Wells, 9/15/03What is fluorescence?Optical and Physical PropertiesDetection and MeasurementChemical properties and Biologicalapplications to be discussed later
Wells, 9/15/03Definition of FluorescenceLight emitted by singlet excited states of molecules followingabsorption of photons from an external sourceRequirements for FluorescenceFluorescent Dye (Fluorophore). A molecule with a rigid conjugatedstructure (usually a polyaromatic hydrocarbon or heterocycle).Excitation. Creation of dye excited state by absorption of a photon.
Wells, 9/15/03Molecular Structure Features and FluorescenceHigh ring density of π electrons increase fluorescence. Aromatichydrocarbons (π to π * absorption) are frequently fluorescent.Increased hydrocarbon conjugation shifts π το π* absorption tothe red and increases the probability of fluorescence.(C3H3) n, n = 3,5,7 Cy3, Cy5, Cy7Protonation (affected by e- withdrawing/donating groups). OHproduces blue shift and decreased Qf in fluorescein. Halogens(e- withdrawing) lower pKa and alkyl groups (e- donating) raise thepKa.Planarity and rigidity affect fluorescence. Increased viscositymay slow free internal rotations and increase fluorescence.C6H5 CH CH C6H5
Wells, 9/15/03Fluorescent PROBES: Tags, Tracers, Sensors, etc.Fluorescent tags generate contrast between an analyte and background, e.g., to identifyreceptors, or decorate cellular anatomy.Fluorescent sensors or indicators change spectral properties in response to dynamicequilibria of chemical reactions, intracellular ions e.g., Ca2+, H+, and membrane potential.
Wells, 9/15/03ANALYTES can evaluated by fluorescence in a wide range ofenvironments, including single molecules in solutions, gels orsolids, cultured cells, thick tissue sections, and live animals.?
Wells, 9/15/03Optical PropertiesSpectral properties of fluorescent probes definetheir utility in biological applications.Fluorescence spectra convey informationregarding molecular identity.Variations in fluorescence spectra characterizechemical and physical behavior.
Wells, 9/15/031 micron spheres viewed under transmitted incandescent “white” lightThe light passing through the sample is diffracted, partiallyabsorbed and transmitted to the detector.
Wells, 9/15/031 micron spheres emitting fluorescenceThe light passing through the sample is diffracted and absorbed, thenFILTERED to detect light originating from molecules within the sample.
Wells, 9/15/03Fluorescence-related TerminologyExcitation – process of absorbing optical energy.Extinction coefficient – efficiency of absorbing optical energy; thisvalue is wavelength dependent.Emission – process of releasing optical energy.Quantum yield – efficiency of releasing energy (0-1), generally in theform of fluorescent light; not wavelength dependent.Stokes shift – difference in energy between excitation and emissionwavelength maxima – the key to contrast generation.Spectrum – distribution of excitation and emission wavelengths
Wells, 9/15/03Spectral Features of Fluorescenceλ2 - λ1 = Stokes shiftF = I0ЄλC0LQfI0(Єλ)a
Absorption and Fluorescence Excitation Spectra:Same or Different?Incident intensity (Io) Transmitted intensity (It)Fluorescence intensity (IF)Absorption spectrum: Scan Io(λ), detect ItFluorescence excitation spectrum: Scan Io(λ), detect IFFluorescenceexcitationFor pure dye molecules in solution, theabsorption and fluorescence excitation spectraare usually identical and can be usedinterchangeably. When more than oneabsorbing or fluorescent species is present inthe sample, they are typically different, as inthe case of protein-conjugatedtetramethylrhodamine dyes, shown right.Absorption500 550 600Wavelength (nm)Iain Johnson [Molecular Probes]
Wells, 9/15/03DETECTION involves only a single observableparameter = INTENSITY.The spectral properties [COLOR] arediscriminated by optical filtering and sometimestemporal filtering prior to measuring the intensity.Therefore, it is extremely important to configurethe filtering and optical collection conditionsproperly to avoid artifacts and faultyobservations!
Wells, 9/15/03Spectral Optimization• Absorption wavelength Optimize excitation by matchingdye absorption to source output (Note: total excitation isthe product of the source power and dye absorption)• Emission wavelength Impacts the ability to discriminatefluorescence signals from probe A versus probe B andbackground autofluorescence• Emission bandwidth Narrow bandwidths minimizeoverspill in multicolor label detection
Wells, 9/15/03Limitations to Sensitivity (S/N)Noise SourcesInstrumentStray light, detector noiseSampleSample–related autofluorescence, scatteredexcitation light (particle size and wavelengthdependent)ReagentUnbound or nonspecifically bound probesSignal LossesInner filter effects, Emission scattering, Photobleaching
Wells, 9/15/03Single T2 genomic DNAmacromolecule stained withYOYO-1 (10 nM loading invery low concentration DNAin agarose). Wide-fieldimage using a 1.4NAobjective and CCD camera.Unfortunately, YOYO-1bleaches very fast.
Wells, 9/15/03Viability2 dyes, 1excitation, 2 emissions, 1 LP filter setcalcien AM (green, live cells), ethidium homodimer (red, dead cells)
Wells, 9/15/03Organelles1 dye, 2 excitation, 2 emissions,switch between 2 filter setsBODIPY FL C5-ceramide accumulation inthe trans-Golgi is sufficient for excimerformation (top panel).Images by Richard Pagano, Mayo Foundation.
Wells, 9/15/03Cytoskeleton3 dyes, 3 excitation, 3 emissionsBPAE cells: microtubules (green, Bodipy-antibody), F-actin (red, Texas Red-Xphalloidin) and nuclei (blue, DAPI). Triple exposure on 35mm film. This can bedone with a triple-band filter set or by 3 filter sets.
Wells, 9/15/031 (UV) excitation “band,” 8 (visible) emission “bands”detected simultaneously on 35mm color filmMacro-image
Wells, 9/15/03Physical Origins of Fluorescence1 Dye excited state created by absorption of a photonfrom an external light source2 Fluorescence photon emitted has lower energy(longer wavelength) than excitation photon. Ratio ofemitted photons/absorbed photons (fluorescencequantum yield) is usually less than 1.03 Unexcited dye is regenerated for repeatabsorption/emission cycles. Cycle can be broken byphotobleaching (irreversible photochemicaldestruction of excited dye)
Wells, 9/15/03Energy Flow: Photons→Electrons→PhotonsDifferent dye moleculesexhibit different excitationand emission “profiles.”Energy isproportionalto 1/λ1 = absorbance of light (excitation)2 = vibronic relaxation, (Stokes shift)3 = emission of light
Wells, 9/15/03Fluorescence Excitation-Emission CycleRelaxationPhosphorescencehνPHS1S1ExcitationhνEXEnergy(hν)T1EmissionhνFLphotoproductsS0S0 = ground electronic stateS1 = first singlet excited stateT1= triplet excited stateRadiative transitionNonradiative transitionIain Johnson [Molecular Probes]
Wells, 9/15/03Molecular Electronic State TransitionsJablonskiDiagramIain Johnson [Molecular Probes]
Wells, 9/15/03• Intermolecular Energy Transfer - characterized by donor-acceptor pairingshort range exchange interactions within interatomic collision diameterlong range interactions by sequential short range exchangeoptical interactions between transition dipoles - decays as 1/r6exciton migration = electron-hole pair migration - decays as 1/r3eximer = complex between excited and ground state of same type moleculesexiplex = complex between excited and ground state of different type molecules• Intramolecular Energy Transferintersystem crossing, e.g., E-type delayed fluorescence, phosphorescenceinternal conversion, e.g., vibronic transitions• Polarized Transitions and Rotational Diffusionangular orientation between excitation and emitted light polarization changeswhen a fluorophore rotates during the excited-state lifetime (τf)Molecular Interactions and FluorescenceexcitationemissionD < 100 Αemission dipoleexcitationtimeexcitation dipole
Wells, 9/15/03Quantitative Fluorometry• Measured fluorescence intensities are products of dye-dependent (ε, QY), sample-dependent (c, L) and instrument-dependent (I0, k) factors• Sample-to-sample and instrument-to-instrument comparisonsshould ideally be made with reference to standard materials suchas fluorescent microspheres• Attempts to increase signal levels by using higher concentrationsof dye may have the opposite effect due to dye–dye interactions(self-quenching) or optical artifacts (e.g. inner filter effects, self-absorption of fluorescence)
Wells, 9/15/03Photometric Output Factors: Absorption• Molar extinction coefficient (aka molar absorptivity).Symbol: ε Units: cm-1 M-1Defined by the Beer-Lambert law log I0/It = ε·c·l• Quantifies efficiency of light absorption at a specificwavelength (εmax = peak value)• Typically 10,000 – 200,000 cm-1 M-1• Not strongly environment dependent
Wells, 9/15/03Photometric Output Factors: Emission• Fluorescence quantum yield– Symbol: QY or φF or QF Units: none• Number of fluorescence photons emitted per photonabsorbed• Typically 0.05 – 1.0• Strongly environment dependent• Other emission parameters: lifetime, polarization
Wells, 9/15/03Quantifying Fluorescence IntensityIF= I0·QY·(1-e–2.303 ε.c.L)·kFor (ε·c·L) < 0.05IF= I0·QY·(2.303·ε·c·L)·k = Fluorescence emission intensity at λEMQY = Fluorophore quantum yieldε = Molar extinction coefficient of fluorophore at λEXc = Fluorophore concentrationL = Optical pathlength for excitationI0= Excitation source intensity at λEXk = Fluorescence collection efficiency**In a typical epifluorescence microscope, about 3% of the total available emittedphotons are collected (k =0.03) [Webb et al, PNAS 94, 11753 (1997)]
Wells, 9/15/03IF will decrease when (ε·c·L) > 0.05Inner Filter Effect Simulation0.00.20.40.60.81.00 0.5 1 1.5 2ε .c.LIntensity1-e-2.303.ε.c.LIF = Io.(1-e-2.303.ε.c.L)IoIain Johnson [Molecular Probes]
Wells, 9/15/03Total Output Limit: Photobleaching• Irreversible destruction of excited fluorophore• Proportional to time-integrated excitation intensity• Avoidance: minimize excitation, maximize detectionefficiency, antifade reagents• QB (photobleaching quantum yield). QF/QB = numberof fluorescence cycles before bleaching. About30,000 for fluorescein.
Photobleaching ReactionsOO1O23Dye* + 3O21Dye +1O2Photosensitized generation of singlet oxygenAnthracene Nonfluorescent endoperoxideThe lifetime of 1O2 in cells is <0.5 µs versus 4 µs in H2O.Why? Iain Johnson [Molecular Probes]
Wells, 9/15/03Photochemistry: everything you didn’t want to know.Cells containing ONLY DAPI may turn green!1st exposure final exposureDIC, t0DAPIchannelfluoresceinchannel
Wells, 9/15/03Avoid dyes when there not essential to the experimentDICfluorescenceDIC + fluorescence
Wells, 9/15/03Antifade ReagentsFixed SpecimensP-phenylene diamineN-propyl gallateDABCOHydroquinoneLive SpecimensMitochondrial particlesBacterial membrane “Oxyrase”Vit. C, ETrolox (water soluble E analogue)OCH3HOH3CCH3CH3C OHOTrolox® is a registered trademark of Hoffman – La Roche
Wells, 9/15/03Guiding Principles for Imaging FluorescenceSelect the proper dye for the job – not always obviousOptimize light collection conditions – use the properoptics, filters, detectors, etc.Minimize excitation exposure – minimize bleaching,reduce sample perturbations, artifactsMaximize the dynamic range – contrast depends on this!Minimize background signal – maximize sensitivity