highest energy of emission 495 nm 520 nm Stokes Shift is 25 nm Fluorescein molecule Fluorescnece Intensity Wavelength

14. Slide 14 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Light Sources - Lasers ArgonAr353-361, 488, 514 nm Krypton-ArKr-Ar488, 568, 647 nm Helium-NeonHe-Ne543 nm, 633 nm He-CadmiumHe-Cd325 - 441 nm (He-Cd light difficult to get 325 nm band through some optical systems) LaserAbbrev.Excitation Lines

15. Slide 15 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Parameters Extinction Coefficient –  refers to a single wavelength (usually the absorption maximum) Quantum Yield –Q f is a measure of the integrated photon emission over the fluorophore spectral band At sub-saturation excitation rates, fluorescence intensity is proportional to the product of  and Q f

16. Slide 16 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Excitation Saturation The rate of emission is dependent upon the time the molecule remains within the excitation state (the excited state lifetime  f ) Optical saturation occurs when the rate of excitation exceeds the reciprocal of  f In a scanned image of 512 x 768 pixels (400,000 pixels) if scanned in 1 second requires a dwell time per pixel of 2 x 10 -6 sec. Molecules that remain in the excitation beam for extended periods have higher probability of interstate crossings and thus phosphorescence Usually, increasing dye concentration can be the most effective means of increasing signal when energy is not the limiting factor (ie laser based confocal systems)

17. Slide 17 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories How many Photons? Consider 1 mW of power at 488 nm focused to a Gaussian spot whose radius at 1/e 2 intensity is 0.25  m via a 1.25 NA objective The peak intensity at the center will be 10 -3 W [ .(0.25 x 10 -4 cm) 2 ]= 5.1 x 10 5 W/cm 2 or 1.25 x 10 24 photons/(cm 2 sec -1 ) FITCAt this power, FITC would have 63% of its molecules in an excited state and 37% in ground state at any one time

18. Slide 18 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Raman Scatter A molecule may undergo a vibrational transition (not an electronic shift) at exactly the same time as scattering occurs This results in a photon emission of a photon differing in energy from the energy of the incident photon by the amount of the above energy - this is Raman scattering. 488 nm excitation 575-595 nmThe dominant effect in flow cytometry is the stretch of the O-H bonds of water. At 488 nm excitation this would give emission at 575-595 nm

19. Slide 19 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Rayleigh Scatter Molecules and very small particles do not absorb, but scatter light in the visible region (same freq as excitation) Rayleigh scattering is directly proportional to the electric dipole and inversely proportional to the 4th power of the wavelength of the incident light the sky looks blue because the gas molecules scatter more light at shorter (blue) rather than longer wavelengths (red)

20. Slide 20 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Photobleaching Defined as the irreversible destruction of an excited fluorophore (discussed in later lecture) Methods for countering photobleaching –Scan for shorter times –Use high magnification, high NA objective –Use wide emission filters –Reduce excitation intensity –Use “antifade” reagents (not compatible with viable cells)

21. Slide 21 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Photobleaching example FITC FITCFITC - at 4.4 x 10 23 photons cm -2 sec -1 FITC bleaches with a quantum efficiency Q b of 3 x 10 -5 FITCTherefore FITC would be bleaching with a rate constant of 4.2 x 10 3 sec -1 so 37% of the molecules would remain after 240  sec of irradiation. In a single plane, 16 scans would cause 6- 50% bleaching

22. Slide 22 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Antifade Agents Many quenchers act by reducing oxygen concentration to prevent formation of singlet oxygen Satisfactory for fixed samples but not live cells! Antioxidents such as propyl gallate, hydroquinone, p- phenylenediamine are used Reduce O 2 concentration or use singlet oxygen quenchers such as carotenoids (50 mM crocetin or etretinate in cell cultures); ascorbate, imidazole, histidine, cysteamine, reduced glutathione, uric acid, trolox (vitamin E analogue)

23. Slide 23 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Excitation - Emission Peaks Fluorophore EX peak EM peak % Max Excitation at 488568 647 nm FITC4965188700 Bodipy5035115811 Tetra-M-Rho55457610610 L-Rhodamine5725905920 Texas Red5926103451 CY564966611198 Note: You will not be able to see CY5 fluorescence under the regular fluorescent microscope because the wavelength is too high.

24. Slide 24 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Fluorescent Microscope Dichroic Filter Objective Arc Lamp Emission Filter Excitation Diaphragm Ocular Excitation Filter EPI-Illumination

25. Slide 25 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Fluorescence Microscope with Color Video (CCD)35 mm Camera

26. Slide 26 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Cameras and emission filters Color CCD camera does not need optical filters to collect all wavelengths but if you want to collect each emission wavelength optimally, you need a monochrome camera with separate emission filters shown on the right (camera is not in position in this photo). Camera goes here Cooled color CCD camera

27. Slide 27 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories

28. Slide 28 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Probes for Proteins FITC488525 PE488575 APC630650 PerCP ™ 488680 Cascade Blue360450 Coumerin-phalloidin350450 Texas Red ™ 610630 Tetramethylrhodamine-amines 550575 CY3 (indotrimethinecyanines) 540575 CY5 (indopentamethinecyanines) 640670 ProbeExcitationEmission

29. Slide 29 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Hoechst 33342 (AT rich) (uv)346460 DAPI (uv)359461 POPO-1 434456 YOYO-1 491509 Acridine Orange (RNA)460650 Acridine Orange (DNA)502536 Thiazole Orange (vis)509525 TOTO-1514533 Ethidium Bromide526604 PI (uv/vis)536620 7-Aminoactinomycin D (7AAD)555655 Probes for Nucleic Acids

30. Slide 30 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories DNA Probes AO –Metachromatic dye concentration dependent emission double stranded NA - Green single stranded NA - Red AT/GC binding dyes –AT rich: DAPI, Hoechst, quinacrine –GC rich: antibiotics bleomycin, chromamycin A 3, mithramycin, olivomycin, rhodamine 800

31. Slide 31 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Probes for Ions INDO-1 E x 350E m 405/480 QUIN-2E x 350E m 490 Fluo-3 E x 488E m 525 Fura -2E x 330/360E m 510

32. Slide 32 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories pH Sensitive Indicators SNARF-1488575 BCECF488525/620 440/488525 [2’,7’-bis-(carboxyethyl)-5,6-carboxyfluorescein] ProbeExcitationEmission

33. Slide 33 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Probes for Oxidation States DCFH-DA(H 2 O 2 )488525 HE(O 2 - )488590 DHR 123(H 2 O 2 )488525 Probe Oxidant ExcitationEmission DCFH-DA- dichlorofluorescin diacetate HE- hydroethidine DHR-123- dihydrorhodamine 123

34. Slide 34 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Specific Organelle Probes BODIPY Golgi505511 NBD Golgi488525 DPH Lipid350420 TMA-DPH Lipid350420 Rhodamine 123 Mitochondria 488525 DiOLipid488500 diI-Cn-(5)Lipid550565 diO-Cn-(3)Lipid488500 Probe Site Excitation Emission BODIPY - borate-dipyrromethene complexes NBD - nitrobenzoxadiazole DPH - diphenylhexatriene TMA - trimethylammonium

35. Slide 35 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Other Probes of Interest GFP - Green Fluorescent Protein –GFP is from the chemiluminescent jellyfish Aequorea victoria –excitation maxima at 395 and 470 nm (quantum efficiency is 0.8) Peak emission at 509 nm –contains a p-hydroxybenzylidene-imidazolone chromophore generated by oxidation of the Ser-Tyr-Gly at positions 65-67 of the primary sequence –Major application is as a reporter gene for assay of promoter activity –requires no added substrates

36. Slide 36 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Multiple Emissions Many possibilities for using multiple probes with a single excitation Multiple excitation lines are possible Combination of multiple excitation lines or probes that have same excitation and quite different emissions –e.g. Calcein AM and Ethidium (ex 488) –emissions 530 nm and 617 nm

37. Slide 37 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Energy Transfer Effective between 10-100 Å only Emission and excitation spectrum must significantly overlap Donor transfers non-radiatively to the acceptor PE-Texas Red ™ Carboxyfluorescein-Sulforhodamine B

38. Slide 38 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Fluorescence Resonance Energy Transfer Intensity Wavelength Absorbance DONOR Absorbance Fluorescence ACCEPTOR Molecule 1Molecule 2

39. Slide 39 t:/classes/BMS524/524lect3.ppt© J.Paul Robinson - Purdue University Cytometry Laboratories Conclusions Fluorescence is the primary energy source for confocal microscopes Dye molecules must be close to, but below saturation levels for optimum emission Fluorescence emission is longer than the exciting wavelength The energy of the light increases with reduction of wavelength Fluorescence probes must be appropriate for the excitation source and the sample of interest Correct optical filters must be used for multiple color fluorescence emission