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Overview

• • • • Fluorescence The fluorescent microscope Types of fluorescent probes Problems with fluorochromes • General applications

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)

Fluorescence
• Chromophores are components of molecules which absorb light • They are generally aromatic rings

Fluorescence • • • • What is it? Where does it come from? Advantages Disadvantages .

Fluorescence T 1. IsC T1 PH IsC S0 [Vibrational sublevels] ABS .Fluorescence Jablonski Diagram Singlet States Triplet States Vibrational energy levels Rotational energy levels Electronic energy levels S2 T2 ENERGY S1 ABS FL I.C.Nonradiative Internal Conversion IsC ..2 .Singlet Electronic Energy Levels FL .Corresponding Triplet States I.2 .C.1.Absorbance S 0.Intersystem Crossing PH .Phosphorescence .

Simplified Jablonski Diagram S’ 1 S1 Energy hvex hvem S0 .

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 .

Fluorescence Excitation Spectra Intensity related to the probability of the event Wavelength the energy of the light absorbed or emitted .

5 nm (HeNe) 700 nm Excitation Emisson .Allophycocyanin (APC) Protein 300 nm 400 nm 500 nm 600 nm 632.

Arc Lamp Excitation Spectra Xe Lamp Irradiance at 0.5 m (mW m-2 nm-1) 102 101 1 Hg Lamp 10−1 200 400 600 800 .

Texas Red PI Ethidium PE FITC cis-Parinaric acid .350 300 nm 457 488 514 400 nm 500 nm 610 632 600 nm 700 nm Common Laser Lines PE-TR Conj.

Fluorescence Stokes Shift – is the energy difference between the lowest energy peak of absorbence and the highest energy of emission Fluorescnece Intensity Fluorescein molecule Stokes Shift is 25 nm 495 nm 520 nm Wavelength .

514 nm 488.Lasers Laser • • • • Argon Krypton-Ar Helium-Neon He-Cadmium Abbrev.Light Sources .441 nm (He-Cd light difficult to get 325 nm band through some optical systems) . 633 nm 325 . 647 nm 543 nm. 568. 488. Ar Kr-Ar He-Ne He-Cd Excitation Lines 353-361.

fluorescence intensity is proportional to the product of ε and Qf .Parameters • Extinction Coefficient – ε 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.

Excitation Saturation • The rate of emission is dependent upon the time the molecule remains within the excitation state (the excited state lifetime τf) Optical saturation occurs when the rate of excitation exceeds the reciprocal of τf In a scanned image of 512 x 768 pixels (400. 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) • • • • .000 pixels) if scanned in 1 second requires a dwell time per pixel of 2 x 10-6 sec.

How many Photons? • Consider 1 mW of power at 488 nm focused to a Gaussian spot whose radius at 1/e2 intensity is 0.25μm via a 1.25 x 10-4 cm)2]= 5.25 NA objective • The peak intensity at the center will be 10-3W [π. FITC would have 63% of its molecules in an excited state and 37% in ground state at any one time .25 x 1024 photons/(cm2 sec-1) • At this power.1 x 105 W/cm2 or 1.(0.

At 488 nm excitation this would give emission at 575-595 nm . • The dominant effect in flow cytometry is the stretch of the O-H bonds of water.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.

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) .

high NA objective Use wide emission filters Reduce excitation intensity Use “antifade” reagents (not compatible with viable cells) .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.

16 scans would cause 650% bleaching .2 x 103 sec-1 so 37% of the molecules would remain after 240 μsec of irradiation. • In a single plane.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.Photobleaching example • FITC .at 4.

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. imidazole. reduced glutathione. cysteamine. hydroquinone. pphenylenediamine are used • Reduce O2 concentration or use singlet oxygen quenchers such as carotenoids (50 mM crocetin or etretinate in cell cultures). trolox (vitamin E analogue) . ascorbate. uric acid. histidine.

Emission Peaks Fluorophore EXpeak % Max Excitation at 488 568 647 nm EM peak FITC Bodipy Tetra-M-Rho L-Rhodamine Texas Red CY5 496 503 554 572 592 649 518 511 576 590 610 666 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.Excitation . .

Fluorescent Microscope Arc Lamp EPI-Illumination Excitation Diaphragm Excitation Filter Ocular Dichroic Filter Objective Emission Filter .

Fluorescence Microscope with Color Video (CCD) 35 mm Camera .

.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).

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Probes for Proteins Probe FITC PE APC PerCP™ Cascade Blue Coumerin-phalloidin Texas Red™ Tetramethylrhodamine-amines Excitation 488 488 630 488 360 350 610 550 540 640 525 575 650 680 450 450 630 575 575 670 Emission CY3 (indotrimethinecyanines) CY5 (indopentamethinecyanines) .

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) 346 359 434 491 460 502 509 514 526 536 555 460 461 456 509 650 536 525 533 604 620 655 .

Hoechst. quinacrine – GC rich: antibiotics bleomycin. chromamycin A3. mithramycin.DNA Probes • AO – Metachromatic dye • concentration dependent emission • double stranded NA .Green • single stranded NA . rhodamine 800 . olivomycin.Red • AT/GC binding dyes – AT rich: DAPI.

Probes for Ions • • • • INDO-1 QUIN-2 Fluo-3 Fura -2 Ex350 Ex350 Ex488 Ex330/360 Em405/480 Em490 Em525 Em510 .

pH Sensitive Indicators Probe Excitation Emission • SNARF-1 • BCECF 488 488 440/488 575 525/620 525 [2’.7’-bis-(carboxyethyl)-5.6-carboxyfluorescein] .

hydroethidine .dichlorofluorescin diacetate .Probes for Oxidation States Probe Oxidant Excitation Emission • DCFH-DA • HE • DHR 123 (H2O2) (O2-) (H2O2) 488 488 488 525 590 525 DCFH-DA HE DHR-123 .dihydrorhodamine 123 .

nitrobenzoxadiazole DPH .borate-dipyrromethene complexes NBD .trimethylammonium .Specific Organelle Probes Probe Site Excitation Emission BODIPY NBD DPH TMA-DPH Rhodamine 123 DiO diI-Cn-(5) diO-Cn-(3) Golgi Golgi Lipid Lipid 505 488 350 350 Mitochondria 488 Lipid 488 Lipid 550 Lipid 488 511 525 420 420 525 500 565 500 BODIPY .diphenylhexatriene TMA .

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 .Green Fluorescent Protein – GFP is from the chemiluminescent jellyfish Aequorea victoria – excitation maxima at 395 and 470 nm (quantum efficiency is 0.Other Probes of Interest • GFP .

g.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. Calcein AM and Ethidium (ex 488) – emissions 530 nm and 617 nm .

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 .

Fluorescence Resonance Energy Transfer Molecule 1 Fluorescence Molecule 2 Fluorescence ACCEPTOR Intensity DONOR Absorbance Absorbance Wavelength .

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 .Conclusions • Fluorescence is the primary energy source for confocal microscopes • Dye molecules must be close to.