confocal course lecture 4
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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 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. UPDATED January 2000 © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 1 t:/classes/BMS524/524lect3.ppt Overview • • • • Fluorescence The fluorescent microscope Types of fluorescent probes Problems with fluorochromes • General applications © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 2 t:/classes/BMS524/524lect3.ppt Excitation Sources Excitation Sources Lamps Xenon Xenon/Mercury Lasers Argon Ion (Ar) Krypton (Kr) Helium Neon (He-Ne) Helium Cadmium (He-Cd) Krypton-Argon (Kr-Ar) © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 3 t:/classes/BMS524/524lect3.ppt Fluorescence • Chromophores are components of molecules which absorb light • They are generally aromatic rings © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 4 t:/classes/BMS524/524lect3.ppt Fluorescence • • • • What is it? Where does it come from? Advantages Disadvantages © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 5 t:/classes/BMS524/524lect3.ppt Fluorescence Jablonski Diagram Singlet States Triplet States Vibrational energy levels Rotational energy levels Electronic energy levels S2 ENERGY T2 S1 IsC T1 ABS FL I.C. PH IsC S0 [Vibrational sublevels] ABS - Absorbance S 0.1.2 - Singlet Electronic Energy Levels FL - Fluorescence T 1,2 - Corresponding Triplet States I.C.- Nonradiative Internal Conversion IsC - Intersystem Crossing PH - Phosphorescence © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 6 t:/classes/BMS524/524lect3.ppt Simplified Jablonski Diagram S’ 1 S1 hvex hvem S0 © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 7 t:/classes/BMS524/524lect3.ppt 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 © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 8 t:/classes/BMS524/524lect3.ppt Fluorescence Excitation Spectra Intensity related to the probability of the event Wavelength the energy of the light absorbed or emitted © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 9 t:/classes/BMS524/524lect3.ppt Allophycocyanin (APC) Protein 300 nm 400 nm 500 nm © J.Paul Robinson - Purdue University Cytometry Laboratories 632.5 nm (HeNe) 600 nm 700 nm Excitation Emisson Slide 10 t:/classes/BMS524/524lect3.ppt Arc Lamp Excitation Spectra Xe Lamp Irradiance at 0.5 m (mW m-2 nm-1) Hg Lamp © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 11 t:/classes/BMS524/524lect3.ppt 350 300 nm 457 488 514 400 nm 500 nm Common Laser Lines 610 632 600 nm 700 nm PE-TR Conj. Texas Red PI Ethidium PE FITC cis-Parinaric acid © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 12 t:/classes/BMS524/524lect3.ppt Fluorescence Stokes Shift Fluorescnece Intensity – is the energy difference between the lowest energy peak of absorbence and the highest energy of emission Fluorescein molecule Stokes Shift is 25 nm 495 nm 520 nm Wavelength © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 13 t:/classes/BMS524/524lect3.ppt Light Sources - Lasers Laser • • • • Argon Krypton-Ar Helium-Neon He-Cadmium Abbrev. Ar Kr-Ar He-Ne He-Cd Excitation Lines 353-361, 488, 514 nm 488, 568, 647 nm 543 nm, 633 nm 325 - 441 nm (He-Cd light difficult to get 325 nm band through some optical systems) © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 14 t:/classes/BMS524/524lect3.ppt Parameters • Extinction Coefficient – refers to a single wavelength (usually the absorption maximum) • Quantum Yield – Qf 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 Qf © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 15 t:/classes/BMS524/524lect3.ppt 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) © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 16 t:/classes/BMS524/524lect3.ppt How many Photons? • Consider 1 mW of power at 488 nm focused to a Gaussian spot whose radius at 1/e2 intensity is 0.25m via a 1.25 NA objective • The peak intensity at the center will be 10-3W [.(0.25 x 10-4 cm)2]= 5.1 x 105 W/cm2 or 1.25 x 1024 photons/(cm2 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 © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 17 t:/classes/BMS524/524lect3.ppt 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 575-595 nm © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 18 t:/classes/BMS524/524lect3.ppt 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) © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 19 t:/classes/BMS524/524lect3.ppt 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) © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 20 t:/classes/BMS524/524lect3.ppt Photobleaching example • FITC - at 4.4 x 1023 photons cm-2 sec-1 FITC bleaches with a quantum efficiency Qb of 3 x 10-5 • Therefore FITC would be bleaching with a rate constant of 4.2 x 103 sec-1 so 37% of the molecules would remain after 240 sec of irradiation. • In a single plane, 16 scans would cause 650% bleaching © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 21 t:/classes/BMS524/524lect3.ppt 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, pphenylenediamine are used • Reduce O2 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) © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 22 t:/classes/BMS524/524lect3.ppt Excitation - Emission Peaks Fluorophore FITC Bodipy Tetra-M-Rho L-Rhodamine Texas Red CY5 EXpeak 496 503 554 572 592 649 % Max Excitation at 488 568 647 nm EM peak 518 511 576 590 610 666 87 58 10 5 3 1 0 1 61 92 45 11 0 1 0 0 1 98 Note: You will not be able to see CY5 fluorescence under the regular fluorescent microscope because the wavelength is too high. © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 23 t:/classes/BMS524/524lect3.ppt Fluorescent Microscope Arc Lamp EPI-Illumination Excitation Diaphragm Excitation Filter Ocular Dichroic Filter Objective Emission Filter © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 24 t:/classes/BMS524/524lect3.ppt Fluorescence Microscope with Color Video (CCD) © J.Paul Robinson - Purdue University Cytometry Laboratories 35 mm Camera Slide 25 t:/classes/BMS524/524lect3.ppt Cameras and emission filters Cooled color CCD camera Camera goes here 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). © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 26 t:/classes/BMS524/524lect3.ppt © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 27 t:/classes/BMS524/524lect3.ppt Probes for Proteins Probe FITC PE APC PerCP™ Cascade Blue Coumerin-phalloidin Texas Red™ Tetramethylrhodamine-amines CY3 (indotrimethinecyanines) CY5 (indopentamethinecyanines) © J.Paul Robinson - Purdue University Cytometry Laboratories Excitation 488 488 630 488 360 350 610 550 540 640 Emission 525 575 650 680 450 450 630 575 575 670 Slide 28 t:/classes/BMS524/524lect3.ppt Probes for Nucleic Acids • • • • • • • • • • • Hoechst 33342 (AT rich) (uv) DAPI (uv) POPO-1 YOYO-1 Acridine Orange (RNA) Acridine Orange (DNA) Thiazole Orange (vis) TOTO-1 Ethidium Bromide PI (uv/vis) 7-Aminoactinomycin D (7AAD) © J.Paul Robinson - Purdue University Cytometry Laboratories 346 359 434 491 460 502 509 514 526 536 555 460 461 456 509 650 536 525 533 604 620 655 Slide 29 t:/classes/BMS524/524lect3.ppt 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 A3, mithramycin, olivomycin, rhodamine 800 © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 30 t:/classes/BMS524/524lect3.ppt Probes for Ions • • • • INDO-1 QUIN-2 Fluo-3 Fura -2 Ex350 Ex350 Ex488 Ex330/360 © J.Paul Robinson - Purdue University Cytometry Laboratories Em405/480 Em490 Em525 Em510 Slide 31 t:/classes/BMS524/524lect3.ppt pH Sensitive Indicators Probe Excitation Emission • SNARF-1 488 575 • BCECF 488 440/488 525/620 525 [2’,7’-bis-(carboxyethyl)-5,6-carboxyfluorescein] © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 32 t:/classes/BMS524/524lect3.ppt Probes for Oxidation States Probe Oxidant • DCFH-DA • HE • DHR 123 (H2O2) (O2-) (H2O2) DCFH-DA HE DHR-123 Excitation Emission 488 488 488 525 590 525 - dichlorofluorescin diacetate - hydroethidine - dihydrorhodamine 123 © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 33 t:/classes/BMS524/524lect3.ppt Specific Organelle Probes Probe BODIPY NBD DPH TMA-DPH Rhodamine 123 DiO diI-Cn-(5) diO-Cn-(3) Site Golgi Golgi Lipid Lipid Excitation 505 488 350 350 Mitochondria 488 Lipid 488 Lipid 550 Lipid 488 Emission 511 525 420 420 525 500 565 500 BODIPY - borate-dipyrromethene complexes NBD - nitrobenzoxadiazole DPH - diphenylhexatriene TMA - trimethylammonium © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 34 t:/classes/BMS524/524lect3.ppt 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 © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 35 t:/classes/BMS524/524lect3.ppt 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 © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 36 t:/classes/BMS524/524lect3.ppt 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 © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 37 t:/classes/BMS524/524lect3.ppt Fluorescence Resonance Energy Transfer Molecule 1 Molecule 2 Fluorescence Fluorescence ACCEPTOR DONOR Absorbance Absorbance Wavelength © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 38 t:/classes/BMS524/524lect3.ppt 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 © J.Paul Robinson - Purdue University Cytometry Laboratories Slide 39 t:/classes/BMS524/524lect3.ppt
