1. Slide 1 t:/powerpnt/course/lect4.ppt 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 October 27, 1998 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:/powerpnt/course/lect4.ppt Purdue University Cytometry Laboratories Overview Fluorescence Types of fluorescent probes Problems with fluorochromes General applications

3. Slide 3 t:/powerpnt/course/lect4.ppt 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:/powerpnt/course/lect4.ppt Purdue University Cytometry Laboratories Light Sources - Lasers ArgonAr353-461, 488, 514 nm Krypton-ArKr-Ar488, 568, 647 nm Helium-NeonHe-Ne633 nm He-CadmiumHe-Cd325 - 441 nm (He-Cd light difficult to get 325 nm band through some optical systems) LaserAbbrev.Excitation Lines

5. Slide 5 t:/powerpnt/course/lect4.ppt Purdue University Cytometry Laboratories Arc Lamp Excitation Spectra Irradiance at 0.5 m (mW m -2 nm -1 )         Xe Lamp Hg Lamp

6. Slide 6 t:/powerpnt/course/lect4.ppt Purdue University Cytometry Laboratories Fluorescence What is it? Where does it come from? Advantages Disadvantages

7. Slide 7 t:/powerpnt/course/lect4.ppt 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

8. Slide 8 t:/powerpnt/course/lect4.ppt 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

9. Slide 9 t:/powerpnt/course/lect4.ppt 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)

10. Slide 10 t:/powerpnt/course/lect4.ppt 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 ) At this power, FITC would have 63% of its molecules in an excited state and 37% in ground state at any one time

11. Slide 11 t:/powerpnt/course/lect4.ppt 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. The dominant effect in flow cytometry is the stretch of the O-H bonds of water. At 488 nm excitation this would give emission at 592 nm

12. Slide 12 t:/powerpnt/course/lect4.ppt Purdue University Cytometry Laboratories Rayleigh Scatter Molecules and very small particles do not absorb, but scatter light in the visible region Rayleigh scattering is directly proportional to the electric dipole and inversely proportional to the 4th power of the wavelength of the incident light e.g. the sky looks blue because the gas molecules scatter more light at shorter (blue) rather than longer wavelengths

13. Slide 13 t:/powerpnt/course/lect4.ppt 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)

14. Slide 14 t:/powerpnt/course/lect4.ppt Purdue University Cytometry Laboratories Photobleaching example FITC - at 4.4 x 10 23 photons cm -2 sec -1 FITC bleaches with a quantum efficiency Q b of 3 x 10 -5 Therefore 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

15. Slide 15 t:/powerpnt/course/lect4.ppt 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)

16. Slide 16 t:/powerpnt/course/lect4.ppt 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.

17. Slide 17 t:/powerpnt/course/lect4.ppt Purdue University Cytometry Laboratories Probes for Proteins FITC488525 PE488525 APC630650 PerCP ™ 488680 Cascade Blue360450 Coumerin-phalloidin350450 Texas Red ™ 610630 Tetramethylrhodamine-amines 550575 CY3 (indotrimethinecyanines) 540575 CY5 (indopentamethinecyanines) 640670 ProbeExcitationEmission

18. Slide 18 t:/powerpnt/course/lect4.ppt Purdue University Cytometry Laboratories Probes for Nucleic Acids Hoechst 33342 (AT rich) (uv) Dapi (uv) PI (uv/vis) Acridine Orange (vis) TOTO-1, YOYO-3, BOBO (vis) Pyrine Y (vis) Thiazole Orange (vis)

19. Slide 19 t:/powerpnt/course/lect4.ppt 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

20. Slide 20 t:/powerpnt/course/lect4.ppt Purdue University Cytometry Laboratories pH Sensitive Indicators SNARF-1488575 BCECF488525/620 440/488525 [2’,7’-bis-(carboxyethyl)-5,6-carboxyfluorescein] ProbeExcitationEmission

21. Slide 21 t:/powerpnt/course/lect4.ppt 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

22. Slide 22 t:/powerpnt/course/lect4.ppt 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

23. Slide 23 t:/powerpnt/course/lect4.ppt 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

24. Slide 24 t:/powerpnt/course/lect4.ppt 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

25. Slide 25 t:/powerpnt/course/lect4.ppt 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

26. Slide 26 t:/powerpnt/course/lect4.ppt Purdue University Cytometry Laboratories Conclusions Confocal Microscopes are designed to use fluorescence 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